Beauveria bassiana on the soil-dwelling, pupal phases of Frankliniella occidentalis in laboratory bioassays and greenhouse experiments. Based on micromorphological observations of fungal conidia during the infection process after treatment of WFT with a B. bassiana suspension, fungal conidia complete the process of surface attachment, germination, and penetration of the body wall of the WFT pupa and enter the host within 60 h of treatment. Given these results, we undertook a controlled greenhouse experiment and applied B. bassiana granules to soil used to cultivate eggplants. Populations of F. occidentalis on eggplants grown in treated soil were 70% lower than those on plants grown in the untreated soil after 8 weeks. Furthermore, when measuring the survival and growth of B. bassiana on granules under different soil moisture conditions, survival was greatest when the soil moisture content was kept at 20%. These results indicate that the application of B. bassiana-impregnated granules could prove to be an effective biological control strategy for use against F. occidentalis under greenhouse conditions.Western flower thrips (WFT) is one of the most important pests of horticultural crops worldwide because it can damage many different crops and transmit various plant viruses. Given these significant impacts on plant production, novel methodologies are required to maximize regulation of WFT to minimize crop losses. One particular approach is to develop control strategies for the non-feeding, soil-dwelling stages of WFT. Control of these stages could be enhanced through the use of granules impregnated with entomopathogenic fungi mixed in the soil. The use of soil-applied fungi contrasts with existing approaches in which entomopathogenic fungi are formulated as oil-based suspensions or water-based wettable powders for foliar applications against the feeding stages of WFT. To examine the efficacy of this approach, we evaluated the effects of a granular formulation of  Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is one of the most important pests of horticultural crops worldwide [Tospovirus (Bunyaviridae) [tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV), which may cause over 90% yield loss in some instances [Western flower thrips (WFT), orldwide ,2,3. Damorldwide . Moreoveviridae) , includinstances ,7,8.The life cycle of WFT contains the foliar-feeding  and soil-dwelling developmental stages . Ninety-eight percent of the WFT that pupate enter the soil although this is dependent on the host plant species ,10. HoweBeauveria bassiana  Vuillemin , Metarhizium brunneum (Metchnikoff) Sorokin, Isaria fumosorosea (Wize) A.H.S. Br. and G. Sm and Lecanicillium lecanii R. Zare and W. Gams targeting Lepidoptera, Coleoptera, Hemiptera, and Diptera [B. bassiana has been shown to be a highly effective biological control agent against the different life stages of WFT under laboratory, greenhouse and open-field conditions [As an alternative option for control, entomopathogenic fungi are well-documented biological control agents and have great potential for controlling agricultural insect pests worldwide . Success Diptera . For exanditions ,21,22.Metarhizium anisopliae preparations resulted in significant infection and mortality in larvae of the sugar beet root maggot, Tetanops myopaeformis [B. bassiana granules on rice nursery soil can be an effective and efficient biological control strategy for the management of rice water weevils [B. bassiana granules to the soil could provide enhanced control of the below-ground stages of WFT through better targeting of infective conidia [Most formulations of entomopathogenic fungi have been developed for foliar applications to control the foliar feeding stages of WFT ,23,24,25aeformis . Applica weevils . The app conidia . B. bassiana strain GZGY-1-3, a native fungal isolate previously shown to be effective against the foliar stages of WFT, to control the pest in the soil. A series of steps were undertaken: (1) laboratory bioassays of the granules to confirm the efficacy of this approach and to define a suitable field application rate; (2) observations of fungal attachment and infection using scanning electron microscopy and fluorescence microscopy to confirm the susceptibility of WFT pupae to this fungal strain and determine the dynamics of the infection process; and (3) an evaluation of fungal granules to control WFT in greenhouse eggplants. We wished to test the overarching hypothesis that the application of B. bassiana granules to the soil would provide effective control of WFT under laboratory and greenhouse conditions.This current research was designed to test the feasibility of using granular formulations of Capsicum annuum L.) in the Mentougou Vegetable Planting Center of Beijing, China, in 2013 . Unsterilized kidney bean pods (Phaseolus vulgaris L. (Fabaceae)) contained within tube-shaped glass jars (0.5 L) with Snap-On lids were used to culture the WFT colonies. A 10-cm diameter hole was cut in each lid and covered with a fine mesh to allow for ventilation. These tubes were maintained under controlled conditions of 25 ± 1 °C, 70 ± 10% RH and a L14:D10 h photoperiod. A method of feeding by age was utilized to maximize colony growth. To do this, bean pods were replaced every three days because WFT take approximately 3 days to complete each larval stage at 25 °C. To prepare an even-aged cohort of WFT pre-pupae for the lab trials, eight bean pods carrying WFT eggs were removed from the colony. Newly emerged first instar WFT were removed and allowed to develop in synchrony for three days and were then transferred to a new jar containing fresh bean pods. After approximately seven days, WFT pre-pupae were collected in the lower part of the rearing device and were used in the experiments that are described below.A colony of WFT was established with adults and collected from pepper fields , was used in the study. This strain was originally isolated from an infected cadaver of Ostrinia furnacalis in Guizhou, China. The fungus was cultured on Sabouraud Dextrose Agar (SDA)  at 26 ± 1 °C for seven days and harvested from the plates in sterile aqueous 0.05% Tween 80 . A suspension containing 1 × 107 conidia/ml was prepared from the stock suspension, which was used in the microscopic studies (to inoculate the pre-pupae) and to inoculate the solid medium when producing granules. The viability of the conidia was confirmed as being over 90%, using protocols described by Goettel and Inglis [d Inglis . Previoud Inglis .B. bassiana was grown through a liquid–solid two-phase fermentation process. During the liquid phase, B. bassiana conidia obtained from agar slant culture media were used to inoculate a spore-forming medium  for strain activation at 26 °C for 5 d until conidiation occurred. Conidia were harvested from the medium, and a suspension containing 1 × 107 conidia/ml was prepared using the methods outlined above, which was used for the microscopic studies (to inoculate the pre-pupae). Three hundred milliliter liquid growing medium  was dispensed to each 1 L Erlenmeyer flask . Each flask was inoculated with the 1 × 107 conidia/ml conidial suspension and incubated on a rotary shaker at 150 rpm 26 °C for 3 d. Mycelia and blastospores were produced in the liquid medium. The solid substrate  was sterilized by autoclaving at 121 °C for 30 min. To inoculate the solid substrate, 1.5 L blastospore culture containing 1 × 1011 conidia/ml was used, and this was then transferred to the bag . Approximately 3 kg of solid substrate was placed in a fermentation gunny bag  with proper ventilation (74 cm × 107 cm) and maintained under controlled conditions of 26 °C for 10 d. A 500 watt electric fan  continuously worked in the first 3 days to ensure that the substrate received ‘proper ventilation’ in the gunny bags. For the first three days, ambient humidity was maintained at over 90% to stimulate mycelium growth. Thereafter, the humidification process was stopped to promote conidiation. After 10 days, the solid medium was placed in a YC-6 commercial ebullated dryer , and conidia were separated from the substrate. The substrate was mixed with vermiculite at a 3:1 ratio to produce granules for the experiments. The viability of the conidia retained on the granules was over 90%, which resulted in a conidial loading of 1 × 108 per gram of granules.To obtain granules for experimentation, B. bassiana granules was investigated against WFT pre-pupae under laboratory conditions. Loam soil was collected from a greenhouse facility at the Langfang Experiment Station of the Chinese Academy of Agricultural Sciences, Langfang, China and dried using an electric constant temperature blast drying oven  at 105 °C for 8 h. B. bassiana granules (1 × 108 conidia per gram) were mixed with soil at six rates: 0, 6.25, 12.5, 25, 50 and 100 g per kg, and 100 g of each soil-granule mixture was placed in each of the 3 Petri dishes for each soil-granule mix (12-cm diameter). Then, 20 pre-pupae were transferred into each Petri dish and a fresh kidney bean pod was added as food. Dishes were covered with a plastic film that was perforated to provide ventilation. Petri dishes were kept in the chamber at 25 ± 1 °C, RH 70 ± 5% and a L14:D10 h photoperiod. The number of adult WFT was counted after 5 days, and the experiment was repeated three times.The efficacy of the B. bassiana, 500 pre-pupae were infected by dipping in a conidial suspension  for 5 s. This concentration is commonly used for spray applications of the fungus against WFT in greenhouses in China. After treatment, 20 pre-pupae were transferred to each of the 6 Petri dishes (7-cm diameter) containing bean leaf discs (7-cm diameter) on moist filter paper; the dishes were covered with plastic film that was perforated to provide ventilation. WFT were then removed from the dishes at prescribed time points, i.e., 2 h, 24 h, 36 h, etc. The Petri dishes were maintained in an environmental chamber at 25 ± 1° C, RH 70 ± 5%, and a L14:D10 photoperiod.To investigate the infection process of WFT pre-pupae by B. bassiana were removed from the Petri dishes 2, 24, 36, 48, 60, and 72 h after inoculation and placed in 5% glutaraldehyde in cacodylate buffer for 12 h at 25 °C. The samples were treated according the method of Wu et al. (2016) [To prepare infected WFT pre-pupae for examination by scanning electron microscopy , 20 WFT exposed to . (2016)  and examSimilarly, inoculated WFT pre-pupae were examined by fluorescence microscopy. A fluorescent dye stock solution was prepared by dissolving 4 mg FDA  in 1 mL of acetone; 35 mL of FDA stock solution was then diluted in 4 mL of deionized water to prepare a fluorescent dye working solution. The working solution for each experiment was freshly prepared and the vessel containing the working liquid was shielded from light by aluminum foil and kept on ice.Beauveria bassiana-treated pre-pupae were removed from each Petri dish 2, 12, 24, 36, 48, and 72 h after inoculation, and were placed on a clean glass slide. A single drop of working solution was placed on the insect which was overlaid with a coverslip. Fifteen treated insects of each group were observed by excitation light of 450–490 nm under a blue filter fluorescent microscope  at 400× magnification.Twenty B. bassiana granules was mixed with 100 g of dried loam soil and water was added to achieve the desired moisture content. Each batch was transferred to a Petri dish which was covered with plastic film. Three replicate batches (dishes) were prepared per treatment. The dishes were transferred to square plastic boxes containing a small amount of water in the bottom to maintain soil moisture levels through the trial and then