Chicken embryos are currently being increasingly used in various areas of research but are frequently not covered by animal protection legislation. In the food industry, it is often even common practice to kill male embryos because they are of no economic use. In both cases, there is a lack of knowledge about the sentience of these chicken embryos, especially their ability to perceive pain. The purpose of this exploratory study was to investigate whether—and if so, on which developmental day—a chicken embryo shows a behavioral change or a specific response to a noxious stimulus, both of which would be indicative of functioning nociception. Two complementary approaches were used for the evaluation: human observation and computer-assisted evaluation using a markerless pose estimation software (DeepLabCut). Through a combination of both approaches, it became apparent that developmental day 15 was the earliest stage at which a response to the applied stimulus was detectable. This result thus represents a contribution to the future improvement of animal welfare as it suggests that from developmental day 15 a chicken embryo in the egg has the capacity to show a nocifensive reaction.Many potentially noxious interventions are performed on chicken embryos in research and in the poultry industry. It is therefore essential and in the interest of animal welfare to be able to precisely define the point at which a chicken embryo is capable of nociception in ovo. The present part III of a comprehensive study examined the movements of developing chicken embryos with the aim of identifying behavioral responses to a noxious stimulus. For this purpose, a noxious mechanical stimulus and a control stimulus were applied in a randomized order. The recorded movements of the embryos were evaluated using the markerless pose estimation software DeepLabCut and manual observations. After the application of the mechanical stimulus, a significant increase in beak movement was identified in 15- to 18-day-old embryos. In younger embryos, no behavioral changes related to the noxious stimulus were observed. The presented results indicate that noxious mechanical stimuli at the beak base evoke a nocifensive reaction in chicken embryos starting at embryonic day 15. The behavior of birds can profoundly differ from the behavior of mammals, especially in terms of indications of pain . For a lBirds show only subtle behaviors of discomfort or pain due to the disadvantage of showing weakness in a social group or as a prey species in general as well as the potential predominance of the flight reflex . In addiGallus gallus domesticus) and evaluated nociceptive responses to procedures that are assumed to be painful or elicit discomfort [Behavioral studies have been conducted in a variety of avian species . Many ofscomfort ,11. The scomfort ,11. In tscomfort ,13,14,15scomfort ,17,18.The results presented are part of a comprehensive study investigating the developmental day at which chicken embryos are capable of nociception and pain perception. The aim of the present part III of the study was to evaluate the acute behavioral responses of chicken embryos at different developmental stages to a noxious mechanical stimulus. The markerless pose estimation software DeepLabCut (DLC) and manual observations were used to analyze embryonic behavior ,20,21. IChicken embryos from ED9 to ED18 were analyzed. An overview of the experimental groups is provided in On ED3, eggs were placed horizontally for two minutes, and 5–7 mL of albumin was withdrawn through a small hole at the pointed pole using a cannula. A small window was cut in the top of the eggshell, and 0.5 mL of penicillin-streptomycin  was added. Eggs were sealed with plastic film and tape. With the eggs in a horizontal position, the incubation proceeded until the desired embryonic day .At the end of the experiments, the embryos were euthanized by an intravenous injection of pentobarbital-sodium , followed by decapitation. Afterward, the sexes of the ED12 to ED18 embryos were identified macroscopically by the assessment of the gonads. For the ED9 embryos, sexing was performed with PCR of genomic DNA samples isolated from pectoral and wing muscle. Screening was performed according to an established protocol  using prTM, Sheldon Manufacturing, Cornelius, NC, USA). In this manner, the inner egg temperature was kept at 37.9 °C ± 0.9 during the entire experiment. To observe the entire embryo, the window in the eggshell was enlarged. Next, the chorioallantoic membrane (CAM) was carefully cut open and removed from the field of view. If necessary, blood vessels were ligated to prevent bleeding. However, to the extent possible, ligating or cutting vessels was avoided to prevent disruption of blood circulation. To gain access to the embryo and improve visibility, the amnion was carefully opened. A Desmarres lid retractor  was carefully placed underneath the beak of the embryo to ensure beak visibility. In the case of ED9 embryos, a small wire loop was used.All experiments were performed between 9:00 am and 7:30 pm by the same two persons to standardize the procedure. To keep the environmental conditions as similar as possible to typical brooding conditions, experiments were conducted in a special heated chamber. The chamber was equipped with a heat mat , a heat lamp  and an air humidifier . Humidity was kept at a constant level of 55.5% ± 4.5. Additionally, the eggs were embedded in warm (38.0 °C) Armor Beads  with a frame rate of 50 frames per second.Pinch) using a manual instrument and a light touch (Touch) as a negative control. Both were applied at the base of the beak. For the ED15 to ED18 embryos, a mosquito clamp  was used to administer the stimulus. To better monitor the applied force, a mosquito clamp combined with an analgesia meter  was used for experiments conducted with ED12 to ED14 embryos. Stimulus 1 (Pinch or Touch) was administered, followed by an observation duration of three minutes. After a second baseline period, stimulus 2 (Touch or Pinch) was administered, followed by another three minutes of observation. Because of their small size, microsurgical anatomical forceps  had to be used to administer the stimulus to ED9 embryos. An additional group of ED18 embryos (ED18 w/Lido) was injected with 0.02 mL of lidocaine  in the upper and lower beak region five minutes before the first baseline. Experiments were then performed according to the above protocol.After preparation, a resting period of three minutes was allotted. Baseline behavior was recorded for two (ED15 to ED18) or three (ED9 to ED14) minutes; subsequently, two stimuli were applied in a randomized order. The stimuli used were a noxious mechanical stimulus  before analysis. For each embryo, four single videos were cut referring to the sections of the experimental design: Baseline Pinch, Baseline Touch, Post Pinch and Post Touch) were analyzed for each embryo. For each labeled body part, DLC created three outputs for each frame of the video: an x coordinate, a y coordinate and a likelihood value. These values were analyzed with custom-written code using MATLAB (MATLAB Version: 9.12.0.1927505 (R2022a) Update 1, MathWorks). In all cases, a likelihood value cutoff of 0.75 was used.To track the body parts of the embryo, the Python-based markerless pose estimation software DLC (version 2.2.1.1) ,21 was uBeak;Head;Limbs;Stationary points on the egg, the Desmarres lid retractor, and the wire loop (for ED9) were used as reference controls.In the analysis, the focus was on the following body parts:As a first step, the labeled data clusters for each analyzed body part were visualized in the x–y coordinate space. This enabled refinement of the dataset through identification of outliers or mislabeled body parts. The videos were then checked for errors, and if any real outlier was found in a frame, its value was manually excluded.d between two points:The distance between the upper and lower boundaries of the beak was calculated in terms of the Euclidian distance The angle between the upper and lower beak was computed by calculating the angle α between two lines, d between identical labels across consecutive frames:The movement of the body parts of interest was calculated in terms of the Euclidean distance Beak Distance, Beak Angle, Movement Eye Corner, Movement Elbow and Movement Metatarsus, the sum of the 1500 frame values of the interval was calculated. In Post Stimulus, this resulted in four intervals: 0–30, 30–60, 60–90 and 90–120 s. The beginning of the first poststimulus interval was defined as the moment from which the clamp was no longer in contact with the beak. The median of the four 30-s intervals prior to the stimulus was considered the baseline. Missing values, which arose after the exclusion of low likelihood values, were manually imputed. For each missing value series, the median was determined for half of the adjacent data and used in place of the missing value. If more than 5% of the data in an interval were missing, the interval was excluded from the analysis. Due to a lack of visibility, one ED14 embryo and one ED18 embryo were completely excluded from the DLC analysis. A precise overview of the number of datasets ultimately included in the analysis is provided in To simplify the analyses, 30 s intervals were evaluated. For each parameter, i.e., p value correction for multiple comparisons. The results with a p value < 0.05 were considered statistically significant. Data analysis was performed using R 4.2.1 (23 June 2022). Detailed information about the statistical analyses, including the corresponding effect size for each reported p value, can be found in Due to the presence of repeated measures, generalized linear mixed effects models with the individual embryo as a random effect were chosen for analysis. Due to the violation of numerous model assumptions , only robust linear mixed-effects models were applied for all analyses (R package-robustlmm). All contrasts (differences) between particular groups were assessed after model-fitting by the estimated marginal means (R package–emmeans) with Tukey’s Pinch, manual observations focused on beak movements. Four different patterns of beak movements were identified from the video material:Beak Shift—a small horizontal shift of the upper and lower beaks against each other;Mandibulation—a small vertical opening of the beak, often executed several times, and reminiscent of a chewing movement;Beak Opening—single, swift, vertical opening of the beak;Wide Beak Opening—single, wide, vertical opening of the beak, accompanied by a characteristic tongue movement.The same video footage as used in the DLC analyses was used for manual observations. Since preliminary observations and data from the DLC analyses indicated that changes in beak position were frequent after In an analogous approach to the one described above, the baseline and poststimulus observations were divided into intervals of 30 s. For manual observations, the 30 s before the stimulus were used as a baseline. For each interval, the occurrences of the described beak movements were counted.Beak Angle) and distance (Beak Distance) between the upper and lower beak were calculated to reflect the opening of the beak as a potential response to a noxious mechanical stimulus applied at the base of the beak. The mechanical stimulation of the beak led to a change in the beak position at embryonic day (ED) 9 and ED12; thus, evaluation with DLC was distorted and could not be interpreted. At ED13 and ED14, Beak Distance did not differ between any time intervals during the two minutes after the control touch stimulus  and the time intervals during the two minutes after the noxious pinch stimulus   did not exhibit reduced beak movements compared to same-age embryos that did not receive the local anesthetic (Beak Distance was still significantly increased in ED18 w/Lido in the first 30 s of Post Pinch (p < 0.0001).To analyze the movements of chicken embryos, the markerless pose estimation software DLC was used. The angle (t Pinch) . At ED15detected . Additioesthetic . Beak DiBeak Angle results are displayed in the Beak Angle showed a similar pattern of changes as Beak Distance. Additionally, significant increases in Beak Angle during Post Pinch were observed from ED15 onward.Post Pinch. On these days, the embryos showed a significant increase in head movements after Pinch compared to after Touch  and during Baseline Pinch . At ED12, head movements increased significantly at 30–60 s after Pinch compared to those 30–60 s after Touch (p = 0.0372). At ED14, head movements also increased significantly in the first 30 s after the stimulus compared to those in the corresponding baseline period. These movements were observed after both stimuli . In addition, a significant difference between head movements in response to Pinch and those in response to Touch was observed at 30–60 s after the stimulus (p = 0.0175). Head movements were significantly reduced in ED18 w/Lido embryos in the first 30 s of Post Pinch compared to those of ED18 embryos in the same period (p < 0.0001). Head movements on ED15 to ED18 are displayed in The medial eye corner was tracked to analyze the head movements of chicken embryos. Changes were particularly observed on ED13 and ED16 to ED18 in the first 30 s of Elbow, Metatarsus and Tarsus (ED9) were analyzed. Significant differences in limb movements between Baseline Pinch and Post Pinch and between Post Pinch and Post Touch were observed only on ED18 (Baseline Pinch and Post Pinch (p = 0.0023) as well as between Post Pinch and Post Touch (p = 0.0096) during the first 30 s after the stimulus. Regarding metatarsus movements, ED18 embryos showed a significant increase between Baseline Pinch and Post Pinch (p < 0.0001) as well as between Post Pinch and Post Touch (p = 0.0002) during the first 30 s after the stimulus. For ED18 w/Lido embryos, no significant differences in limb movements were observed between Baseline and the first 30 s of Post Stimulus. There was also no significant difference between the ED18 embryos and the ED18 w/Lido embryos. Other significant changes in limb movements were observed at specific time intervals during development.To track limb movements, the movements of the  on ED18 . An incrPost Pinch in embryos from ED15 to ED18. To characterize beak movements in further detail, manual observations were performed. The focus of the manual observations was on four behaviors: Beak Shift, Mandibulation, Beak Opening and Wide Beak Opening. An overview of the percentage of animals that exhibited each behavior at specific time intervals is shown in In particular, DLC analysis identified changes in beak movement during Beak Opening was rarely displayed during Baseline and was observed in only 10.0% of animals from ED9 to ED18. Beak Opening was particularly rare on ED9 and ED12 to ED14. Before ED12, a maximum of 10.0% of animals exhibited this behavior within a single time interval; up to ED14, a maximum of 20.0% of animals exhibited this behavior within a single time interval. Starting from ED15, an increasing frequency (31.3%) of Beak Opening was observed after the application of the noxious stimulus. At ED16, 87.5% of embryos showed Beak Opening in the first 30 s of Post Pinch. Additionally, 50.0% of ED17 embryos and 62.5% of ED18 embryos showed this behavioral response to Pinch. During these days, at least twice as many embryos showed Beak Opening during Post Pinch as those during Post Touch.Wide Beak Opening, characterized by visible tongue movement, was observed only sporadically during Baseline on all developmental days. This behavior was observed in only one animal each on ED13, ED14 and in ED18 w/Lido embryos during baseline. Moreover, this specific beak movement was not observed during Post Pinch and Post Touch for ED9 to ED13 embryos and was observed only once during Post Pinch on ED14. On ED15 and ED16, this behavior was increasingly observed. A total of 18.8% (ED15) and 25.0% (ED16) of embryos exhibited Wide Beak Opening in the first 30 s of Post Pinch. A total of 81.3% and 87.5% of embryos on ED17 and ED18, respectively, showed more Wide Beak Opening in the first 30 s of Post Pinch. However, this behavior was never observed during Post Touch or corresponding baseline periods at these ages.Beak Shift was observed from ED12 onward, but it did not appear to be associated with Pinch. Mandibulation was also observed across all embryonic days. Changes were observed in Mandibulation at all times in Post Pinch and Post Touch and regularly during both baseline periods.Beak Opening and Wide Beak Opening were the most noticeable Post Pinch responses, the focus of comparisons with the additional control group that received local anesthetic (ED18 w/Lido) was on these two movements, as the application of lidocaine reduced these behaviors. In the ED18 w/Lido group, 40.0% of the embryos reacted with Wide Beak Opening to the noxious mechanical stimulus; in the ED18 embryos without a lidocaine injection, 87.5% exhibited this behavior. Beak Opening was observed in 20.0% of the ED18 w/Lido animals and 62.5% of the untreated ED18 embryos. Neither Mandibulation nor Beak Shift appeared to be associated with a specific reaction in any time interval, similar to embryos without lidocaine treatment. In other words, no noticeable increase or decrease in these behaviors was observed after a stimulus.Since In this exploratory study, we investigated the movements of chicken embryos in response to a noxious stimulus at different developmental stages. We used DeepLabCut, a Python-based markerless pose estimation software, as well as manual observations to determine their responses.Recently, the use of artificial intelligence and deep learning systems in behavioral studies has increased, and the availability of free software such as DLC allows such techniques to be used by researchers with less sophisticated programming experience ,29,30. IPain behavior in general is influenced by a variety of factors specific to the stimulus or the affected animal. For example, noxious agents can differ in duration (acute or chronic), source  and severity (mild to severe), each of which may provoke a different reaction ,10,31. SNociceptive reflexes have evolved as protective mechanisms . A noxioBeak Angle and Beak Distance showed the most noticeable changes after the stimulus. Both parameters, Beak Angle and Beak Distance, quantified beak movements. A significant increase in beak movements was detected immediately after Pinch from ED15 to ED18. As the increase in beak movements during Post Pinch was significant compared to those during Baseline Pinch and Post Touch, we assumed that the increase in beak movements was a reaction to the noxious stimulus and was not a random movement of the chicken embryos.We analyzed the movements of chicken embryos in response to a noxious stimulus applied to the beak from ED9 to ED18. Consistent with the assumption that a response to a stimulus is expected at the site of stimulus application, as was shown for well-innervated regions such as the beak , our DLCBeak Opening (starting on ED16) and Wide Beak Opening (starting on ED17) were recurring movements in response to the noxious stimulus. Individual, slow beak openings have been described in connection with the penetration of the air sac membrane shortly before hatching, at the end of day 18 [Further differentiation of the movements through manual observation revealed that f day 18 . This def day 18 , and no Mandibulation in our study. Likewise, the movement was randomly observed across time intervals and had no clear connection to any of the stimuli. However, a similar behavior was observed in adult chickens as a response to low atmospheric pressure stunning before slaughter [Mandibulation is also a sign of stress in chicken embryos.Hamburger and Oppenheim also described a behavior that they called beak clapping, which involves rapid opening and closing of the beak in sequences that occurred at irregular intervals . The deslaughter . In thislaughter . As in tWide Beak Opening and Beak Opening by approximately half. Furthermore, local anesthetics are known to be effective in birds [Application of the local anesthetic lidocaine did not yield a significant reduction in the beak movements of chicken embryos on ED18 according to the DLC analysis. However, in the manual observations, the application of lidocaine reduced the percentage of embryos that responded to stimuli with in birds ,40,41 anin birds  or brachin birds . Howeverin birds . This isOverall, stress could not be completely eliminated within the experimental setup; thus, its potential influence on behavior must be considered. The fenestrated egg does not represent a completely typical environment for the embryo because of the increased exposure to environmental influences, such as light. Additionally, the invasiveness of the preparation itself can induce stress, which is known to alter the behavior of birds . We attePost Pinch compared to those during Baseline Pinch or Post Touch were inconsistent over several EDs or time intervals and are therefore likely due to random movements, which have been described previously in the literature [Post Pinch compared to those during the first 30 s of Baseline Pinch and Post Touch on only ED18, suggesting that these movements may represent an actual response to the noxious stimulus.We were also interested in whether limb movements changed after the noxious stimulus; however, we did not detect any overarching pattern until ED17. Occasional significant differences in limb movements during terature ,47,48,49terature . Wu et aterature . In the Post Pinch, the observed movement changes in ED15 to ED18 embryos were most likely a response to the noxious mechanical stimulus and can therefore be interpreted as nocifensive behavior. The results of our current movement analysis in combination with the corresponding results of the cardiovascular changes [We observed the movements of chicken embryos from ED9 to ED18 before and after noxious stimulation. During  changes  and the  changes  in chick