kept at 25 °C. Soil samples were collected from the dishes at random every day for 8 weeks using a 1-cm diameter core sampler. To assess the level of soil colonization by B. bassiana, the number of colony-forming units (CFU) per gram of dried soil was determined. Three grams of each soil sample was weighed into a 250 mL flask which contained 97 mL of sterile aqueous 0.05% Tween 80. The contents were mixed thoroughly on a rotary shaker at 195 RPM/min for 2 h. A dilution series was prepared from the resulting suspension, and 300 μL samples from each dilution were plated onto PDA medium supplemented with streptomycin; three replicate plates were prepared from each dilution. Plates were incubated at 25 °C for 4 days. Plates with between 10 and 100 colonies were selected, and colonies were counted and used for data analysis [The soil moisture content  was adjusted to 10%, 15%, 20%, 25%, and 30%, respectively. Ten grams of analysis . ConcurrB. bassiana against F. occidentalis were conducted using eggplants (Solanum melongena L. (Solanaceae)) in a greenhouse facility at the Langfang Experiment Station of the Chinese Academy of Agricultural Sciences, Langfang, China . Briefly, eggplant seedlings were transplanted at the three-leaf stage in a total of 13 double-rows, 14 plants per row. Two weeks after transplanting, groups of four adjacent eggplants were covered with cages constructed with fine-mesh thrips-proof screening (length × width × height = 70 cm × 110 cm × 2 m). The cages were fitted with a zipper to allow the plants to be accessed and enable F. occidentalis populations to be monitored. At the beginning of the experiment, 200 adult WFT were added to each cage to establish a breeding population. One hundred and fifty grams of B. bassiana granules per cage were applied to the soil surface and were incorporated into the top 1 cm of soil within the row using a hoe. This concentration of B. bassiana granules was shown to cause 80% mortality of WFT in the laboratory bioassays described earlier. The eggplant was watered by drip irrigation. The soil moisture content was measured by a soil humidity sensor , and the experimental soil was maintained at 20% to simulate the optimal conditions for B. bassiana activity. This level of soil moisture was also suitable for eggplant growth. The granular treatment and an untreated control were replicated three times within the greenhouse. Experiments examining the effectiveness of the granular formulation of A five-point sampling method was used to count the number of WFT. Specifically, leaves were selected by visually dividing the eggplants in a cage into three equal vertical strata. One leaf was randomly sampled from each stratum of each plant, giving a total of 12 leaf samples from each cage and all WFT on the leaves (upper and lower leaf surfaces) were counted in situ. This ensured uniform sampling among plants and an accurate representation of the WFT population on each plant . The inip = 0.05 to determine significance. For colonization of B. bassiana tests, a two-way repeated-measures ANOVA (‘proc GLM’ procedure in SAS 9.4) (first factor: the soil moisture content (five levels), second factor: sampling date (eight levels)) was used to compare the CFUs of B. bassiana between treatments over time. For the greenhouse trials, a two-way repeated-measures ANOVA (‘proc GLM’ procedure in SAS 9.4) , second factor: Sampling date (seven levels)) was used to compare the density of F. occidentalis between treatments over time. Differences in mean F. occidentalis densities between treatments were compared by t-test on each sampling date. All data were analyzed using SAS 9.4 at a 0.05 level of significance.For the screening tests, mortality data were corrected with Abbott’s formula , normaliB. bassiana granules exerted significantly greater control efficacy against WFT. Mortality of F. occidentalis increased with increasing concentrations of B. bassiana (B. bassiana-containing granules was mixed with 1 kg of soil (83.33 ± 3.33%). There were significant differences between the five groups of treatments . Mycelia of B. bassiana were observed on the surface of WFT cadavers when dead thrips were examined under a stereomicroscope 4 days after infestation. These results confirmed that B. bassiana granules could be applied to the soil to control the soil-dwelling stages of WFT.Compared with the control, bassiana . The higScanning electron microscopy (SEM) and fluorescence microscopy revealed that conidia of strain GZGY-1-3 became attached to the body surface of WFT pre-pupae within 2 h A1,A2. AfB. bassiana CFUs recovered from inoculated soil varied among the different soil moisture levels tested. The highest CFU levels (5.86 × 107 CFU/g) were obtained in the 20% soil moisture treatments by week 2 , and with time after treatment . Interestingly, there was also a significant interaction between the soil moisture content and treatment time .The number of y week 2 , represeB. bassiana survival and persistence and WFT populations were significantly lower on plants grown in soil treated with the GZGY-1-3 granules than in the untreated control at all time points after plants were infested (p = 0.1117). After the application of B. bassiana granules, populations in the treatment group decreased significantly  within the first week. The granules continued to suppress WFT for eight weeks relative to the control. Both the treatment and control groups showed an overall upward trend in WFT numbers after four weeks, and WFT populations were highest after seven weeks . After eight weeks, WFT numbers declined by 67% in the B. bassiana treatment and there was a significant difference between the two populations . There were significant differences between treatments and times and there was a significant interaction of B. bassiana between time after treatment .In the greenhouse experiments, soil moisture levels were maintained at 20% throughout to simulate optimal conditions for infested . There wB. bassiana granules to the soil surface suppressed F. occidentalis population growth. Moreover, this research clarified the time taken for B. bassiana to infect the pupal stages of WFT and the influence of soil moisture conditions on B. bassiana survival and soil colonization. Our results confirm earlier reports that B. bassiana granules are an effective formulation of this entomopathogen for F. occidentalis control in soil and validated the potential of this alternative management approach [In this study, applications of approach .B. bassiana and F. occidentalis. We observed that the surface of the insect body becomes covered with hyphae, suggesting that B. bassiana strain GZGY-1-3 was pathogenic to WFT [B. bassiana suspension, confirming the utility of fluorescence microscopy as a viable means of observing the fungal infection process in WFT [Scanning and fluorescence electron microscopy were instrumental in observing the interactions between c to WFT ,34. A las in WFT . We obses in WFT ,37.Beauveria bassiana infection is moderated by external environmental factors, such as ambient humidity and temperature, which has limited use of B. bassiana in large-scale field settings. When environmental conditions are unsuitable, the conidia are easily inactivated, reducing their capacity to regulate pest populations [ulations . Importaulations ,39. In culations . B. bassiana [B. bassiana [B. bassiana in our trial to determine whether the B. bassiana granules were capable of infecting and controlling WFT. We found that the survival of B. bassiana conidia and the growth of the fungus from granules were significantly reduced when the soil moisture content was below 20%. Moisture is an important factor affecting spore germination, mycelial growth and the pathogenicity of bassiana ,41. It hbassiana . MortaliB. bassiana as part of an integrated management approach. For instance, entomopathogenic fungal granules could be applied to the soil to eliminate the soil-dwelling stages of WFT, allowing insecticides to be applied for the additional management of WFT populations on the foliage when required [In our greenhouse trials, granular treatments suppressed WFT population growth. Infection of WFT pre-pupae probably occurred as the insects moved into the soil. The formation of conidia on WFT cadavers could serve as a source of fresh inoculum capable of infecting susceptible insects in the same environment. The granular formulation clearly provided adequate protection against WFT in our experimental system and may be useful in other greenhouse vegetable production systems. Although a single biological control agent may not be expected to control an entire target pest population, IPM methods that combine multiple agents are likely to succeed . Our resrequired ,24. Morerequired .B. bassiana granules to the soil surface can successfully suppress WFT under greenhouse conditions. More in-depth research to devise viable application strategies for fungal granules and their integration with other biocontrol strategies could lead to a valuable alternative strategy for managing WFT. In conclusion, our research demonstrates that the application of


OTOG gene variant. We analyzed 98 hearing loss-related genes in patients with hearing loss originally from the Okinawa Islands using next-generation sequencing. We identified a homozygous variant of the gene encoding otogelin NM_001277269(OTOG): c.330C>G, p.Tyr110* in four families. All patients had moderate hearing loss with a slightly downsloping audiogram, including low frequency hearing loss without equilibrium dysfunction. Progressive hearing loss was not observed over the long-term in any patient. Among the three patients who underwent newborn hearing screening, two patients passed the test. OTOG-associated hearing loss was considered to progress early after birth, leading to moderate hearing loss and the later stable phase of hearing loss. Therefore, there are patients whose hearing loss cannot be detected by NHS, making genetic diagnosis of OTOG variants highly useful for complementing NHS in the clinical setting. Based on the allele frequency results, hearing loss caused by the p.Tyr110* variant in OTOG might be more common than we identified. The p.Tyr110* variant was reported in South Korea, suggesting that this variant is a common cause of moderate hearing loss in Japanese and Korean populations.We describe the clinical features of four Japanese families with moderate sensorineural hearing loss due to the  OTOG, which encodes a protein expressed in sensory cells of the inner ear, could thus be an important complement to newborn hearing screening for individuals with a family history of hearing loss who may benefit from early medical intervention. A Japan-based team led by Akira Ganaha from the University of Miyazaki studied seven patients with hearing loss from four unrelated families living on the Japanese archipelago of Okinawa. Each had two copies of a recessive mutation in OTOG, a variant previously documented only in South Korea. Of the three who underwent newborn hearing tests, only one was diagnosed with hearing deficiency at birth.People born with mutations in a gene linked to moderate hearing loss don’t always show problems during newborn hearing screening and may benefit from genetic screening. Testing for this gene,  A large majority of recessive variants are associated with congenital, nonprogressive, and severe-to-profound hearing loss3. Compared with severe-to-profound hearing loss, mild-to-moderate hearing loss in children has been less well characterized. OTOG (DFNB18B) is a causative gene of mild-to-moderate nonsyndromic hearing loss5. OTOG encodes otogelin, which is a noncollagenous protein specific to the