Coexistence of TGM1 and FLG mutations in a newborn with congenital ichthyosis is not well described in the literature. Early genetic testing and counseling are crucial for accurate diagnosis and appropriate management. Further exploration of associated problems, including hearing loss and developmental delay, is warranted in patients with these mutations. Infant with collodion membrane just after birth. Work‐up led to the discovery of a unique combination of genetic mutations (FLG and TGM 1), contributing to this condition. Conservative management with bland emollients and humidity were used for this infant. This case report describes a rare combination of mutations leading to congenital ichthyosis, along with other clinical findings, including hearing loss and developmental delay, in the affected patient.2A 34‐week gestational age male infant, born secondary to premature rupture of membranes presented with a collodion membrane at birth. Maternal history was significant only for Hepatitis C. There was no other contributory family history. Other than shortened femurs noted on prenatal ultrasound, the pregnancy was uncomplicated. The infant did not require significant resuscitation after delivery. On exam, the infant had diffusely shiny, tight parchment‐like skin; cracking skin in the neck and knee folds; and bilateral ectropion Figure .2.1The infant was transferred to a tertiary care center for pediatric subspecialty care. Genetics, dermatology, and ophthalmology were consulted. The infant was admitted to the NICU in a humidified isolette and placed on broad spectrum antibiotics until the blood culture was negative for 48 hours. Due to concern for skin integrity, a tunneled catheter was placed for venous access. The infant was weaned from the humidified isolette and intravenous fluids as his enteral feeds were increased. Congenital ichthyosis gene panel was performed, and the infant was heterozygous for the c.944 G > A mutation and the c.1025 G > T mutation on TGM1. He also had a c.4768 C > T mutation on the FLG gene. Due to concern for both ichthyosis vulgaris and lamellar ichthyosis, the dermatologist recommended use of bland emollients for skin care. The ophthalmologist recommended use of lubricating eye ointment until the infant was able to fully close his eyes. The infant's skin and eyes were monitored closely by ophthalmology and dermatology specialists. His course was prolonged by slow feeding due to prematurity. Hearing screen was not passed prior to discharge. The infant was gavage fed until he was able to take all enteral nutrition by bottle.2.2As the collodion membrane healed, the infant developed brown plaques overlying the trunk and most prominent on the scalp Figure . By 42 wAt 19 months of age, the patient had persistent eczematous patches in the axillae, trunk, and face with some scaly plaques on the scalp. These areas had markedly improved with the use of bland emollients when compared to initial presentation. The patient also has developmental delay, though it is unclear if this is related to the genetic mutations and has not previously been associated with either mutation. The infant does suffer from conductive hearing loss of the left ear, which has been rarely associated with congenital ichthyosis and ichthyosis vulgaris.3An infant born with collodion membrane is rare, occurring in 1:50,000–1:100,000 live births.The FLG gene encodes filament‐aggregating protein profilaggrin.Huang et. al showed that 28% of participants with ARCI had abnormal hearing results and 16% had hearing aids.Few studies have evaluated hearing loss in ARCI, but a 2014 survey by In conclusion, while this infant with two mutations showed substantial improvement in skin condition over time, the persistence of eczematous patches and scaly plaques underscores the importance of continued follow‐up and monitoring. Furthermore, the presence of developmental delay and hearing impairment serves as a reminder of the need for comprehensive long‐term care and support for these patients.Written informed consent was obtained from the legal guardian to publish this report in accordance with the journal's patient consent policy.Zackary Shearer: Conceptualization; data curation; formal analysis; writing – original draft; writing – review and editing. Gwenevere White: Conceptualization; formal analysis; writing – review and editing. John Zachary Steed: Conceptualization; writing – review and editing. Carla Brown: Conceptualization; formal analysis; writing – review and editing. Tara Venable: Formal analysis; writing – review and editing. Megan Baber: Conceptualization; data curation; investigation; writing – review and editing.The authors recieved no financial support for the research, authorship, and publication of this article.All authors declare that they have no conflicts of interest.


HcgA‐G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S‐adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.In the biosynthesis of the iron‐guanylylpyridinol (FeGP) cofactor, 6‐carboxymethyl‐5‐methyl‐4‐hydroxy‐2‐pyridinol ( 2 to an organic substrate. HcgA and HcgG are involved in the biosynthesis of the FeGP cofactor. In vitro biosynthesis experiments indicated the functions of these two radical S‐adenosyl methionine enzymes. HcgA biosynthesizes the initial pyridinol precursor, and HcgG contributes to the formation of the CO and acyl ligands.The FeGP cofactor is the prosthetic group of [Fe]‐hydrogenase, which catalyzes reversible hydride transfer from H The iron‐guanylylpyridinol (FeGP) cofactor is the prosthetic group of [Fe]‐hydrogenase and contains a low spin FeII in complex with an acyl ligand from a pyridinol derivative, the pyridinol nitrogen and two CO ligands  encode the proteins responsible for biosynthesis of the FeGP cofactor.1) to form the 3‐methylated precursor (2).2 to form 3.3 is adenylylated by HcgE and then forms a thioester bond with a cysteine residue from HcgF to form 4.4 is further converted to the acyl ligand of the FeGP cofactor by unknown reactions.1 is unknown.1 or completion of the iron complex, including formation of the CO and acyl ligands.The S‐adenosyl methionine (SAM) enzyme family and is similar to [FeFe]‐hydrogenase maturation enzymes (HydE and HydG), biotin synthase and methylornithin synthase.5CX2C motif rather than the standard motif (CX3CX2C) for [4Fe‐4S]‐cluster binding.HcgA is a member of the radical hcgBΔhcgC mutated strain of Methanococcus maripaludis, which cannot biosynthesize precursor 3.M. maripaludis ΔhcgBΔhcgC mutant and the standard reaction mixture. Incubation of 3 in the in vitro biosynthesis assay under H2 and CO produced the FeGP cofactor, where the active [Fe]‐hydrogenase holoenzyme is formed by binding of the FeGP cofactor to the apoenzyme in the assay. Using this method, we demonstrated that 1, 2 and 3 are the precursors of the FeGP cofactor biosynthesis.3 is converted to the acyl ligand and that the CO ligands are formed from external CO gas or from an unknown CO‐donating compound in the cell extract. Furthermore, these experiments revealed that an electron donor is needed for biosynthesis.Recently, we developed an in vitro biosynthesis assay for the FeGP cofactor using the cell extract from the Δ1 and that HcgG contributes to the formation of the CO and acyl ligands to complete biosynthesis of the FeGP cofactor.Here, we report the function of the radical SAM enzymes, HcgA and HcgG, in biosynthesis of the FeGP cofactor using in vitro complementation assays. Our data indicated that HcgA catalyzes biosynthesis of hcgA and ΔhcgG strains of M. maripaludis (Table S1). We confirmed the absence of the respective proteins in the cell extracts of the ΔhcgA and ΔhcgG strains by proteome analysis (Table S2). Accordingly, the cell extracts of the mutated strains did not show [Fe]‐hydrogenase activity. The in vitro biosynthesis assay solution contained the standard reaction cocktail and the following tested materials: the cell extract of the M. maripaludis ΔhcgA or ΔhcgG strain, the purified Hcg enzymes and/or the biosynthesis precursors . The standard reaction cocktail contained ATP/MgCl2, dithiothreitol (DTT), sodium dithionite, [Fe]‐hydrogenase apoenzyme from Methanocaldococcus jannaschii, Fe2+ and SAM. The gas phase of the standard assay was 50 % H2/50 % CO. To evaluate the in vitro biosynthesis of the FeGP cofactor, we determined the enzyme activity of the [Fe]‐hydrogenase that was constructed in the assay. For details, see the Experimental Procedures section in the Supporting Information.In this study, we used the cell extract from the ΔM. maripaludis in Escherichia coli as a His‐tagged protein using a helper plasmid (pRKISC) to improve iron‐sulfur cluster biosynthesis .Escherichia coli resulted in the formation of inclusion bodies,in trans in M. maripaludis using an expression vector with the Methanococcus voltae histone promoter . Gel permeation chromatography indicated that the molecular mass of the purified HcgG was 98 kDa . The UV/Vis spectrum of HcgG showed the presence of absorbance peaks at 350, 420, and 600 nm  of the ΔhcgA strain with HcgA . The MS/MS signals of this compound are also identical to those found in chemically synthesized 1 . When HcgC was added to the assay, the 184 m/z compound disappeared and a 198.0760 m/z signal was produced, which is identical to the signal of 2  was produced .Based on a previous labeling study, it was predicted that β‐alanine or aspartate reacts with 2,3‐dihydroxy‐4‐oxo‐pentanoate to form hcgG strain of M. maripaludis. The addition of 3 into the in vitro biosynthesis assay did not complement the biosynthesis activity  should be addressed to the authors.Supporting InformationClick here for additional data file.


To study the prevalence of risk for hearing impairment in neonates with congenital syphilis in a newborn hearing screening program.post-hoc tests were used, comparing the groups that passed and failed the hearing screening that had RIHL, using a significance level of p of <0.5.The study design is retrospective, documentary, and is cross-sectional. The sample consisted of newborns who were born between January 2019 and December 2021 and who underwent neonatal hearing screening in a public maternity hospital. Demographic data and the presence and specification of risk indicators for hearing impairment (RIHL) were collected. In retest cases, the results and the final score were also collected. For data analysis, the Kruskal–Wallis and Conover-Iman p  < 0.05) between them.Among the RIHL observed in the sample, prematurity was more frequent in newborns who passed the screening (55.26%) than in those who failed the test (45.67%). Congenital syphilis was the ninth most frequent RIHL (8.04%) among the newborns who passed the test and the 15th factor (3.03%), with the highest occurrence in those who failed the hearing screening. When comparing the two groups (pass and fail), we found significant differences (Congenital syphilis was the ninth risk indicator for the most common hearing impairment and, in isolation, did not present a risk for failure in neonatal hearing screening. Notably, congenital syphilis can cause late hearing loss during child development. Thus, there is an indication of audiological monitoring of these neonates. Treponema Pallidum that can be transmitted via the placenta or at the time of delivery when the infection in the pregnant woman is not properly treated at any stage of pregnancy. As it is often asymptomatic, the detection of congenital syphilis in children depends on tests performed in the maternity ward. This detection is crucial because, when not properly treated, it can cause metabolic alterations, neurological alterations, prematurity, low birth weight or low weight during the child's first years of life  for >5 days, among others, must be monitored after hearing screening, regardless of failure or not in the procedure , 8. MoreTherefore, identifying risk indicators for hearing impairment is paramount for children's hearing health and consequently strengthens the promotion, prevention, and follow-up activities in the Comprehensive Care Network for children in the health system. Thus, the objective of the study was to study the prevalence of risk for hearing impairment in neonates with congenital syphilis in a newborn hearing screening program (NHSP).The present study has a retrospective, documental, cross-sectional study design that was carried out at a public maternity hospital's Newborn Hearing Screening Sector. This study was approved by the Committee of Ethics of the University Hospital Onofre Lopes, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil, number 4,648,404. This maternity hospital is a reference in maternal and child health in the state because it has Neonatal and Maternal Intensive Care Unit beds and is responsible for the birth of approximately 3,600 newborns a year .The sample comprised neonates who were born in the maternity hospital between January 2019 and December 2021 and who had neonatal hearing screening. Those born in other institutions were excluded from the survey, making the size of the final sample 7,879 records. This sample corresponded to 66.69% of live births during the study period at this maternity hospital.® AccuScreen, which was duly calibrated, and the exam was the Transient Stimulus Evoked Otoacoustic Emissions (TEOAE).NHS was performed during the mother's and neonate's hospitalization or at the institution's outpatient clinic by one of the two audiologists in the program. The equipment used was the MadsenThe probe test of the equipment was performed every day before the test was applied to the infants. In this way, it was inspected and connected. Afterward, the infants' data were entered into the equipment's software.The infants were comfortably positioned on their mother's lap in natural sleep so that their ears were accessible for inserting the probe into the external auditory meatus. The audiologist had performed a prior inspection of the acoustic meatus to identify the presence of cerumen or other residues at the entrance of the meatus. Subsequently, the audiologist would insert the auricular tip into the appropriately sized probe for each external acoustic meatus so that it would be in a proper position. During this insertion procedure, the audiologist carefully pulled the external acoustic meatus backward and slightly downward with slight pressure. Afterward, the audiologist visually checked whether the fit was correct using a horizontal bar that highlighted this functionality. When starting the TEOAE test, this equipment first performs the probe calibration, and as soon as it completes the calibration, the test starts automatically in each ear, with both performed one after the other. In this equipment, the pass response is determined by a statistical algorithm based on the weighted average, which ensures detection with proven high specificity and sensitivity. The “pass” result in the screening according to the parameters in the equipment manual presents the following parameters: eight peak frequency responses, an artifact rate of <20%, and probe stability >80%. If one of these parameters is not reached, it is considered a “failure” in the screening.The NHS was carried out in two stages. In the first test call, it was considered a “pass” when the newborn obtained the presence of otoacoustic emissions in both ears and “fail” when the emissions were not present in at least one ear. When “failure” occurred, a retest was scheduled at the maternity clinic after ~15–30 days. If there was a new failure in the retest, the baby was referred for an audiological diagnosis at the Hearing Health Service (SSA) or Center Specialized in Rehabilitation (CER) to perform an audiological diagnosis. If the newborn had a risk indicator for hearing impairment (RIHL), they were also referred to the reference service for hearing monitoring, which consists of audiological evaluation and monitoring of hearing and language development. Finally, when the newborn had no risk indicator and the result was “pass,” the child was discharged with guidance, and the caregiver received verbal guidance on the child's auditory and language development. The flowchart is shown in Data collection from medical records occurred from October 2021 to February 2022 through the consultation of the care records and the sector's database.Owing to the absence of a computerized database, the NHS sector organizes the data necessary for evaluating the neonatal hearing screening program in an Excel spreadsheet. The data extracted were the date of screening, birth date of the neonate, neonate's gender, municipality where the mother lives, presence and specification of RIHL, screening result , and management . In cases of retesting, data on attendance (yes or no), the result , and the final management  were also collected.A survey of the patient's clinical history was carried out, considering all risk indicators for hearing impairment as identified in the program and recorded by the medical team responsible for caring for the parturient woman and the neonate. These indicators include altered APGAR scores, arbovirus infections during pregnancy, craniofacial anomalies involving the ear and the temporal bone, severe perinatal anoxia, heredity factors, hyperbilirubinemia, congenital infections , postnatal bacterial or viral infections , being small for gestational age, weight <1,500 g, prematurity, parental concerns, syndromes often associated with hearing impairment, use of ototoxic drugs, use of mechanical ventilation, and staying in the Neonatal Intensive Care Unit (NICU) for more than 5 days.All risk indicators were collected from each newborn's medical records at the maternity hospital. Therefore, the identification and diagnosis criteria for each of the criteria followed the good medical practices of this maternity hospital in line with the guidelines recommended by the Ministry of Health of Brazil. With regard to congenital syphilis, the parameters adopted for its diagnosis and treatment followed the Clinical Protocol and Therapeutic Guidelines of the Ministry of Health .It should be noted that, until the year 2019, maternity followed the Brazilian Ministry of Health's Guidelines for Newborn Hearing Screening 2012) , similar , similapost-hoc statistical tests compared the groups that passed and failed hearing screening that had RIHL, using a significance level of p of <0.5.In the data analysis, the percentage distribution of categorical variables and measures of central tendency and dispersion of continuous variables were performed. For statistical analysis, the Kruskal–Wallis and Conover-Iman's In the studied period, 11,927 neonates were born at the maternity hospital, of which 7,879 (66.69%) underwent NHSP. Of the screened neonates, 96 (1.14%) reported congenital syphilis as the only RIHL and 90 (1.07%) reported congenital syphilis associated with other RIHL, totaling 186 neonates with congenital syphilis.Of these, 183 (98.39%) neonates passed the test and retest, two (1.07%) did not show up for the retest, and the screening was not concluded, and one (0.54%) neonate did not pass the test and retest and was referred to the Specialized Care Service for a complete audiological diagnosis. In addition to congenital syphilis, this neonate was premature, had been in the ICU for >5 days, and used ototoxic medication.post hoc test, we found a significant difference (p  < 0.05) between them.The statistical analysis showed that, among the RIHL observed in the sample, prematurity was most prevalent in the group of neonates who were evaluated (55.26%) and those who failed the test (45.67%) ; congeniThe analysis also showed that the percentage of RIHL incidence throughout the sample during the 3 years observed, when the 10 risk indicators for hearing impairment with the highest incidence were found, with prematurity, ototoxic medication, and an ICU stay of >5 days being the most common .Furthermore, statistical analysis showed that, in the presence of syphilis, the main accompanying risk factor is ototoxicity, followed by prematurity and an ICU stay of >5 days .Congenital syphilis is a RIHL, according to national and international scientific recommendations. In the sample studied, no neonate with isolated congenital syphilis failed the neonatal hearing screening test. Even so, these infants should be referred for hearing monitoring and medical follow-up since they may develop signs and symptoms later in life, regardless of the first evaluation and/or treatment in the maternity hospital , 11.It was observed that the coverage of neonatal hearing screening performed in the program studied did not reach the rate recommended by the Joint Committee on Infant Hearing , stayingRegarding the RIHL, prematurity, hyperbilirubinemia, use of ototoxic medication, and a stay at the NICU for more than 5 days were the most frequent in the sample, both in neonates who passed or failed the screening, which does not differ from what has been found in other recent studies –16. ThesAs for congenital syphilis, it was noticed in the sample studied that congenital infection was the most frequent, but in isolation, it did not present a risk for failure in neonatal hearing screening. In Brazil, over the past 5 years, a steady increase in the number of cases of syphilis in pregnant women, congenital syphilis, and acquired syphilis has been observed . This inTreponema Pallidum, which can cause hearing loss and other neurological problems , we can find another possible cause of this coverage. Despite the limitations of the study's sample on coverage of the NHS and risk factors, a statistical analysis was performed to reduce impacts and highlight the main points that require careful attention from management on children's health, as discussed below.Furthermore, the lack of data control and the need for unified computerization of data hinder the follow-up of these children in the stages of monitoring and diagnosis of services .Public policy efforts to prevent and combat syphilis are also necessary to continually reduce the incidence rate of syphilis in pregnant women and cases of congenital syphilis. These efforts also play a crucial role in promoting accurate detection and adequate treatment for infected mothers and children. This approach seeks to minimize the impact of congenital syphilis, particularly on hearing and child development outcomes.A study conducted by Brito et al.  revealedMoreover, it is crucial to emphasize the need for reinforcing the healthcare networks within the health system that oversee the follow-up of children with congenital syphilis during outpatient visits, either in primary and specialized care settings. This approach aims to prevent families from slipping through the cracks and to ensure that early childhood hearing loss is promptly detected without any hindrance .Finally, it was observed that there are two limiting conditions in the study: neonates who did not undergo outpatient testing and/or retesting due to non-attendance or opting for testing at a different facility and neonates who need to undergo hearing diagnosis from an alternative hearing healthcare service. These groups were excluded from the study, resulting in the absence of corresponding results for this aspect.It is suggested that further research be done on the presence of congenital syphilis after neonatal hearing screening to verify whether the presence of this risk indicator is, in fact, a risk for the development of future hearing loss. There should also be studies that can observe the potential of two or more indicators for hearing loss in childhood.If there is a low risk for failure in neonatal hearing screening in newborns with congenital syphilis and, in future follow-up studies, a low risk of the presence of hearing impairment in infants with congenital syphilis treated at birth can lead to changes in management and guidelines in the care network for these infants compared to others with a higher prevalence of failure and of triggering hearing loss that, currently, are not included in the public policies in Brazil and elsewhere in the world.Public health practices have evolved over time, and in Brazil, a notable change has occurred where newborns identified and diagnosed with congenital syphilis at birth have access to treatment before being discharged from the hospital; this positive development contrasts with the treatment observed in previous decades and even in the present situation in certain regions around the world. Therefore, this study can help alert the scientific society and managers in the health area to the importance of identifying the epidemiological data of each reality in view of the public policies adopted to adapt identification and follow-up behaviors in health.This study shows a greater risk of screening failure in the presence of two or more risk indicators (co-occurrence), hence the importance of monitoring these newborns more closely after neonatal hearing screening. The strengths of the study also include a large sample and a variety of RIHL. However, the incidence percentage of passing and failing on the NHS shows similar risk factors. This result points to the need to study not only isolated risk factors but also their co-occurrence, considering local characteristics such as socioeconomic ones.Among the RIHL observed in the sample, prematurity was more frequent in the newborns who passed the screening (55.26%) than in those who failed the test (45.67%). Since the maternity hospital in the study is a high-risk hospital and a reference in the state, the volume of patients with risk conditions increases, and it is more common to have those born prematurely. Owing to this, we can observe a homogeneous distribution of this risk indicator in the sample.A lack of standardized protocols is evident in neonatal hearing screening practices , 34. DesIn addition, the maternity hospital in this study uses TEOAE for hearing screening across all sectors. The Joint Committee on Infant Hearing  advocateCongenital syphilis was the ninth most frequent risk indicator for hearing loss and, in isolation, does not present a risk for failure in neonatal hearing screening since no neonate was referred for audiological diagnosis. Notably, congenital syphilis can cause late hearing loss during child development; thus, audiological monitoring of these neonates and studies that address this issue are recommended.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by University Hospital Onofre Lopes of the Federal University of Rio Grande do Norte. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants' legal guardians/next of kin because we collect the information in the Institution's database, without access to personal information.TO, MD, AN-A, and AS performed the collection, analysis, tabulation of data and writing, and revision of the manuscript. GO, GS, and RV performed analysis data and scientific writing. SB conceived and guided the study and was responsible for analyzing data, writing, and revising the manuscript. All authors contributed to the article and approved the submitted version.