inner ear6. Hearing loss in OTOG knockout mice is bilateral and highly progressive immediately after birth, ranging from mild-to-profound7. Five causative variants in OTOG have been reported in four families to date8. Although only four families have been reported to date with OTOG mutations, the onset and long-term course of hearing loss and complications of disequilibrium have not been clarified.To date, over 100 causative genes have been identified for nonsyndromic deafness, of which 70% follow a recessive inheritance patternOTOG, c.330C>G, p.Tyr110*, in four families. In this study, we investigated the onset and long-term clinical features of hearing loss patients with the OTOG p.Tyr110* variant. Based on the allele frequency in the 1000 Genomes Project and in this study, hearing loss caused by the OTOG gene might be more common than reported previously.We analyzed 98 genes related to hearing loss using next-generation sequencing (NGS) in patients originally from the Okinawa Islands. NGS revealed the presence of a homozygous variant in exon 4 of Seven affected patients from four unrelated families were investigated Fig. . ClinicaPrior to enrollment, all subjects provided written informed consent. Our research protocol was approved by the Ethics Review Boards of the University of Miyazaki and University of the Ryukyus.10. Briefly, genomic DNA was extracted from whole-blood cells using the QIAamp DNA Blood Mini Kit . For targeted resequencing, a customized HaloPlex Target Enrichment Panel  was used to enrich each DNA sample from the families for 98 candidate genes for hearing loss, including OTOG. Each enriched DNA samples were sequenced with the MiSeq System . Variants from sequencing data were called using the SureCall pipeline (Agilent Technologies). For whole-exome sequencing analysis, the SureSelect Human All Exon V6 Kit  was used for capture, and the HiSeq2500  was used for sequencing. Reads were aligned to CRC37 using Burrows-Wheeler Aligner. Variants were called using the GATK Unified Genotyper and ANNOVA (http://annover.openbioinfomatics.org/en/latest/).Targeted resequencing for hearing loss (family 48) or whole-exome sequencing  was performed as described previouslyOTOG gene was performed in all subjects. PCR was performed as follows: initial denaturation at 94 °C for 5 min; 35 cycles of 94 °C for 40 s, 64 °C for 40 s, and 72 °C for 1 min; and a final extension at 72 °C for 5 min. PCR was performed on a programmable thermal cycler . PCR products were purified using the Wizard SV Gel and PCR Clean-Up System  and sequenced directly using an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems). The sequences were aligned and compared using the BLAST program with known human genome sequences available in the GenBank database.To confirm the coding sequence and variant, Sanger sequencing of the OTOG  in 100 healthy unrelated Okinawan individuals used as controls. The genotypes of the p.Tyr110* variants were detected by digestion of the PCR product with the restriction enzyme BsaXI .We surveyed the substitutions in exon 4 of OTOG gene, c.330C>G, p.Tyr110*, in seven patients with moderate sensorineural hearing loss  is a causative gene of nonsyndromic mild-to-moderate hearing loss that follows an autosomal recessive inheritance pattern. Five causative variants in OTOG have been reported in four families to date8 . The p.Tyr110* variant was reported in only the Japanese population in the 1000G with an allele frequency of 0.5%. In this study, the allele frequency of the p.Tyr110* in Okinawan control individuals was identified in 0.5% (1/200), with the same allele frequency in 1000G and that of p.H723R in SLC26A4. Which means that one out of 40,000 may have mild-to-moderate hearing loss due to the p.Tyr110*. Based on these results, hearing loss caused by p.Tyr110* in OTOG might be more common than we identified. However, only eight families, including our four families, with hearing loss caused by OTOG variants have been reported to date, and this is the first report of hearing loss caused by this variant in the Japanese population. There is a possibility of underestimation of the prevalence of hearing loss caused by the OTOG gene in that the patients with the variant might be excluded from the genomic research because of mild phenotype12.Among the hereditary hearing loss, the c.235delC in 13. The common variant p.Tyr110* identified in all four families in this report is considered to be the result of a founder effect by a common ancestor. The same variant was reported from Korea in 20185, which also suggests that the variant has common ancestor in Korea5. Altogether, it is possible that p.Tyr110* may be a common cause of moderate hearing loss in Japanese and Korean5 populations.The Okinawa Islands are the southwestern-most islands of the Japanese archipelago. Previous studies suggested that there were substantial ancestral differences between the Okinawa Islands and the main islands of JapanOTOG in seven patients of four families with moderate hearing loss in the Japanese population. Hearing loss was considered to occur immediately after birth, showing a moderate and slightly downsloping audiogram, and was nonprogressive thereafter. None of the families had symptoms of vestibular disorders. NM_001277269(OTOG): c.330C>G, p.Tyr110* was considered to be a highly frequent causative variant of mild-to-moderate hearing loss in Japanese and Korean5 populations. The present results with a long-term follow-up will improve the genetic counseling of patients with mutations in OTOG.In conclusion, we identified the variant c.330C>G, p.Tyr110* in


Phalacrocorax aristotelis), we tested the effect of parasitism on maintenance costs as well as the relationship between proxies for RMR and DEE. We found a positive relationship between parasite load and our RMR proxy in females but not males, and no relationship between proxies for RMR and DEE. This provides evidence for increased maintenance costs in individuals with higher parasite loads and suggests the use of an allocation energy management strategy, whereby an increase to RMR creates restrictions on energy allocation to other activities. This is likely to have fitness consequences as energy allocated to immunity is traded off against reproduction. Our findings demonstrate that understanding energy management strategies alongside fitness drivers is central to understanding the mechanisms by which these drivers influence individual fitness.Parasites often prompt sub-lethal costs to their hosts by eliciting immune responses. These costs can be hard to quantify but are crucial to our understanding of the host's ecology. Energy is a fundamental currency to quantify these costs, as energetic trade-offs often exist between key fitness-related processes. Daily energy expenditure (DEE) comprises of resting metabolic rate (RMR) and energy available for activity, which are linked via the energy management strategy of an organism. Parasitism may play a role in the balance between self-maintenance and activity, as immune costs can be expressed in elevated RMR. Therefore, understanding energy use in the presence of parasitism enables mechanistic elucidation of potential parasite costs. Using a gradient of natural parasite load and proxies for RMR and DEE in a wild population of breeding European shags ( Summary: Demonstration of the sub-lethal costs of endo-parasitism for a wild population of seabirds via increased maintenance metabolism. Parasites cause major fitness consequences to a huge array of taxa . Often, Energy can be a limited resource, and individuals are required to allocate energy to the demands of competing life-history traits to maximize fitness . TherefoParasitism may play a central role in the relationship between self-maintenance and activity because a primary predicted cost of parasitism is increasing RMR, through immune-associated costs . Hosts sUnderstanding the impacts of natural parasite burdens on individual metabolic rate is crucial, with experimental studies suggesting that endo-parasites and elevated immune function incur significant costs to hosts . HoweverPhalacrocorax aristotelis (Linnaeus 1761)] to understand the effect of endo-parasites on maintenance costs as well as the relationship between maintenance and total energy expenditure under high energetic demands during the breeding period. Specifically, we investigate two questions: (1) Does parasite load relate to RMR? We expect that individuals with higher parasite loads will have higher immune costs and that this will be reflected in elevated RMR. (2) Does DEE relate to RMR and what can this tell us about the allocation between energy to reproduction and self-maintenance of this species? We predict that individuals will be energetically constrained by rapidly growing chicks during the chick-rearing period under the allocation model and therefore any increase in RMR will not result in an increase in DEE.Here, we measure proxies of both DEE and RMR in individuals with known natural endo-parasite loads in a free-living population of breeding European shags  using bater see . Thus, aater see .All models were fitted separately for males and females due to non-independence of nest pairs. To answer the first question of whether parasite load is related to elevated RMR, we modelled T3 concentration using linear mixed effects models (LMMs). Parasite load, brood size (number of chicks), brood age (age of the oldest chick in the brood at the time of sampling), year, and adult age and body mass were explanatory variables and we controlled for variation between birds and repeated sampling by including individual as a random factor. We fitted models containing all combinations of the fixed effects, and interactions between parasite load and brood age and parasite load and brood size  using LMMs. Adult age, adult body mass, brood age (at the time of sampling), brood size and year were also included as explanatory variables . We conthttp://www.R-project.org/) and model selection for both sets of models was based on Akaike's information criterion (AIC) (All models were fitted using the lme4 package in R (on (AIC) .A total of 87  individuals were blood sampled and endoscoped over 3 years (of which 27 were sampled in more than one year). Whilst the sample size in 2015 is clearly smaller than the other years, removal of these data had no impact on the conclusions of the analyses and were therefore included. The best-supported model for females showed a positive relationship between parasite load and T3 concentration, an effect of year, and an interaction between brood age and parasite load in that there was a more positive relationship between parasite load and T3 when broods were younger see . When yeDEE was estimated in a subset of the birds  to gain insight into the relationship between RMR and DEE. The best-supported model for females was the null model with no relationship found between T3 and estimated DEE see . For malMeasuring both natural parasite burdens and metabolic rates in free-ranging animals is challenging. In particular, estimating metabolic rate requires the use of proxies, the potential limitations of which have been extensively documented e.g. . Our stuWe found no relationship between our proxy for RMR and parasitism in males despite the strong relationship found in females. Males are hypothesized to experience the immunosuppressive qualities of testosterone ; thus, tThis study is the first to use T3 concentration as a proxy for RMR in the context of parasitism effects. This enables us to gain greater insight into the energy use of free-ranging animals and complements our previous findings and other approaches. It is likely that energy management is not fixed across time but varies according to particular conditions, as seen in At the scale of a lifetime, life-history theory predicts that trade-offs between reproduction and self-maintenance change with age. As the probability of future reproduction declines with age, it is predicted that resources will be increasingly allocated to current reproduction rather than maintenance . There iWe found no evidence for a relationship between proxies for RMR and DEE, indicating that DEE does not vary with maintenance costs. Despite parasite-induced behavioural costs, European shag estimated DEE does not correlate with parasite load , suggestIt is unclear why species vary in their energy management strategy, although, during energetically demanding periods, the capacity to increase expenditure can impose energy limits, in terms of foraging constraints, making an allocation strategy more likely . Under l