CSTB) is encoded on chromosome 21. Thus, individuals who have Down syndrome, a genetic condition caused by having an additional copy of chromosome 21, have an extra copy of an endogenous inhibitor of the enzyme. Individuals who have Down syndrome are also at significantly increased risk of developing early-onset Alzheimer’s disease (EOAD). The impact of the additional copy of CSTB on Alzheimer’s disease development in people who have Down syndrome is not well understood. Here we compared the biology of cathepsin B and CSTB in individuals who had Down syndrome and Alzheimer’s disease, with disomic individuals who had Alzheimer’s disease or were ageing healthily. We find that the activity of cathepsin B enzyme is decreased in the brain of people who had Down syndrome and Alzheimer’s disease compared with disomic individuals who had Alzheimer’s disease. This change occurs independently of an alteration in the abundance of the mature enzyme or the number of cathepsin B+ cells. We find that the abundance of CSTB is significantly increased in the brains of individuals who have Down syndrome and Alzheimer’s disease compared to disomic individuals both with and without Alzheimer’s disease. In mouse and human cellular preclinical models of Down syndrome, three-copies of CSTB increases CSTB protein abundance but this is not sufficient to modulate cathepsin B activity. EOAD and Alzheimer’s disease-Down syndrome share many overlapping mechanisms but differences in disease occur in individuals who have trisomy 21. Understanding this biology will ensure that people who have Down syndrome access the most appropriate Alzheimer’s disease therapeutics and moreover will provide unique insight into disease pathogenesis more broadly.Cathepsin B is a cysteine protease that is implicated in multiple aspects of Alzheimer’s disease pathogenesis. The endogenous inhibitor of this enzyme, cystatin B (The online version contains supplementary material available at 10.1186/s40478-023-01632-8. APP) is located on Hsa21, and duplication of this gene (dupAPP) is sufficient to cause EOAD in individuals who do not have DS [People who have Down syndrome (DS) are at much greater risk of developing early-onset Alzheimer’s disease (EOAD) than individuals from the general population, by the age of 55 around half of all people with DS will have been diagnosed with clinical dementia . DS is c have DS –5. Howev have DS –11 and t have DS –16. ThesCTSB) with AD risk [Cathepsin B is a lysosomal cysteine protease, that has been implicated in important aspects of AD development, and a recent genome-wide association study has demonstrated an association of variation in the Cathepsin B gene  is encoded on Hsa21. This endogenous inhibitor does not target all lysosomal proteases, implicated in Alzheimer’s disease, it does not impact the activity of Cathepsin D, which has recently been suggested to have a role in disease development via its function in amyloid-β and tau clearance [CSTB influences the changes to cathepsin B that occur during AD pathogenesis in people who have DS is not well understood. We hypothesize that trisomy of chromosome 21, will raise CSTB protein abundance in the brain and this will modulate the activity of cathepsin B in individuals who have DS when they develop AD compared to people with EOAD from the general population. Here we use a combination of studies in human post-mortem brain material and preclinical model systems to address this knowledge-gap.The abundance and activity of cathepsin B has been reported to be raised in DS fibroblasts but its activity in the brain is not well understood . It is nlearance . How theSee Tables All experiments were undertaken in accordance with the Animals (Scientific Procedures) Act 1986 (United Kingdom), after local institutional ethical review by the Medical Research Council, University College London and results are reported in accordance with ARRIVE 2.0 guidelines.Mice were housed in individually ventilated cages (Tecniplast) with grade 5, autoclaved dust-free wood bedding, paper bedding and a translucent red “mouse house”. Free access to food and water was provided. The animal facility was maintained at a constant temperature of 19–23 °C with 55 ± 10% humidity in a 12-h light/dark cycle. Pups were weaned at 21 days and moved to standardised same-sex group housing with a maximum of 5 mice per cage.AppNL−F , Dp1Tyb (Dp(16Lipi-Zbtb21)1TybEmcf, MGI5703798) Dp(10)2Yey (Dp(10Prmt2-Pdxk)2Yey, MGI:4461400) and Dp(17)3Yey (Dp(17Abcg1-Rrp1b)3Yey, MGI:4461398). All mouse strains were maintained by backcrossing males and females to C57BL/6J mice (imported from the Jackson Laboratory) and for all strains used backcrossing had taken place for more than ten generations. For Fig. AppNL−F/+ animals over two-generations to generate all required genotypes from the second generation 2Yey), AppNL−F/NL−F, Dp(10)2Yey;AppNL−F/NL−F). All other mouse cohorts  identifier: 2. Cells were collected at 70% confluency by trypsinisation (Gibco™ Trypsin-Ethylenediaminetetraacetic acid (EDTA) (0.25%), Thermo Fisher Scientific) and mechanical agitation, pelleted, and washed 3 times in phosphate buffered saline (PBS) prior to homogenization. Experiments were undertaken between passage number 7 and 17. All experiments were blinded for trisomy of Hsa21 or disomic status.Human fibroblasts from individuals who had DS and age and sex-matched disomic controls (kindly supplied by the Coriell Biorepository) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 100 units/ml of penicillin–streptomycin (Thermo Fisher Scientific), at 37 °C with 5% COg for 5 min. Protein concentration of the resultant supernatant was determined by Bradford assay. For CatB assays, 40–100 μg homogenate was diluted in 50 μl CB lysis buffer from the Cathepsin B Activity Assay Kit (Fluorometric) (ab65300) and loaded into 96 well microplates (Greiner Bio-One flat black clear bottom). For CatD, 5–10 μg homogenate was loaded. 2 μl CatB inhibitors N-Acetyl-l-leucyl-l-leucyl-l-methioninal (ALLM)  or Z-Phe-Phe-FMK , and 3 μg Pepstatin A (Sigma) (an inhibitor of aspartyl proteases including CatD) were used for negative controls. The substrate (Ac-RR) labelled with amino-4-trifluoromethyl coumarin (AFC) was used for the CatB assay; and for CatD, GKPILFFRLK(Dnp)-D-R-NH2 labelled with 7-methoxycoumarin-4-acetic acid (MCA) was used. Plates were incubated at 37 °C in the dark and fluorescence activity measured at Ex/Em λ 400/505 nm (CatB) and Ex/Em λ 328/460 nm (CatD) every 90 s over a period of 135 min on a Tecan fluorometric plate reader.Cathepsin B (CatB), and cathepsin D (CatD) endo-peptidase activity was determined using commercial assay kits . Samples were homogenized in lysis buffer as per the manufacturer’s instructions, incubated on ice for 10–30 min, then centrifuged at 15,000× The rate of reaction, proportional to enzymatic activity, was calculated using the slope of the linear phase of the reaction for a minimum of 10 cycles and expressed as a % of the equivalent slope for the control samples. For fibroblast samples and samples of human temporal cortex, an inhibited control was run for each individual sample used. For samples of mouse cortex, a representative inhibited control for each genotype was used. To control for non-specific activity, any activity detected in the appropriate inhibited control reaction was subtracted from the total activity observed in the uninhibited samples prior to analysis. Means of technical replicates were calculated for each individual sample, with biological replicate being used as the experimental unit. For CatB assays 4, 10 and 2–6 technical replicates were used for human post-mortem temporal cortex, fibroblasts and mouse cortex samples respectively. For CatD assays 2 technical replicates were run for each human post-mortem temporal cortex sample.For BMV109 staining of human fibroblasts, cells were incubated in Cellvis 6 Well glass bottom plates with high performance #1.5 cover glass (Fisher Scientific). For control wells to be pre-treated with cathepsin inhibitors, CA-074ME  was added 30 min prior to the BMV109 preincubation. BMV109  and LysoTracker™ Green DND-26  were then added for 1 h and followed by 3 washes in PBS. Live cell imaging was then conducted by ZEISS LSM 980 confocal microscope . A diode laser 488 was used for imaging LysoTracker Green (excitation 504 nm/emission 511 nm) and a diode laser 639 was used for imaging BMV109 (excitation 633 nm/emission 670 nm).After confocal imaging, fluorescent sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was conducted by following Edgington-Mitchell et al.  with minHuman fibroblasts were lysed in M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) supplemented with cOmplete protease and PhosSTOP phosphatase inhibitors (Roche). Human temporal cortex samples were lysed in CB Cell Lysis Buffer from the Cathepsin B Activity Assay Kit . Mouse cortex samples were lysed in RIPA buffer  plus complete protease inhibitors  by mechanical disruption.Samples were denatured in NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) and NuPAGE Sample Reducing Agent in a 95 °C heat block for 5 min. Samples were then loaded into NuPAGE Novex 4–12% Bis–Tris Protein Gels in MOPs or MES SDS running buffer (both Thermo Fisher Scientific) and SDS-PAGE was conducted by the application of 200 V for 50 min or 120 V for 1.5 h. The proteins in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane by Trans-Blot® Turbo™ Transfer System (Bio-Rad Laboratories) at 25 V, 2.5 A, for 7 min. The membranes were blocked with Intercept (PBS) Blocking Buffer (LI-COR Biosciences) for 1 h at room temperature. The membranes were then incubated in primary antibodies in Intercept (PBS) Blocking Buffer (LI-COR Biosciences) overnight with agitation at 4 °C. The membranes were washed three times in PBS with 1% Tween20 (PBST) for 10 min before application of IRDye 680RD or 800CW secondary antibodies (LI-COR Biosciences) for 1 h at room temperature: both at 1:10,000 dilutions in Intercept (PBS) Blocking Buffer (LI-COR Biosciences). After antibody probing, membranes