CDKL5 gene that encodes a serine/threonine kinase. CDD is characterised by the early onset of seizures and impaired cognitive and motor skills. Loss of CDKL5 in vitro and in vivo affects neuronal morphology at early and late stages of maturation, suggesting a link between CDKL5 and the neuronal cytoskeleton. Recently, various microtubule (MT)-binding proteins have been identified as interactors of CDKL5, indicating that its roles converge on regulating MT functioning. MTs are dynamic structures that are important for neuronal morphology, migration and polarity. The delicate control of MT dynamics is fundamental for proper neuronal functions, as evidenced by the fact that aberrant MT dynamics are involved in various neurological disorders. In this review, we highlight the link between CDKL5 and MTs, discussing how CDKL5 deficiency may lead to deranged neuronal functions through aberrant MT dynamics. Finally, we discuss whether the regulation of MT dynamics through microtubule-targeting agents may represent a novel strategy for future pharmacological approaches in the CDD field.CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy caused by mutations in the X-linked  CDKL5) gene is known since 1998, when it was identified in a transcriptional mapping approach to identify disease-causing genes in the Xp22 region . N. N18]. NAs mentioned, dynamic instability is largely influenced by a plethora of proteins that bind MTs. Classical MT-associated proteins (MAPs) constitute an important group of proteins that bind along the MT lattice, influencing its polymerisation, stability and bundling. MAP1 family proteins, such as MAP1A, MAP1B and MAP1S, are high molecular weight complexes formed of heavy and light chains (LC) . The capThe +TIP family comprises another large group of diverse proteins that share the common feature of associating with the growing MT plus-end, where they control MT dynamics and functioning . The pluFunctionally, the +TIPs can affect MT dynamics in opposing ways with the EBs suppressing the frequency of MT catastrophes and the CLIPs promoting MT rescue . The +TIThe importance of the proper regulation of the MT network for neuronal functions has been demonstrated through the pharmacological manipulation of MT dynamics. Indeed, axon formation can be artificially induced treating unpolarised neurons with the MT-stabilising drug, taxol . On the As mentioned, several links have recently been established between CDKL5 and MTs. Indeed, CDKL5 has recently been found to interact with the two +TIPs, CLIP170 and EB2 ,10,11. TCDKL5 mutations, CDKL5 functions have been studied in vitro and in vivo in proliferating cells, primary neurons, in mouse models carrying inactivating deletions of the Cdkl5-gene and in human induced pluripotent stem cells (iPSC)-derived neurons [Since the discovery of the first patients with  neurons . The oveCdkl5-KO neurons, which in both cases is accompanied by altered MT dynamics [In primary low-density cultures of hippocampal neurons, CDKL5 controls axon specification and outgrowth ,36. Axondynamics ,40. The dynamics . Interesdynamics , is capadynamics . It thusIn the past, CDKL5 was proposed to affect axon formation through its interaction with Shootin1 . ShootinCdkl5-KO brains [Cdkl5-KO primary hippocampal neurons [CDKL5 deficiency was first shown, through silencing studies, to influence dendritic arborisation in vitro in primary cultures of rat cortical neurons and in vivo in migrating neurons derived from silenced neural progenitor cells . Such deO brains ,47,48 an neurons ,40. The  neurons .The control of CDKL5 on dendrite morphology was found to occur through Rac1, which is a critical regulator of actin remodelling. Recombinant CDKL5 was capable of pulling out Rac1 from neuronal lysates, an interaction that was further stimulated upon prior neuronal stimulation with brain-derived neurotrophic factor (Bdnf). Further, Bdnf activation of Rac1 was impaired in CDKL5 silenced cells, thus placing Rac1 downstream the kinase . The exaCdkl5-KO neurons, but are restored to normal life-times upon MAP1S silencing [Another possible way in which CDKL5 regulates MT dynamics during dendritic arborisation may be represented by the phosphorylation on MAP1S, recently identified as a bona fide substrate of CDKL5 ,12 Figu. When philencing .Various studies in vitro and in vivo converge on a role of CDKL5 in controlling dendritic spine morphology and excitatory functions. Neurons devoid of CDKL5 are characterised by an increased number of filopodia-like protrusions and a reduced number of mushroom-shaped spines ,53. The Plasticity-related changes in spine morphology are widely known to rely on remodelling of the actin cytoskeleton, which is highly enriched in dendritic protrusions . MTs werIqgap1-KO mice display cognitive defects and impaired long-term potentiation (LTP) [Regarding a possible role of CDKL5 in regulating spine morphogenesis through MT-related events, it is tempting to speculate that more +TIPs may be involved in regulating spine morphogenesis and functions creating other distinct links between MTs and the actin cytoskeleton. This would allow an expansion in the repertoire of possible signalling events underlying structural plasticity. While EB2 is broadly expressed in the neuronal soma, its levels are very low in the post-synaptic compartment . Accordion (LTP) . IQGAP1 on (LTP) ,69; it ion (LTP) , is an ion (LTP) . DLG5 ison (LTP) .Considering that MTs constitute fundamental tracks for the motor-based transport of cargoes, their invasion into dendritic spines is likely to provide a way of transporting cargoes into and out of these structures. The nature of which cargoes enter spines through MTs is still not clear. However, synaptotagmin has been found to be transported in a kinesin-dependent manner into dendritic spines . FurtherThe role of CDKL5 in regulating MT dynamics is consistent with our previous report that described CDKL5 as a centrosomal protein . The cenCDKL5 include missense mutations, nonsense, frame-shift and splicing variants, and large genomic deletions [The above studies linking CDKL5 to MT-related functions have been addressed in cells or tissues silenced for CDKL5 or with no expression of the protein. This reflects well the current view that CDD is caused by the absence of functional CDKL5 or by the expression of hypomorphic derivatives. Indeed, CDD-causing mutations in eletions . The pateletions ,85,86,87eletions ,88,89. CDKL5, have been identified in individuals with varying degrees of macrocephaly and learning disabilities [The existing animal models of CDD have addressed the consequences of the absence of CDKL5 either in the whole body or in distinct brain areas ,47,48,90bilities , suggestADNP have been linked to autism and intellectual disability, and Adnp haploinsufficiency in mice is linked to altered axonal transport and dendritic spine alterations [Alterations of the MT cytoskeleton have been linked to neurodegenerative and neurodevelopmental disorders such as schizophrenia and autism and major depression disorder ,93. Effoerations . The NAPerations . In pre-erations . In clinerations  and dailerations . The possibility of targeting of proteins that have been functionally linked to CDKL5 might hold an even higher therapeutic potential for CDD. Interestingly, the neuroactive steroid PREG was found to directly bind CLIP170, inducing its open active conformation, as well as MAP2 . NotablyTo conclude, the last findings of CDKL5 functions converging on MT dynamics are likely to be of great relevance not only for a deeper understanding of the neuronal dysfunctions linked to CDKL5 deficiency but also for the development of disease-modifying pharmacological approaches. Importantly, they may also lead to the identification of clinical diagnostic biomarkers. However, before considering MT-targeting drugs for clinical applications for CDD, we still need to understand in more detail how precisely CDKL5 deficiency affects MT dynamics.


Cathepsin H is a member of the papain superfamily of lysosomal cysteine proteases. It is the only known aminopeptidase in the family and is reported to be involved in cancer and other major diseases. Like many other proteases, it is synthesized as an inactive proenzyme. Although the crystal structure of mature porcine cathepsin H revealed the binding of the mini-chain and provided structural basis for the aminopeptidase activity, detailed structural and functional information on the inhibition and activation of procathepsin H has been elusive. Here we present the crystal structures of human procathepsin H at 2.00 Å and 1.66 Å resolution. These structures allow us to explore in detail the molecular basis for the inhibition of the mature domain by the prodomain. Comparison with cathepsin H structure reveals how mini-chain reorients upon activation. We further demonstrate that procathepsin H is not auto-activated but can be trans-activated by cathepsin L. Cathepsin H is a lysosomal cysteine protease of the papain superfamily involved in protein degradation. Overexpression of cathepsin H was reported to be associated with several pathological states including carcinoma and melanoma –3. For pUpon activation, cathepsin H displays a distinct proteolytic activity among the papain superfamily, functioning predominantly as an aminopeptidase that cleaves a single N-terminal residue from a polypeptide chain . CathepsHere we report a structural and functional analysis of procathepsin H to understand its inactivation and activation. Our crystal structures of human procathepsin H wild type (PDB code: 6CZK) and C26S mutant (PDB code: 6CZS) at 2.00 Å and 1.66 Å provide high resolution molecular basis of the binding of the mini-chain to the mature domain and understanding how the prodomain inhibits the mature domain. Superposition with previous structures suggests how the mini-chain reorients and positions upon activation. In addition, we demonstrate that procathepsin H is not capable of autoactivation and requires other proteases such as cathepsin L for its activation.Purified procathepsin H protein was not autoprocessed under the acidic crystallization condition and we were able to determine the crystal structure of procathepsin H at 2.00 Å resolution . ProcathProcathepsin H closely resembles the other papain family cysteine proteases, particularly procathepsin L (PDB code: 1CS8) , with anGlycans could be assigned unambiguously in procathepsin H for the two glycosylation sites, Asn79P on the mini-chain of the prodomain and Asn115 on the mature domain. Similar glycosylation of Asn115 was also observed in cathepsin H . HoweverSimilar to procathepsin L , the pro3-Arg32P-X3-Phe36P-X2-Asn39-X3-Ile43P-X3-Asn47P)  [35 [35-1. Tlgorithm , GLYCAM_lgorithm , TIP3P algorithm .


Although considerable progress has been made in the last 30 years in the treatment of cleft palate (CP), a multidisciplinary approach combining examinations by a paediatrician, maxillofacial surgeon, otolaryngologist and speech and language pathologist followed by surgical operation is still required. In this work, we performed an observational cross-sectional study to determine whether the CP grade or number of ventilation tubes received was associated with tympanic membrane abnormalities, hearing loss or speech outcomes.Otologic, audiometric, tympanometric and speech evaluations were performed in a cohort of 121 patients (children > 6 years) who underwent an operation for CP at the Vall d’Hebron Hospital, Barcelona from 2000 to 2014.p = 0.820). The degree of hearing loss (p = 0.616), maximum impedance (p = 0.800) and tympanic membrane abnormalities indicative of chronic otitis media (COM) (p = 0.505) among examined patients revealed no statistically significant association with the grade of CP. However, an association was identified between hypernasality and the grade of CP (p = 0.053), COM (p = 0.000), hearing loss (p = 0.000) and number of inserted ventilation tubes.The most and least frequent CP types evaluated according to the Veau grade were type III (55.37%) and I (8.26%), respectively. A normal appearance of the membrane was observed in 58% individuals, of whom 55% never underwent ventilation ear tube insertion. No statistically significant associations were identified between the CP type and number of surgeries for insertion of tubes (Although the placement of tympanic ventilation tubes has been accompanied by an increased rate of COM, it is still important to assess whether this is a result of the number of ventilation tubes inserted or it is intrinsic to the natural history of middle ear inflammatory disease of such patients.Our results do not support improvements in speech, hearing, or tympanic membrane abnormalities with more aggressive management of COM with tympanostomy tubes. Clefts of the lip, alveolus and palate are the most common congenital malformations of the head and neck and the second most common of all congenital malformations in general . Both clMalformations affecting the soft and hard palate influence the functionality of the Eustachian tube, interfering with the ear ventilation and drainage from the ear . TherefoThe list of specialists required for the proper treatment of CP is not complete without the identification of ear, nose and throat (ENT) manifestations associated with CP. Follow-up examination involves a maxillofacial surgeon who monitors the development of dentition and palate, an otolaryngologist who evaluates the middle ear status, an audiologist who monitors hearing loss, and a speech and language pathologist. Recommendations for surgical correction and treatment of middle ear pathology are based on otologic and audiometric evaluations. To date, there is still a lack of evidence indicating that a greater degree of alteration of the palate is associated with increased velopalatal and Eustachian tube dysfunction and consequently an increased rate of chronic otitis media (COM) and increased hearing loss , 12. In In the short term, patients treated early with ear tubes exhibit better audiometric results as well as better articulation and speech development compared with patients treated conservatively, whereas no significant differences have been observed regarding mid- or long-term outcomes. Since early placement of tympanic ventilation tubes has been the standard in the care of CP children and often accompanied by an increased rate of tympanic membrane abnormalities indicative of COM, it is important to assess whether this is a result of the insertion of multiple tympanostomy tubes or it is intrinsic to the natural history of middle ear inflammatory disease of such patients .Here, we studied a cohort of CP patients who underwent operation at the Vall d’Hebron Hospital in Barcelona to determine how CP type or degree may affect the presence of tympanic membrane abnormalities indicative of COM among patients who underwent operation for this type of malformation. The impact of CP type and number of ventilation tubes received on otologic, audiologic, and speech outcomes was investigated. Therefore, the state of the ear was reviewed by otoendoscopy/otomicroscopy, and audiometry was performed in external consultations. This information was related to the type of CP not with the type of surgery because we agreed that this variable was uniform in all cases.This research is an observational cross-sectional study on a group of patients. Otologic, audiometric, tympanometric and speech evaluation were conducted for a cohort of children (> 6 years) with cleft repair between 2000 and 2014. All individuals were followed by the ENT service of our centre. The type and degree of velopalatal malformation, presence/absence of hearing loss, and number of surgeries for insertion of ear tubes were recorded. The indication used in our service for the placement of the tympanostomy tubes is the presence of persistent seromucosa otitis media (with at least 3 months of evolution) associated with conductive hearing loss greater than 30–35 dB (predominantly in the low frequencies) and/or a delay/alteration in the acquisition of language to a greater or lesser degree.In addition, patients were classified according to the presence/absence of tympanic membrane abnormalities indicative of COM. The procedures described within the present study are part of routine clinical care.The inclusion criteria were age greater than or equal to 6 years, repair of cleft palate +/− cleft lip between 2000 and 2014, repair performed in our centre, follow-up by ENT in our centre, and complete otoscopic, audiometric, impedance and speech evaluation at the time of the study.The exclusion criteria were malformative syndrome affecting the facial mass and/or accompanied by alteration of the Central Nervous System, isolated cleft lip, cleft repair at outside hospital, no ENT follow-up, or no otoscopic, audiometric impedance and/or speech testing at the time of this study.Tympanic membranes were evaluated using microscopic otoscopy. The term chronic otitis media refers to intractable middle ear or mastoid tissue pathology behind an intact or perforated tympanic membrane. The presence of cholesteatoma, deep attic retraction pockets, persistent membrane perforations (> 3 months), atelectasis or severe adhesive membranes were considered as COM. Ears that had a lower degree of shrinkage or persistent otitis media with effusion (> 3 months) without further tympanic alterations were considered abnormal but not as COM. In the same way, the presence of tympanostomy tubes without the presence of other changes in the tympanic membrane, apart from these at the time of examination, was not considered COM. Finally, the presence of isolated myringosclerosis was not considered abnormal, as this finding has not been suggested as a risk factor in the development of COM.Pure-tone audiometry was performed on all patients using an audiometer MAICO MA-51 (CE certificate 0124)  and headphones  properly calibrated in accordance with the manufacturer’s manuals. Five pure tone averages (PTA) at 500, 1000, 2000, 4000, and 8000 Hz were calculated. The degree of hearing loss was evaluated as classified by Northern and Downs . BrieflyMiddle ear impedance was measured using Titan .Type A tympanograms with peak pressures within {plus minus} 50 daPa and compliance of 0.2–1.4 cc were considered normal because it is a normal curve where the pressure in the middle ear corresponds to the atmospheric and therefore to that of the external auditory canal. However, the tympanograms Ad, As, B and C are pathological curves.Speech parameters assessed included nasal resonance and articulation errors. Hypernasality was ranked on a scale of 0–3, which corresponds to the absence of nasal resonance (0), mild (1), moderate (2) and severe (3) hypernasality. Speech articulation was evaluated by demonstrating the presence or absence of errors.The continuous variables of the study were summarized by descriptive statistics: mean and standard deviation (sd). Similarly, categorical variables were summarized by frequency statistics: number of cases and percentage.The following relationships were evaluated using Chi-squared or Fisher’s exact test: Veau classification-COM, Veau classification-hypoacusis, Veau classification-abnormal impedance, Veau classification-hypernasality, Veau classification-number of ventilation tube surgeries, number of ventilation tube surgeries-COM, and number of ventilation tube surgeries-hypoacusis.Multivariate analysis by using the multiple linear regressions was performed to assess the association with Veau classification, age, number of ventilation tubes inserted, degree of hearing loss (independent variables) and COM (dependent variable).The SPSS program (version 16.0)  for data management and the Stata program (version 13.1)  were used for the remainder of the calculations.A level of significance α = 0.05 was established in all tests performed.Otoscopic evaluation was performed on 121 individuals, including 64 males and 57 females. Ages ranged from 6 to 31 years with an average of 13.21 years (sd = 5.94); 50% were older than 12 years. Tympanic membranes were viewed in each of the 242 ears; 58% of the subjects had a normal membrane. Our data revealed 42% individuals with various abnormalities, including severe adhesiveness (18.20%), simple perforation (3.30%), ventilation tubes in situ (3.30%), cholesteatoma (2.10%) and others  (15%) Fig. .Fig. 1OtThe types of palate were evaluated according to Veau grade . The mosp = 0.820)  Table . Our datp = 0.616)  Fig. . Our datp = 0.800)   Fig. . Patient5) Table . AlthougNo conclusive or statistically significant results were obtained regarding the relationship of the laterality of the unilateral fissure with the presence of hearing loss, COM or language impairment (data not shown).Normal appearance of the membrane was observed in 58% individuals, and a significant portion of these individuals (55%) never underwent ventilation tube insertion Fig. .p = 0.000) (Tables There was a significant association between the number of tubes inserted or hearing loss and COM  Tables  and 4. N) Tables . The preIn general terms, the multivariate analysis showed that the presence of COM is more frequently associated with a greater cleft palate type, number of surgeries for the placement of ventilation tubes and degree of hearing loss Table .Table 5Mp = 0.053)  Table . Up to 4Overall, our data suggested that the type of cleft malformation did not lead to an increased rate of otologic or audiometric alterations but rather depended on the number of surgeries for insertion of ear tubes.The association of CP with ear pathology and hearing loss has been studied for greater than a century. CP is believed to be associated with COM of infants and children due to poor Eustachian tube function and other anatomic problems. The current study aimed to determine whether Veau classification or number of ventilation tubes is associated with the presence of COM, hearing loss or speech alteration.Despite the results obtained, we cannot be sure whether the positive relationship between COM and the number of surgeries is related to surgery or with the natural course of a more aggressive ear pathology that makes it necessary to place a greater number of ventilation tubes in this