were subjected to another three 10-min washes with PBST, and images were then taken by Odyssey DLx Fluorescence Imaging System (LI-COR Biosciences). The density of protein bands was analysed with ImageJ. β-actin or GAPDH was used for the normalisation of CSTB and CatB protein levels. Relative intensities were calculated by dividing the lane density by the healthy ageing or WT within gel average density.The primary antibodies are rabbit polyclonal anti-cystatin-B , anti-cathepsin B  and anti-β-actin mouse monoclonal antibody . The secondary antibodies were IRDye 800CW Goat anti-Rabbit IgG (H + L)  and IRDye 680RD Goat anti-Mouse IgG (H + L)  (LI-COR Biosciences).Human fibroblasts were cultured on coverslips in 12 or 24-well plates. Cells were first washed with PBS, then fixed with 4% paraformaldehyde (PFA) for 10 min. After three PBS washes, cells were permeabilised with PBS with 0.1% Triton X-100 (Sigma-Aldrich). Cells were then blocked for 1 h at room temperature in blocking solution containing 5% goat serum, Tween-20 (Bio-Rad), and Glycine (Sigma-Aldrich) to prevent non-specific antibody binding. The cells were then stained with primary antibodies overnight at 4 °C in a humidified chamber. After primary antibody incubation, cells were washed with PBS with 0.1% Triton X-100, three times prior to one-hour secondary antibody incubation at room temperature. After secondary antibody incubation, cells were subjected to another three washes with PBST and mounted with Invitrogen ProLong Gold Antifade Mountant with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Thermo Fisher Scientific). Images were then taken by ZEISS LSM 980 confocal microscope .The primary antibodies are rabbit polyclonal anti-cystatin-B , anti-cathepsin B  and mouse anti-LAMP1 . The secondary antibodies were Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488  and goat-anti-rabbit-IgG-H–L-highly-cross-adsorbed-secondary-antibody-polyclonal .To deparaffinise the samples, slides were first treated with xylene for three 5-min intervals, followed by 20–30 dips in 100%, 90% and 70% ethanol, and then 20–30 dips in water. Antigen retrieval was then carried out by placing the slides in 10 mM citrate buffer, pH 6, and heated in a microwave for 5 min at high power. After cooling for 3 min, the slides were heated for 2 min at high power, followed by another 3 min at high power after cooling. The slides were then washed in water for 10 min with agitation, followed by 3 washes in PBS with 0.2% triton X-100, for 10 min each with agitation. The samples were then preincubated with blocking buffer  for 1 h at room temperature in a humid chamber. Primary antibodies  and rabbit anti-Cathepsin B ) were added to the blocking solution and incubated overnight at 4 °C in the humid chamber. After overnight incubation, the samples were washed in PBS and 0.2% triton for 3 times, 10 min each, and then secondary antibodies  and donkey anti-Goat IgG H&L Alexa Fluor 488 ) diluted in blocking buffer were added to the samples and incubated for 1 h at room temperature in the humid chamber. The samples were then washed in PBS and 0.2% triton for 3 times, 10 min each, and DAPI was added for nuclear counterstain. The samples were incubated in DAPI solution for 5 min, then washed in PBS. Slides were incubated with TrueBlack Plus Lipofuscin Autofluorescence Quencher  for 20 min, followed by washing in PBS. Then samples were mounted in ProLong Antifade Mountant (Thermo Fisher Scientific) and imaged by ZEISS LSM 980 confocal microscope .For each case two slides were stained and analysed. For each slide, 10 fields were chosen randomly and imaged using a 20 × objective lens with a 426.67 μm × 426.67 μm total size per image. The images were exported as TIF files. To count the cells, the Cell Counter plugin (FIJI for ImageJ) was used. Any cell with red or green pixels within a proximity of 20 µm to a DAPI stained nucleus was identified as a cathepsin B or IBA1 positive cell.g for 5 min. The cell pellet containing approximately 1 × 106 cells was then washed with PBS prior to transferring to a 1.5 mL Eppendorf tube. Cells were then centrifuged at 500× g for 2–3 min. The supernatant was removed, and the cell pellet resuspended in ice-cold CER I. The tube was then vortexed on the highest speed for 15 s and the resuspended cells incubated on ice for 10 min. Ice-cold CER II buffer was added and the tube vortexed again for 5 s. The tube was incubated on ice for 1 min and then vortexed again for 5 s. The cell lysate was then centrifuged for 5 min at maximum speed  at 4 °C. The supernatant, which contains the cytoplasmic fraction was transferred to a new pre-chilled tube and stored at −80 °C for further use. The remaining pellet fraction containing the nuclei was resuspended in ice-cold NER and vortexed again. The sample was placed on ice and the 15-s vortexing step was repeated every 10 min for 40 min in total. The tube was then centrifuged at maximum speed  for 10 min at 4 °C. The supernatant that contained the nuclear extract fraction was transferred to a new pre-chilled tube.Nuclear and cytoplasmic sub-fractionations of human fibroblasts were carried out by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (CER) (Thermo Fisher Scientific) as per the manufacturer’s instructions. In brief, human fibroblasts were resuspended by trypsinisation (Gibco™Trypsin–EDTA (0.25%) (Thermo Fisher Scientific)) and then centrifuged at 500× Cytoplasmic and nuclear fractions were then subjected to western blotting and labelled with rabbit monoclonal anti-GAPDH  antibody and rabbit polyclonal anti-LSD1 antibody .Individual post-mortem brain donor, independent cell-line or individual animals were used as the unit of replication. Unique identification numbers were assigned to all samples to ensure the study remained blind and randomised during data acquisition and analysis. Statistical analysis was carried out using SPSS Statistics 22 (IBM) for ANOVA and Prism8 (GraphPad) for simple linear regression and Mann–Whitney U tests. When ANOVA was used for analysis, for studies of human post-mortem brain, gender and case type  were used as variables and age at death and post-mortem interval (PMI) as covariates; for mouse tissue studies genotype and sex of animal were used as variables. For fibroblast experiments, as the group sizes were small the non-parametric Mann–Whitney U test was used for analysis with disomy/trisomy of Hsa21 status as the variable. The mean of technical replicates was used for biochemical datasets and cellular immunofluorescence data. Repeated measures ANOVA was used for immunofluorescence data from cases of human post-mortem brain, slide 1 and slide 2 treated as repeated measures.To determine the colocalisation between two proteins  in an image , Pearsonp-values less than 0.05 are considered to be statistically significant.Graphs were plotted using Prism8 (GraphPad), data are presented as mean ± Standard Error of the Mean (SEM), APP, PSEN1 or PSEN2), Alzheimer’s disease in individuals who had DS (AD-DS), and healthy ageing disomic individuals. All cases were sourced from the South West Dementia Brain Bank (SWDBB), University of Bristol, UK and were matched as far as possible for demographics . No significant difference in PMI was found between case type  = 0.487, p = 0.620). No significant difference was observed in the number of cases that had a APOE 2/3, 2/4 alleles compared with 3/3 alleles or with 3/4, 4/4 alleles  = 1.945, p = 0.745). However, APOE 4/4 homozygotes were only observed in the EOAD group, likely contributing to the early development of disease in these individuals. The majority of cases of EOAD and AD-DS were Braak neurofibrillary tangle stage VI [In this study we compared cases of EOAD . To determine if trisomy 21 results in an increase in CSTB protein in the brains of people who have AD-DS, we compared the abundance of the protein between these individuals and individuals from the general population with EOAD or who were ageing healthily, by western blot. The abundance of CSTB was significantly higher in cases of people who had AD-DS than in those from the general population who had EOAD or those that did not have dementia  , by western blot using two independent antibodies (Ab-3 and ab92955). We found no significant change in the abundance of mature cathepsin B protein between the subgroups of cases with either antibody . To control for the non-specific activity, we measured the rate of cleavage in each sample inhibited by FMK(z-FA-FMK), a cysteine protease inhibitor. This non-specific activity was then subtracted from the total activity to identify the proportion of the activity specific to cathepsin B. Using this method, we observed that cathepsin B activity is elevated in the temporal cortex of people who had EOAD compared with healthy controls, but that no difference in cathepsin B activity was observed in individuals who have AD-DS compared to healthy ageing controls Fig. h. MoreovTo understand if the change in cathepsin B activity was specific, we assayed the activity of another major lysosomal protease (cathepsin D); the activity of which is not regulated by CSTB . We measCSTB, raises the abundance of CSTB protein in the brains of individuals who have AD-DS. This may cause the specific decrease in cathepsin B activity observed in the brains of people who have AD-DS compared to the raised activity of the enzyme which occurs in people who have EOAD.These data indicate that having three copies of p = 0.007), with activity being significantly higher in EOAD compared with healthy ageing (pairwise comparison with Bonferroni p = 0.008) but not different between AD-DS and healthy ageing (pairwise comparison with Bonferroni p = 1.000) , with activity being significantly lower in EOAD compared with healthy ageing (pairwise comparison with Bonferroni p = 0.028), activity did not significantly differ in AD-DS compared with healthy ageing (pairwise comparison with Bonferroni p = 0.070) and no difference in the activity was observed between AD-DS and EOAD (pairwise comparison with Bonferroni p = 1.000)  with EOAD  