type of patient. However, we can confirm that those patients with tympanic and/or middle ear alterations present worse hearing compared with patients with a relatively normal otoscopy. In a previous study, the rate of type A and B tympanograms, the proportion of discharge during myringotomy, and the indwelling period of the first ventilating tube insertion also influenced the probability of additional ventilation tube insertion in CP patients .Little information is known about the natural history of CP patients who have not been subjected to palatoplasty or any intervention at the otologic level. Based on the vocal quality characteristics of children with CP, a comprehensive speech and language pathology intervention is recommended . HoweverThe type of palatine defect plays an important role in the development of mucosal otitis media (OM) and has been observed in patients with major defects requiring additional surgeries for the insertion of tympanostomy tubes . It is nThe number of tympanostomy tubes inserted appears to be related to the abnormal mid-term and long-term tympanic findings. However, atrophy, retraction, and COM may also appear as sequelae in children with persistent mucosal OM who never had tympanostomy tubes . The lonFocal atrophy and tympanosclerosis are two well-documented types of complications of ventilation tube insertion. The tympanic membrane with tympanosclerosis is considered in numerous abnormal studies despite its small tendency to progress to COM with a minimal effect on hearing. Persistent perforations (9.13%) may also occur, particularly after insertions of repeated tubes, interventions at very early age, or the insertion of permanent drainage tubes, such as Goode’s T-tubes .Cholesteatoma appears in studies as a complication of ventilation tube insertion, especially after insertion of T-tubes. The relationship between COM and cholesteatoma is clear in CP patients, with rates ranging from 0.90 to 9.20% for cholesteatoma. In our case, the frequency of cholesteatomatous COM was 2.06% as it was only observed in 5 patients with this type of pathology. The incidence of COM in this type of patients is variable (11–37%). Persistent tympanic perforation (1.30–19%), tympanic retraction (11.50–36.80%), tympanic sclerosis (11–37%), secondary cholesteatoma (0–3.80%) and otorrhea (up to 11.50%) present with great variability based on the revised study. In our study, we exclusively focused on membrane changes, such as retraction and ‘pexia’, that could be considered to be risk factors for the possible progression to COM when tympanosclerosis is not considered to be an abnormal appearance of the tympanic membrane.The involvement of COM in CP remains debated. A recent study on 105 CP Turkish patients demonstrates a statistically significant relationship between the middle ear disease, function of Eustachian tube, treatment outcomes, middle ear pressure, CP type and bilateralism of the disease . A studyp = 0.546), the need for an increased number of ventilation tubes inserted (p = 0.820) and grade of hearing loss (p = 0.616). Although these results did not imply a difference between the groups, patients with a grade III or IV Veau fissure exhibit an increased frequency of COM  and a greater degree of hearing loss (up to 38% of patients with grade IV fissures had hearing loss). Similarly, patients with more complex palatine fissures (grades III and IV) require a greater number of surgeries for insertion of ventilation tubes (approximately 50% of patients compared with 30% for patients with Veau grade I or II). Given the lack of significance of the results obtained, we conclude that further studies would be necessary to determine whether this finding was influenced by sample size or whether the relationship described was not significant.In our study, we observed no statistical association between CP type and COM. Accordingly, no statistically significant relationship was noted between palate type and COM  exhibited mild hearing loss (49.17%). In addition, 34.30% of the ears evaluated exhibited normal hearing (0–25 dB), 14.04% had moderate hearing loss and only 2.47% of the ears had severe hearing loss. Profound hearing loss was not noted in any patient. It seems likely that patients who received a greater number of ventilation tubes displayed an increased rate of COM and worse audiometric results in the long term. Up to 86% of the patients in our study who had COM underwent operation for at least two occasions of transtympanic drainage. Only 17.24% of these patients had strictly normal hearing.In our study, most ears examined (Our results demonstrate that the CP type did not affect articulation, speech development or hearing loss and was also not associated with COM. By contrast, these parameters seemed to vary depending on the number of tympanostomy tubes inserted. These results do not support improvements in speech, hearing, or tympanic membrane abnormalities indicative of COM with tympanostomy tubes. Although the placement of tympanic ventilation tubes has been often accompanied by an increased rate of COM, it is important to assess whether this is a result of the number of ventilation tubes inserted or it is intrinsic to the natural history of middle ear inflammatory disease of such patients, so more studies are needed.In the case of children, CP is believed to be associated with COM due to poor Eustachian tube function and other anatomic problems, so understanding Eustachian tube dysfunction is an important component of CP management.There are many reasons to suggest that mucous OM in these children should be treated conservatively in early childhood. Given that conservative management of mucosal OM is relatively safe and has no long-term sequelae and that other forms of auditory rehabilitation, such as a hearing aid, are available, clinicians could consider more conservative management in light of these data; however, more studies are needed. Nevertheless, the improvement is not exclusively the result of palatal surgery but a combination of factors, including surgical correction, developmental factors, treatment of middle ear pathology and a multidisciplinary approach.


Our in vitro studies showed that E09241 increased the alkaline phosphatase (ALP) activity of mouse osteoblasts, promoted mineralization, and increased the expression of osteogenic differentiation-related genes. In addition, we observed that E09241 inhibited RANKL-induced osteoclast differentiation and reduced the expression of osteoclast differentiation-related proteins nuclear factor of activated T cells c1 (NFATc1) and matrix metalloproteinase 9 (MMP-9). More importantly, E09241 exerted therapeutic protection against bone loss in ovariectomized rats in vivo. This protective effect was confirmed to be achieved by inhibiting bone resorption and promoting bone formation in vivo. Mechanistically, E09241 regulates OPG expression through canonical Wnt/β-catenin signaling. Our findings suggest that E09241 is a promising small-molecule compound for treating osteoporosis with a dual effect on osteoblasts and osteoclasts.The ratio of osteoprotegerin (OPG) to the receptor activator of NF-κB ligand (RANKL) is a key determinant in the regulation of bone metabolism. The study was performed to screen novel anti-osteoporotic drugs regulating OPG/RANKL ratio and evaluate their effect on bone metabolism. According to the screening results and  Osteoporosis is a systemic metabolic bone disease characterized by a gradual decrease in bone mass, deterioration of the bone microstructure, and an increased risk of fracture . The etiBone remodeling is regulated by the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) . RANKL, In this study, we identified E09241 as an OPG/RANKL upregulator, which increases OPG expression but does not affect RANKL expression, by using an OPG/RANKL ratio high-throughput screening (HTS) system. We then explored the effect of E09241 on bone metabolism and revealed that E09241 critically affected the OPG/RANKL ratio and promoted osteoblast differentiation while inhibiting osteoclast differentiation. Furthermore, E09241 rescued rats from ovariectomy-induced bone loss. Further study revealed that E09241 regulated OPG expression through canonical Wnt/β-catenin signaling. Our findings demonstrate the potential role of E09241 in osteoporosis treatment and highlight OPG/RANKL as a promising target for osteoporosis.in vitro assays and 1‰ DMSO  was used as a solvent control named 0 μM E09241.Alpha modification of Eagle’s medium (α-MEM), McCoy’s 5A medium, and Dulbecco’s modified eagle medium (DMEM) were purchased from Thermo Fisher Scientific . Alizarin red S, ascorbic acid, and β-glycerophosphate were obtained from Sigma-Aldrich Inc. . RANKL was obtained from PeproTech . All antibodies were purchased from Abcam . Compound E09241 was obtained from the National Laboratory for Screening New Microbial Drugs, Peking Union Medical College . Compound E09241 was dissolved in dimethylsulfoxide (DMSO) in all 2 atmosphere.MC3T3-E1 mouse calvarial preosteoblasts were obtained from the Cell Center of the Chinese Academy of Science . Human osteosarcoma cell lines U-2OS, murine monocytic cell lines RAW 264.7, and murine mesenchymal C3H10T1/2 cells were purchased from the Cell Center of Basic Medicine, Chinese Academy of Medical Sciences . MC3T3-E1, U-2OS, RAW264.7, and C3H10T1/2 cells were cultured in α-MEM, McCoy’s 5A, DMEM, and MEM, respectively, with 10% FBS . Osteogenic differentiation of MC3T3-E1 cells was induced using an osteogenic induction medium containing α-MEM supplemented with 10% FBS, 50 mg/ml ascorbic acid, and 10 mM β-glycerophosphoric acid. All cells were cultured at 37°C in a 5% CORenilla luciferases. After treatment by compounds for 24 h, the cells were lysed and the luciferase activity was determined by the Dual-Luciferase Reporter Assay System (Promega) with a microplate reader . The activity of a compound in the regulation of the OPG/RANKL ratio was calculated with the following formula: the regulatory activity of the OPG/RANKL ratio = the ratio of firefly to Renilla luciferase activities of test compound/the ratio of firefly to Renilla luciferase activities of vehicle control. A total of 20,000 synthetic compounds from the National Laboratory for Screening New Microbial Drugs were screened. The regulatory activity ≥150% was considered as primarily positive, and these compounds were retested in triplicate to calculate EC50 values.An HTS assay was performed to identify OPG/RANKL upregulator as described previously . U-2OS c4 cells/well and induced with osteogenic differentiation medium. After 12 days of induction, the cells were sonicated on ice and the supernatants were incubated with a solution containing 1.0 mg/ml p-nitrophenyl phosphate (pNPP), 0.5 mM magnesium chloride, and 1 M diethanolamine buffer at 37°C for 30 min and terminated with 3 M NaOH. The absorbance was read at 405 nm using a microplate reader (PerkinElmer). Total protein content was determined using a bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific). The ALP levels were normalized to the total protein content, and the experiments were performed in triplicate.Alkaline phosphatase (ALP) activity assay was performed according to previous reports . MC3T3-EMC3T3-E1 cells were seeded in six-well plates and treated with osteogenic differentiation medium for 21 days. After treatment, the cells were fixed with 4% paraformaldehyde and stained with 40 mM alizarin red S  at room temperature and images were taken.3 cells/well with DMEM containing 50 ng/ml RANKL and treated with various concentrations of E09241. The cells were fixed and stained using a Leukocyte Acid Phosphatase kit  according to the manufacturer’s instructions. The tartrate-resistant acid phosphatase (TRAP)-positive cells with more than three nuclei were counted as osteoclasts. The osteoclasts were visualized with an optical microscope and photographed.RAW264.7 cells were seeded in 96-well plates at a density of 3 × 10Total RNA from the cells was extracted with TRIzol reagent , and reverse transcription for mRNAs was carried out with reverse transcriptional kits (TransGen Biotech). The sequences of the primers were as follows: GAPDH, 5′-AGGTCGGTGTGAACGGATTTG-3′ and 5′-GGGGTCGTTGATGGCAACA-3′; Runx2, 5′-CCGCCTCAGTGATTTAGGGC-3′ and 5′-GGGTCTGTAATCTGACTCTGTCC-3′; ALP, 5′-CCAACTCTTTTGTGCCAGAGA-3′ and 5′-GGCTACATTGGTGTTGAGCTTTT-3′; and Bglap, 5′-CAATAAGGTAGTGAACAGAC-3′ and 5′-CTTCAAGCCATACTGGTCT-3′.MC3T3-E1 cells were plated in six-well plates. The cells were transfected with 50 pmol scramble siRNA  or β-catenin siRNA  using Lipofectamine 2000 (Invitrogen). After 6 h, the medium was exchanged with fresh medium containing E09241 and incubated for 48 h. Cells were then harvested for western blotting assays.The cells were washed with PBS, and protein extracts were prepared in radio immune precipitation assay (RIPA) lysis buffer. Equal amounts of protein extracts were electrophoresed by 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The blots were blocked with 5% (w/v) skimmed milk in PBS-T buffer for 1 h and immunoblotted with primary antibodies at 4°C overnight. Then, blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature and visualized with an electrochemical luminescence reagent (ECL) detection system . Data quantification and statistical analysis were conducted with ImageJ . The membrane protein was prepared using cell membrane protein and Cytoplasmic Protein Extraction Kit .The levels of OPG and RANKL proteins in the cell supernatants were determined using the corresponding ELISA kit . The levels of OPG, RANKL, CTX-1, and ALP in rat serum were measured using ELISA kits .Renilla plasmid using Lipofectamine 2000 (Invitrogen). After 24 h of transfection, the cells were treated with various concentrations of E09241. The firefly luciferase activity and Renilla luciferase activity were measured using the Dual-Luciferase Reporter Assay System (Promega).The cells were co-transfected with pTOPflash (Upstate) or pFOPflash (Upstate), and pRL-TK Twenty-four 7-month-old female Sprague-Dawley rats (290–310 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . Six female rats were subjected to resection of some fat tissue close to the ovaries (sham), and 18 underwent bilateral ovariectomy as previously described . One weeThe femurs taken from rats were dissected free of soft tissue, fixed in 4% paraformaldehyde, and distal femurs were analyzed using a micro-PET/CT scanner . The scanner was set at a resolution of 10 μm, with a tube voltage of 50 kV, and tube current of 400 μA. Three-dimensional reconstruction was conducted with the Inveon analysis workstation. One millimeter below the center of the epiphyseal line with 50 slices was selected as the region of interest (ROI) for the analysis, and the following bone properties were generated: bone mineral density (BMD), relative bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), bone surface/bone volume (BS/BV), and trabecular separation (Tb.Sp).The femurs were fixed in 4% paraformaldehyde, decalcified with 10% EDTA (pH 7.2), and then stained with H&E and TRAP to visualize osteoblasts and osteoclasts. The numbers of osteoblasts per bone surface (N.Ob/BS) and osteoclasts per bone surface (N.Oc/BS) were analyzed by Image Pro Plus 6.0.p values of <0.05.All data are presented as mean values ± SD. Differences between values were analyzed using one-way ANOVA analysis with Tukey’s multiple comparison tests. Statistical significant was assigned to N-(4-methylpyridin-2-yl)furan-2-carboxamide (Renilla luciferase activities, with an EC50 of 1.23 μM (E09241, the systematic name of which is 5-chloro-boxamide , was ide 1.23 μM . We init 1.23 μM . In addi 1.23 μM . WesternRANKL has two forms including membrane-bound form and soluble form, and the membrane-bound form RANKL is essential for osteoclastogenesis. In order to examine the effect of E09241 on different RANKL forms, RANKL levels in cells, cell membranes, and culture supernatants were detected, respectively. As shown in The effect of E09241 on the differentiation of osteoblast MC3T3-E1 cells was determined by an ALP activity assay and alizarin red S staining. E09241 increased the ALP activity of MC3T3-E1 cells in osteogenic induction medium with E09241, especially at 1 μM, for 12 days . MoreoveRunx2, ALP, and Bglap in MC3T3-E1 cells for 12 days. The mRNA expression of Runx2 was increased by treatment with 1–10 μM E09241, and ALP mRNA expression was increased by 1–10 μM E09241 (Bglap mRNA expression was markedly induced by treatment with 0.1–10 μM E09241.We analyzed the effect of E09241 on the mRNA levels of osteogenic differentiation-related genes, including M E09241 . In addiin vitro.To investigate whether E09241 may affect osteoclast differentiation, we treated RAW264.7 cells with 1–10 μM E09241 as well as 50 ng/ml RANKL for 3 days. Our results ,B showedTo explain the direct effect of E09241 on osteoclast differentiation, OPG, RANKL, and RANK levels in E09241-treated RAW264.7 cells were then detected. The results showed that E09241 significantly increased OPG levels and decreased RANK levels but had no obvious effect on RANKL levels in RAW264.7 cells ,D. NucleWe explored the role of E09241 in an OVX osteoporosis rat model. The micro-CT images of OVX rats showed significant trabecular bone loss compared with the sham-operated group , whereasin vivo in each group. The results showed that osteoclast formation was significantly elevated in OVX rats compared to sham-operated rats (in vitro studies.To analyze how E09241 attenuated OVX-induced bone loss, we subsequently evaluated bone formation and bone resorption ted rats ,B. Howevted rats ,B. Moreoted rats . In addiin vitro and in vivo, we further explored how E09241 might regulate the expression of OPG. Increasing evidence has shown that canonical Wnt/β-catenin signaling plays a significant role in regulating bone homeostasis (Given that the upregulation of OPG/RANKL by E09241 is mainly through upregulation of OPG expression eostasis . β-Cateneostasis ; we hypoSince β-catenin accumulates in the nucleus and activates LEF/TCF-mediated gene transcription, thereby affecting the expression of downstream target genes , we firsin vitro results, we propose that E09241 significantly increased the ratio of OPG/RANKL by mainly increasing OPG expression.The homeostasis of bone metabolism is maintained by the balance between osteoblast bone formation and osteoclast bone resorption, and the relative levels of OPG and RANKL determine the processes of osteogenesis and osteoclastogenesis . RANKL, in vitro at 1–10 μM. Osteoclast formation is triggered by a sequence of RANKL-induced signaling that results in the activation of transcription factors, such as NFATc1, and NFATc1 is a master gene for osteoclastic differentiation (Osteoporosis may occur when the bone resorption of osteoclasts is greater than the bone formation of osteoblasts. Thus, strategies for treating osteoporosis are based on stimulating osteoblast activity or inhibiting osteoclast activity. To investigate the effects of E09241 on osteoblasts, we examined the osteogenic differentiation of MC3T3-E1 cells after E09241 treatment. We found that E09241 promoted osteoblast differentiation, as shown by increases in both ALP activity and mineralized nodules in the osteoblasts treated with E09241. Runx-2 is a major marker of osteoblast differentiation . ALP is ntiation . NFATc1 ntiation . We evalin vitro results, our in vivo data in the OVX rat model demonstrated that E09241 effectively suppressed ovariectomy-induced bone loss. Based on the micro-CT image of the trabecular bone structure, our study demonstrated that both low- and high-dose E09241 treatment may prevent trabecular bone structure deterioration induced by ovariectomy. Interestingly, the bone parameters by micro-CT showed that E09241 significantly increased Tb.Th but showed no obvious effect on Tb.sp and Tb.N, indicating that E09241 mainly prevented ovariectomy-induced bone loss by thickening trabecular bone.Consistent with the in vivo, which suppressed bone resorption. In addition, E09241 markedly increases the number of osteoblasts in vivo, thereby resulting in enhanced bone formation. More importantly, we detected the levels of CTX-1 and ALP, which are markers of bone resorption and bone formation, respectively. Our results demonstrated that E09241 reduced serum CTX-1 level and increased serum ALP level, which further confirmed that E09241 attenuated OVX-induced bone loss by inhibiting bone resorption and promoting bone formation.In view of the protective effect of E09241 on bone mass, it is necessary to clarify how E09241 contributed to inhibition of OVX-induced bone loss. We found that E09241 reduced OVX-induced increase in the number of osteoclasts Wnt/β-catenin signaling plays critical roles in bone metabolism . In addiOpg−/− mice. And, further studies will be done to obtain more insights as to the precise mechanism of E09241 on osteoblastic differentiation and osteoclastic differentiation.In this study, we found that E09241 enhanced osteoblast activity and decreased osteoclast formation, thereby resulting in an increase in bone volume. We proposed that mechanistically E09241 regulated OPG expression through canonical Wnt/β-catenin signaling, which upregulated the ratio of OPG/RANKL and therefore affected osteoblastic differentiation and osteoclastic differentiation. Moreover, our findings revealed that E09241 could decrease RANK levels in RAW264.7 cells, which might explain the direct effect of E09241 in RAW264.7 cells. But we do not know whether the decreased RANK level induced by E09241 is related to OPG upregulation. Thus, it is still necessary to explore whether the effect of E09241 on bone metabolism is OPG dependent, which might be conducted by detecting the effects of E09241 on bone metabolism in OPG knockout osteoblasts and In summary, the present study demonstrates that a novel small molecule E09241, which increases OPG expression but does not affect RANKL levels, protects against ovariectomy-related bone loss in rats. The effect of E09241 on OPG expression and bone metabolism may be mediated by Wnt/β-catenin signaling. Our findings suggest that E09241 is a potential compound with dual activity for preventing bone loss and osteoporosis.XHa, SG, WJ, and SS conceived and designed the experiment. XHa, SG, NL, XW, and PL performed the experiment. XHa, YX, and XHe analyzed the data. XHa, SG, WJ, and SS drafted the manuscript. YX, WJ, and SS reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A deeper knowledge of the activity of the forearm muscles during activities of daily living (ADL) could help to better understand the role of those muscles and allow clinicians to treat motor dysfunctions more effectively and thus improve patients’ ability to perform activities of daily living.In this work, we recorded sEMG activity from 30 spots distributed over the skin of the whole forearm of six subjects during