and healthy ageing . In this sub-analysis, cathepsin B activity was significantly different between case types  = 6.792, + microglia, the number of cathepsin B+ cells, the proportion of cathepsin B colocalised with IBA1+ microglia and the relative proportion of IBA+ microglia that contain cathepsin B, in samples of temporal cortex from people who had EOAD, AD-DS or healthy ageing  and matched disomic individuals 2Yey mouse model, which has an additional copy of 38 genes, carried on an internal duplication on Mmu10 of the region of homology with Hsa21. This region includes Cstb and thus the Dp(10)2Yey model carries three copies of this gene. We undertook the study in mice from a cross of this duplicated region with a mouse model (AppNL−F/NL−F) in which the endogenous mouse App gene is partially humanised and carries AD causal point mutations, resulting in the accumulation of human amyloid-β within the brain of the model 2Yey mice; but was not altered in littermates by the expression of human APP/amyloid-β Fig. a, b. We CSTB on Hsa21 when in three copies might be sufficient to modulate the activity of cathepsin B in mouse brain, we measured activity in the cortex of 3-month of age Dp1Tyb mouse model of DS, which has an additional copy of 148 Hsa21 homologous genes, including App, located on Mmu16 and the Dp(17)3Yey mouse model of DS, which has an additional copy of 17 Hsa21 homologues located on Mmu17. Enzyme activity was not altered by the presence of either segmental duplication, we note neither of these models carries an additional copy of Cstb. These data indicate that three copies of Cstb is not sufficient to modulate cathepsin B activity in a mouse model of AD-DS, consistent with our previous findings from a segmental duplication model of Cstb [To determine if genes other than  of Cstb , nor is Our data demonstrate that three copies of Hsa21 raises the abundance of CSTB protein in the brains of individuals who have AD-DS, in the Dp2(10)Yey mouse model of DS, and in fibroblasts isolated from children who have DS compared with matched disomic controls, consistent with our primary hypothesis. Increased abundance of this endogenous inhibitor of cysteine cathepsins could modulate key aspects of neurobiology by the inhibition of the activity of cathepsin B. This may be particularly relevant in AD pathogenesis in which abundance and activity of the enzyme is upregulated and has been suggested to impact on a number of key disease mechanisms, including APP processing, amyloid-β catabolism, and the neuroinflammatory response to pathology; particularly via the activation of inflammasomes –37.+ cells, and the activity of cathepsin B is significantly increased in people who have EOAD compared with those undergoing healthy ageing, supporting the proposed role of the enzyme as a potential therapeutic target in AD. This is consistent with reports of elevated cathepsin B associated with amyloid plaques in LOAD [Here, we demonstrate that the number of cathepsin B in LOAD , 39 and  in LOAD , 41. We  in LOAD , indicat+ cells is similarly increased in individuals who have AD-DS, we find that the activity of the enzyme is significantly lower than in matched individuals who have EOAD consistent with our hypothesis; this indicates that trisomy of Hsa21 impairs enzyme activity during end-stage disease. These changes occur independently of neurofibrillary tangle stage, or the colocalisation of the enzyme with IBA1+ microglia and may result from the additional copy of CSTB carried by individuals who have DS.Although the number of cathepsin BWe observe no difference in the activity of cathepsin B between individuals who have AD-DS and euploid individuals who do not have AD and are ageing healthily, in contrast to the upregulation in activity we observed in disomic individuals who had EOAD. The majority of individuals who have DS develop early onset-AD in mid-life , thus weCSTB which encodes cystatin B. In these model systems we observed that the abundance of CSTB protein is increased but that cathepsin B enzyme activity is not robustly altered. We also observed this in the cortex of a DS-AD mouse model 2Yey) at 3-months of age, which expresses human amyloid-β but has yet to accumulate significant aggregated peptide [APP mouse models have indicated an increase in cathepsin B protein abundance [Cstb was sufficient to increase cathepsin B activity in the mouse brain, but here we find conversely that an additional copy of Cstb is not sufficient to decrease cathepsin B activity. This is consistent with our previous report that duplication of the Cstb gene is also not sufficient to alter cathepsin B activity in the cortex of a mouse model, and underlies that loss and gain of function of a gene do not always have inverse consequences [To investigate the role of increased CSTB further, we used mouse and human cellular preclinical models, that have an additional copy of  peptide , 33. Prebundance . Howeverbundance . In thisequences .A previous study has reported a small but significant increase in cathepsin B activity in fibroblasts isolated from infants and children who had DS and typically developing controls . We noteOur data indicate that the raised abundance of CSTB protein is not sufficient to modulate cathepsin B activity in a range of model systems, perhaps because of the homeostatic regulation of the interaction of enzyme and inhibitor. However, our preclinical models do not recapitulate all aspects of end-stage (Braak VI) AD, notably the development of tau neurofibrillary pathology, a neuroinflammatory reaction to neuropathology and changes to the blood brain barrier, all of which may contribute to the dramatic increase in cathepsin B activity we observed in individuals who have EOAD. Thus, further research is required to determine if raised CSTB may contribute to altered AD-associated cathepsin B activity in the context of these key aspects of the disease. This is important, as the changed AD-associated cathepsin B activity we observe, could contribute to differences in the progression of AD in people who have DS. Moreover, understanding why the activity is different in the presence of an additional copy of Hsa21 could provide significant novel insight into the underlying biology. Furthermore, our data indicate that targeting cathepsin B activity in people who have DS  is unlikely to have the same therapeutic effect as in individuals who have AD in the general population and that alternative targets should be considered for this important group of individuals who have a greatly elevated risk of early-onset disease.Here we find that cathepsin D activity is not significantly altered in the brains of individuals who have AD-DS compared to disomic healthy ageing controls; although our study may be underpowered. A previous study had shown that cathepsin D activity is robustly decreased in fibroblasts isolated from individuals who have DS compared to disomic controls . MoreoveOur data indicate that disomic individuals who have EOAD have decreased cathepsin D activity, this suggests that decreased enzyme activity may be attributable to the early development of AD in mid-life rather than a direct effect of three copies of Hsa21 and these processes may differ in EOAD and LOAD. This could relate to the elevated abundance of amyloid-β or APP-C-terminal fragment, which have been proposed to inhibit cathepsin D activity , 52 and + and CatB+ cells we observed in people who have AD-DS compared with disomic individuals who were ageing healthily may have a neurodevelopmental cause and not be the direct result of AD.Here we study a case series of individuals who had EOAD and did, or did not, have DS, compared with healthily ageing individuals from the general population. As outlined, the demographics of the groups differed. In particular, the healthy ageing group was older than the AD-DS and EOAD groups, and the Braak tangle stage of the AD-DS and EOAD groups were not perfectly matched. To address these limitations, we included age at death as a covariant in our analysis and performed a sub-analysis of our Braak VI stage cases. In addition, here we only studied 10 individuals of each case-type and thus intra-individual variability may confound our results, and confirmation of our key findings in independent cases would be beneficial. It was not possible to study aged-matched cases of human-post-mortem material from individuals who have DS but did not have AD, because of the early development of AD neuropathology in people who have DS . Thus, wHere we tested our hypothesis that raised CSTB protein leads to reduced cathepsin B activity, as suggested by our human post-mortem analysis, in both human cellular and mouse models using a range of technical approaches. As outlined above, both preclinical systems have limitations and do not model all aspects of the biology of the human brain.+ cells and the abundance of cathepsin B protein does not differ between AD-DS and EOAD cases. However, the abundance of CSTB is significantly higher in the temporal cortex of cases of AD-DS compared with EOAD or healthy ageing. We also find that in both a human cellular and mouse model of DS, CSTB protein levels are increased but this is not sufficient to change cathepsin B activity. Research to understand these similarities and differences will both ensure that individuals who have DS have access to the most appropriate AD therapeutics and provides unique insight into the biology of AD pathogenesis by understanding how an additional copy of genes on Hsa21 can modulate disease.Our data indicate both similarities and differences between EOAD in people who have and do not have DS. We find that cathepsin B activity is lower in people with DS when they develop AD compared with disomic individuals. In contrast, EOAD is associated with a similar decrease in cathepsin D activity in people with and without an additional copy of Hsa21. We also find that the number of cathepsin BAdditional file 1. Supplementary Figure 1: changes to cathepsin B and cathepsin D activity in a sub analysis of EOAD and AD-DS Braak VI cases.


Mapping mouse grooming episodes to neural activity shows that striatal cells deep in the brain collectively represent key aspects of self-grooming. Related research article Minkowicz S, Mathews MA, Mou FH, Yoon H, Freda SN, Cui ES, Kennedy A, Kozorovitskiy Y. 2023. Striatal ensemble activity in an innate naturalistic behavior. eLife12:RP87042. doi: 10.7554/eLife.87042.Are you sitting still right now, or are you moving? Occasionally, our brains compel us to scratch an itch, comb our fingers through our hair, or stroke our chin without even thinking about it. These actions may seem inconsequential to us, but they are examples of a unique type of behavior called self-grooming. Found in species spanning most of the animal kingdom – from monkeys to prairie voles and even fruit flies – self-grooming can serve as a powerful lens for revealing what the brains of monkeys and fruit flies might have in common.Self-grooming is commonly defined as a behavior used to care for the outside of the body . While tNow, in eLife, Ann Kennedy and Yevgenia Kozorovitskiy of Northwestern University and colleagues – including Samuel Minkowicz as first author – report a new method for identifying grooming episodes in video footage, and then show that discrete groups of striatal neurons show activity at the beginning and end of such episodes in mice, as well as throughout episodes .Historically, grooming episodes were identified by hand-labeling high-speed videos , which wIn addition to capturing video footage, Minkowicz et al. used probes embedded in the striatum of mice to record neuronal activity and investigate how neurons in the striatum collectively represent grooming. The recordings showed that when a mouse is relatively still, striatal cells fire sparsely and randomly. However, during a grooming episode, the cells appear to coalesce into clusters of neurons that fire simultaneously, in line with recent recordings of striatal neurons during other types of movement . These ‘The findings suggest that grooming, and perhaps other spontaneous behaviors, are represented by small ensembles of striatal neurons as discrete, unitary objects that are strung together to form sequences. This is potentially at odds with other work that has found that the striatum represents continuous aspects of movement like velocity and vigor , althoug


Background: With the advent of cochlear implants, tactile aids for the profoundly deaf became obsolete decades ago. Nevertheless, they might still be useful in rare cases. We report the case of a 25-year-old woman with Bosley–Salih–Alorainy Syndrome and bilateral cochlear aplasia. Methods: After it was determined that cochlear or brainstem implants were not an option and tactile aids were not available anymore, a bone conduction device (BCD) on a softband was tried as a tactile aid. The usual retroauricular position and a second position close to the wrist, preferred by the patient, were compared. Sound detection thresholds were measured with and without the aid. Additionally, three bilaterally deaf adult cochlear implant users were tested under the same conditions. Results: At 250–1000 Hz, sounds were perceived as vibrations above approximately 45–60 dB with the device at the wrist. Thresholds were approximately 10 dB poorer when placed retroauricularly. Differentiation between different sounds seemed difficult. Nevertheless, the patient uses the device and can perceive loud sounds. Conclusions: Cases where the use of tactile aids may make sense are probably very rare. The use of BCD, placed, e.g., at the wrist, may be useful, but sound perception is limited to low frequencies and relatively loud levels. Cochlear implants have been the standard method of treatment of profound or even severe hearing loss ,2 for seIn the light of this undeniable success, older methods and former ideas, such as tactile aids ,5,6, havAs the largest centre for cochlear implantation at this time in our country, we have never used or prescribed tactile aids until, amazingly, this very year, when we learned that they may still be of some limited value in some very rare cases.The parents of a 25-year-old woman diagnosed with Bosley–Salih–Alorainy syndrome ,9,10,11 The patient had bilateral cochlear aplasia, which is known to occur in some, but not in all, patients with Bosley–Salih–Alorainy syndrome ,11. FiguIn the audiometric assessment, we found a bilaterally normal impedance audiometry and observed normal otoscopic findings, but no otoacoustic emissions could be evoked in either ear. In pure tone audiometry, no hearing was found in either ear, at any of the audiometric frequencies, and neither in the air conduction (AC) measurements (125–8000 Hz) nor in the bone conduction (BC) measurements (250–800 Hz) up to the audiometer limits, i.e., up to 120 dB HL for the AC measurements and up to 80 dB HL for BC measurements.Cognitive and behavioural abnormalities have also been described in Bosley–Salih–Alorainy syndrome ,11, and In the absence of both cochleae, cochlear implantation was not an option. Anatomically, a brain stem hearing implant ,13 was cWith the options thus severely limited, we searched for tactile aids but did not find any that were commercially available anymore. In this situation, we performed a trial with a Cochlear Baha 6 max   bone conWe tried the sound processor on a softband in the usual position behind the ear, and indeed the patient could perceive loud sounds in the order of magnitude of 70 dB to 90 dB HL in the frequency range of 250 to 1000 Hz as vibrations. In contrast, no sound detection was found without the device, at least not up to the maximum levels available with our sound field audiometer. The audiometer was an Equinox 2.0  and it was connected to a Genelec type 8030C loudspeaker . The frequency-dependent maximum sound field levels of the system are shown in We felt that perception thresholds as high as 70 to 90 dB HL were not satisfactory. In order to improve the range, we tried another position: above the wrist of the subject . Indeed,The patient took the device home for a trial and now uses it on an irregular, almost but not quite daily basis, for up to several hours a day. She clearly prefers the position close to the wrist over any placement on the head. For practical reasons (limitation of the movement of her hand), she prefers a position behind the wrist, as shown in As we had no prior experience of our own, it was unclear to us whether the absolute perception thresholds found were typical and, specifically, if the difference between the head and the hand positions was real and could also be found in other subjects. A literature search was rather unfruitful, as research on vibratory sensations seems to be mostly concentrated on low to very low frequencies of often 300 Hz or even lower, and to sensations at the hands, e.g., .In order to learn more about these vibratory thresholds, we asked three cochlear implant users to help us with a limited additional evaluation. Two of the subjects were male, one was female. The age range was 23–85 years. All three had bilateral profound deafness and air conduction hearing thresholds well above the maximum sound field levels our audiometer could emit at any frequency. All of their bone-conduction hearing thresholds were above the maximum output limits of our audiometer, but one control subject was able to perceive the vibration of the bone vibrator at 250 Hz and 500 Hz, as shown in Their perception thresholds were measured under sound-field conditions using narrow-band noise signals, with their cochlear implant sound processors taken off. Measurements were performed with the BC sound processor mounted on a softband and placed either in the usual position behind the ear on their non-implanted side, or the wrist, in a position as similar as possible to the one preferred by the patient. Vibratory sensation rather than auditory perceptions were expected and also reported by all three subjects. For all subjects, a third measurement was performed without any sound processor.Above the individual thresholds, sounds could be reliably detected and very rough temporal patterns  were reliably perceived. Any further discrimination seemed to be next to impossible, at least without training.Cochlear implants have been used since 1990 at our department and, until recently, the use of tactile aids was not even seriously considered. Somewhat to our surprise, we found that there may still be justifiable applications, although they are probably very rare. It is interesting to note that a very different application of tactile aids than the one reported here may also be useful. Specifically, it has been found that a multisensory approach, i.e., combined auditory and tactile stimulation, can improve speech understanding in noise .The bone conduction sound processor used as a tactile aid does indeed allow the perception of acoustical signals. However, it is limited to relatively loud sounds and to low frequencies, and the sensation is vibrotactile. By itself, it is certainly unsuitable for any but possibly the simplest forms of oral communication. It is unclear how much it can help, e.g., by supporting lip reading, as the limited cognitive capabilities of our patient did not allow a closer examination. We would expect at most a limited help. Nevertheless, the patient does use the device and it seems to be helpful to detect some of the louder acoustical signals in her surroundings.Bone conduction devices such as the one used here were developed to elicit auditory sensations via the BC pathway, the cochlea and, ultimately, the auditory nerve. We have no indication that this is happening here. All subjects reported clearly tactile sensations and no hearing sensations through the BC device. This holds true for the position at the head as well as at the wrist. Consequently, we believe that we have measured purely tactile sensations.The placement of the BC processor does affect perception thresholds. The placement behind the wrist seems, in this respect, to be better than the normal placement of BC hearing aids behind the ear, not only in our patient, but mostly also in the small sample tested. Certainly, each placement has also its own practical challenges, such as, e.g., limitations of own movements, visibility, or risk of contact to clothing. It is conceivable that other placements than the two reported here may be better.Although BC devices can be used as tactile aids to perceive acoustic signals, it is important to stress that in most cases this is one of the last resorts. If at least one cochlea with an intact auditory nerve is present, cochlear implantation should clearly be considered first and will probably lead to much better results in most cases. Even in patients with bilateral cochlear aplasia and bilateral auditory nerve aplasia, the use of tactile aids is one of the last solutions to be evaluated. Auditory brainstem implants should be considered first, even though results are generally poorer than with cochlear implants . As withCases where the use of tactile aids may make sense are probably very rare. The use of bone conduction devices, placed preferably close to the wrist, may be useful, although the perception of sound signals is limited to low frequencies and relatively loud levels, and sound discrimination must be expected to be very poor.