the performance of 21 representative simulated ADL from the Sollerman Hand Function Test. Functional principal component analysis and hierarchical cluster analysis (HCA) were used to identify forearm spots with similar muscle activation patterns.The best classification of spots with similar activity in simulated ADL consisted in seven muscular-anatomically coherent groups: (1) wrist flexion and ulnar deviation; (2) wrist flexion and radial deviation; (3) digit flexion; (4) thumb extension and abduction/adduction; (5) finger extension; (6) wrist extension and ulnar deviation; and (7) wrist extension and radial deviation.The number of sEMG sensors could be reduced from 30 to 7 without losing any relevant information, using them as representative spots of the muscular activity of the forearm in simulated ADL. This may help to assess muscle function in rehabilitation while also simplifying the complexity of prosthesis control. The forearm and the hand are connected by the wrist, together forming a functional unit in which there are about 30 muscles that work in concert in a highly complex way , allowinSurface electromyography (sEMG) is a non-invasive approach that allows both structural and functional characterization of the neuromuscular system, as well as making it possible to quantify variations of this system in different situations. sEMG is applied in many fields such as motor control of human movement, myoelectric control of prosthetic and orthotic devices, and rehabilitation –5. In reThe central nervous system must control a structure that is in general vastly more complex than necessary to execute any particular task. In this sense, muscular activation patterns or muscular synergies , 13 appeMyoelectric prostheses are usually controlled with sEMG –27. HoweIn this work, we recorded sEMG activity from different areas of the whole forearm skin during the performance of the ADL included in the SHFT, a standardized test for hand function evaluation. The aim was to identify skin zones with similar muscle activation patterns in order to determine the minimum number of electrodes required to characterize the muscle activity during simulated ADL without losing any relevant information.Six able-bodied subjects  age of 34.5 (8.2) years) gave their informed written consent before participating in this study, approved by the ethics committee of our University.Twenty-one simulated ADL were selected, all of them tasks included in the SHFT. Some of the simulated ADL were adapted in order to ensure their repeatability. The activity 14 from the original SHFT was separated in two different activities. In the first one, the piece of paper was folded (A14) and in the second one the folded paper was inserted into an envelope (A15). Others were slightly adapted, for example, reducing the number of objects to be manipulated  or indicating the direction and the degrees to be turned in each case , measured in each session: flexion and extension of the wrist, flexion and extension of the fingers, pronation of the forearm, ulnar deviation of the wrist, and elbow flexion. MVCs for the different movements were recorded without the use of any external device. In a comfortable posture, the subjects were asked to exert maximum effort (recorded with a dynamometer) without the help of other muscles than those of the forearm and hand. In addition, the beginning and end of the sEMG records were cut to eliminate the first and last moments in which muscle activity did not exceed 5% in any of the spots. Finally, to make records comparable in duration, all the records were interpolated to 1000 data.i) ADL repeatability: The confidence intervals (CI) of the 10 muscular activity records (2 repetitions × 5 sessions) of spot 30 were computed for each simulated ADL . Statistics per ADL were considered for the analysis of repeatability.ii) Reduction of temporary muscle activity data: The data from all repetitions at each spot for each simulated ADL were averaged, and used in the subsequent analysis, so that a total of 3780 signals (21 ADL × 6 subjects × 30 spots) of 1000 temporary data were considered. The Principal Component Analysis (PCA) was one of the first methods adapted to functional data. The fundamental idea of this extension is to conserve all the benefits of the PCA as a tool for the reduction of the dimension, conditioning them to functional data. Thus, the Functional Principal Component Analysis (FPCA)  could priii) Similarity of patterns of muscle activation: Conglomerate or Hierarchical clustering analysis  is a muli)ADL repeatability: Statistics of the CI of the muscular activity records at spot 30 for each simulated ADL are shown in Table ii)Reduction of temporary muscle activity data: 17 FPCs explained 91% of the total variance. RVs are displayed in Fig. iii)Similarity of patterns of muscle activation: Fig. Figure In this work we identified forearm areas with similar muscle activation patterns by means of FPCA and cluster analysis, which could be used to characterize the muscle activity during relevant simulated ADLs. Consequently, one of the main contribution of the proposed approach is the focus on goal-directed actions.The tasks used in this study were taken from the SHFT. The selection of the tasks was originally based on the percentage use of the most common handgrips during ADL and hence reflects an accurate representativeness of hand function in day-to-day life. Muscle activity on 30 different forearm spots was obtained for each of the 21 representative simulated ADLs by merging sEMG signals recorded in five different sessions This was possible because of the high repeatability observed for the simulated ADLs considered , thanks to the proposed standardization. Unscrewing a lid and leaving it on the Table (ADL 9) was the least repeatable activity, with a mean CI value of 6% of MVC.Seventeen RVs explained 91% of the total variance. This new set of variables provided the same information but in a more easily interpretable way: each of the 30 original functions of 126,000 temporary data was expressed as a common mean function plus a linear combination of 17 FPCs, with specific coefficients (RVs) for each original function. These 17 RVs obtained from the FPCA have been used in a hierarchical cluster analysis to group spots with similar activation patterns.From the observation of the distances in each step in the hierarchical cluster analysis, seven groups may be established based on the moment in which the distance between the clustered groups in a step becomes high in comparison to the previous steps. Therefore, we could reduce the number of EMG sensors from 30 to 7 without losing any relevant muscle activity information during the performance of simulated ADL. This is very interesting, as there are 20 muscles superimposed on each other in the forearm, thus making it practically impossible to isolate the sEMG signal of each muscle. Protocols of muscular function assessment of the forearm in rehabilitation can benefit from this. The measurement of the amplitude of the sEMG signals from the seven resulting groups may provide a reasonably complete quantitative picture of the patient’s rehabilitation outcome. Furthermore, given that the number of available electrodes could be limited and that a smaller number of EMG signals may simplify the assessment, knowing the similarity of the sEMG signals with different levels of hierarchy, as provided with the method used, is also quite interesting. This also applies for prosthesis control, where the number of EMG signals to be used is limited by the complexity of the controller.When observing the dendrogram obtained from the cluster analysis, different numbers of clusters could be chosen. Two main hierarchy levels can be observed: one involves the flexor muscles of the wrist and digits (spot groups 1 to 3), and the other includes their extensor muscles (spot groups 4 to 7). In the next hierarchy level, flexors bifurcate into wrist flexors (groups 1 and 2) and digit flexors (group 3); it is known that digit flexors also contribute to wrist flexion . And somIf we were to divide the classification a step further we would get the seven groups described in the results section: Group 1 is defined by spots 1, 3, 15, 23 and 24. Anatomically, it could be recording mainly the muscle activity of the flexor carpi ulnaris (FCU). The FCU is a very powerful muscle that acts as a flexor and ulnar deviator of the wrist , being rTherefore, we propose using seven groups of spots for characterizing the muscular activity of the forearm during simulated ADL, in order to substantially reduce the number of spots to be registered, and to maintain muscular-anatomical coherence. The signals from these seven spots would be related to seven different movements: (group 1) wrist flexion and ulnar deviation; (group 2) wrist flexion and radial deviation; (group 3) digit flexion; (group 4) thumb extension and abduction/adduction; (group 5) finger extension; (group 6) wrist extension and ulnar deviation; and (group 7) wrist extension and radial deviation.When assessing muscle function in rehabilitation, some spots may be unavailable because of the simultaneous usage of other equipment, such as sensors for kinematics measurement. When choosing the representative spots of each group to be recorded from among those available, the percentage of muscle activity measured Fig.  should bFPCA is one of the most popular multivariate analysis techniques for extracting information from functional data, reducing the dimensions of a data set in which there are a large number of interrelated variables, while still holding as much of the total variation as possible . While FThe EMG recordings have been carried out with an 8-channel sEMG device, which required the repetition of the same activities for each subject in five different sessions to allow the measurement of the high number of spots chosen. Although reproducibility error has been checked to be small for the spot 30 between sessions, the use of high-density surface EMG might be considered in upcoming works.The current study has been limited to six healthy, able-bodied subjects, and the results could be verified in further studies with a higher number of subjects and including impaired subjects. However, muscular groupings obtained in this study may be used as a first approximation, and may be used as guide for future validation for subjects with hand impairments or amputees.As future work, studies could be conducted to relate EMG to kinematics during the performance of ADL, by using these seven spots as representative of the muscular activity of the forearm in ADL. A further step would be to evaluate kinematics and muscular synergies during specific functional tasks.This study aimed to identify skin zones with similar muscle activation patterns in order to determine the minimum number of electrodes required to characterize the muscle activity during simulated ADL without losing any relevant information. The results indicate that we could reduce the number of sEMG sensors from 30 to 7 and use them as representative spots of the muscular activity of the forearm in simulated ADL. The simulated ADL performed are included in the SHFT, which uses the most common handgrips in day-to-day life. Hence, the simulated ADL chosen in this study may reflect an accurate representativeness of the hand function. This result may help to assess muscle function in rehabilitation as well as simplify the complexity of prosthesis control.