Allosteric targeting is progressively gaining ground as a strategy in drug design. Its success, however, depends on our knowledge of the investigated system. In the case of the papain-like cysteine peptidase cathepsin K, a major obstacle in our understanding of allostery is represented by the lack of observable conformational change at the active site. This makes it difficult to understand how binding of effectors at known allosteric sites translates into modified enzyme activity. Herein, we address this issue by a computational approach based on experimental data. We analyze the conformational space of the papain-like family and the positioning of cathepsin K within it using principal component analysis and molecular dynamics simulations. We show that human cathepsin L-like endopeptidases  adopt similar conformations which are distinct from their non-animal counterparts and other related peptidases. Molecular dynamics simulations show that the conformation of cathepsin K is influenced by known allosteric effectors, chondroitin sulfate and the small molecules NSC13345 and NSC94914. Importantly, all effectors affect the geometry of the active site around sites S1 and S2 that represent the narrowest part of the active site cleft and the major specificity determinant in papain-like endopeptidases. The effectors act by stabilizing pre-existing conformational states according to a two-state model and thereby facilitate or hinder the binding of substrate into the active site, as shown by molecular docking simulations. Comparison with other related enzymes shows that similar conformational variability and, by implication, allostery also exist in other papain-like endopeptidases. The extent of conformational change associated with allosteric regulation ranges from movement of entire domains to minimal changes in the protein backbone . While cThe cysteine peptidase cathepsin K is becoming established as a model enzyme for regulation of papain-like peptidases via sites distant from the active site. However, lack of significant conformational change in X-ray structures has thus far hindered our understanding of this system. The enzyme itself is a critical enzyme in bone homeostasis  and has In our work, we have been investigating allosteric mechanisms by combining computational and experimental approaches. Using the statistical coupling analysis we identified the protein sector of papain-like peptidases , an evolDespite these data, the acceptance of cathepsin K as a model for allosteric regulation in papain-like peptidases has been hindered due to the absence of conformational change in X-ray structures of cathepsin K in complexes with allosteric effectors and orthosteric inhibitors. Herein we address this issue by a computational approach based on the significant amount of available structural data. Up-to-date over 50 X-ray structures of cathepsin K alone and over 400 structures of papain-like peptidases are available from the Protein Data Bank, hence a large enough sample is available for thorough analysis. We investigate the conformational space of the papain-like family, the position of cathepsin K within it, we examine the conformational variability of the molecule alone and in complexes with known effectors and simulate the binding of a peptide substrate into the active site in the presence of different effectors. Based on these results we provide a molecular model for allosteric regulation of cathepsin K and provide an outlook of similar mechanisms in other related enzymes for which experimental data are not yet available.As the starting point of this work, we aimed to obtain a perspective of the total conformational variability within the papain-like family. For this purpose we investigated its conformational space using principal component analysis (PCA) on the complete ensemble of X-ray structures currently available from the Protein Data Bank , plants and animals. Within the animal group, which contains mostly mammalian enzymes, cathepsin H lies separate from the rest, whereas the endopeptidases are clustered together, with the exception of two structures of procathepsin L2 from the mealworm Tenebrio molitor which cluster with cathepsin H. Within the endpopeptidase group  is contained within the first two principal components (PC1 and PC2) which account for 50.4% and 13.5% of sample variance, respectively, enabling its representation in a straightforward two-dimensional diagram. The plot of PC1 versus PC2  shows thstidiosa , whereas protein . Distincse group , cathepsStructural variability within the family, as identified by PCA, is shown in The results of PCA also showed dispersion of PC values calculated for the same protein, indicating structural variabilility of the associated regions within individual proteins. To analyze the variability of cathepsin K, we examined RMS fluctuations in 55 non-redundant cathepsin K structures  and creaThus, taken together, the results of PCA indicate that the loops surrounding the non-primed sites of the active site (loops 1 and 3) are flexible, which may be important for the functional properties of cathepsin K and other papain-like peptidases. However, at least in the case of cathepsin K, no convincing evidence of conformational variability could be obtained from X-ray data. Lack of observable change could be due to crystal contacts or to crystallization conditions that usually involve high salt concentrations, to which cathepsin K is known to be sensitive . Since mFor further insight into the conformational fluctuations in cathepsin K, the ensemble of its X-ray structures was also analyzed by normal mode analysis. Fluctuations corresponding to the first five  normal modes are illustrated in Results in the previous section showed that conformational variability exists in the family of papain-like peptidases, but only limited variability could be observed in cathepsin K by crystallographic analyses. To investigate the conformational space not reachable by X-ray crystallography, we performed MD simulations of cathepsin K alone and in complexes with substrate or allosteric effectors. For comparison, the analysis was also extended to other animal endopeptidases in their unbound states. The calculated trajectories were analyzed using PCA and mapped onto the conformational space defined by the X-ray ensemble in the previous section. Overall, trajectories contained between 100 and 240 ns of total simulation time  were investigated by MD simulations in the same manner. Similar to the former, all enzymes sampled a significantly greater portion of conformational space in comparison to their X-ray structures . CompariIn the continuation, we analyzed MD simulations of cathepsin K in complexes with different ligands. These included the octapeptide substrate AGLKEDDA bound into the active site to simulate the bound mode of the enzyme, allosteric effectors NSC13345 and NSC94914, and C4S bound to each of the three known binding sites on cathepsin K . The parFor structural evaluation of the observed differences in conformational space, RMS fluctuations were first calculated for each simulation . As expeFor direct comparison between the simulations and to X-ray data, average MD conformers were calculated from each trajectory. Their positions within the conformational space are shown in In addition to the comparison of average MD conformers, we also attempted to identify individual conformations in the simulations of cathepsin K in complexes with NSC13345 and NSC94914, respectively, which had RMSF values significantly higher than 2 Å  at multiUltimately, the action of effectors results in structural change of the active site and altered enzyme activity. The results in the previous sections indicate that only minor conformational changes occur in cathepsin K, however, these can be sufficient to modify enzyme activity, especially when keeping in mind that modulation of cathepsin K usually involves a few-fold decrease or increase in enzyme activity and not full inhibition or activation , 17, 24.Km with only minor variations observed in the catalytical rates of the enzyme [in vitro [Experimental data had collectively shown, that all effectors predominantly affect e enzyme , 17, 24.in vitro , which aIn a similar fashion, docking of the substrate AGLKEDDA to average MD conformers of cathepsin K in complexes with allosteric modifiers was performed and analyzed . Small mTrypanosoma cruzi [Trypanosoma brucei [Leishmania mexicana [Based on the data presented in the previous sections, we also sought to examine the variability of other human cathepsin endopeptidases  and its potential effects on their activity. Unfortunately, no allosteric effectors of family members other than cathepsin K have yet been characterized at the structural level. However, there have, been several indications of their existence. GAGs, in particular, have been shown to modulate the activity and stability of not only cathepsin K but also of cathepsins S and B , 29 as wma cruzi , rhodesaa brucei  and cystmexicana . Moreovemexicana . IdentifAs above, we perfomed MD simulations followed by analysis of interatomic distances and docking of substrate molecules to X-ray and average MD structures. Appropriate substrates for each enzyme were constructed according to their specificity profiles , 27, 35.These observations were mirrored in the results of docking experiments in which we compared the poses of substrate molecules docked into the active sites of X-ray and average MD conformers of each peptidase, where X-ray conformers represent the bound state, since they were derived from X-ray structures of enzyme/inhibitor complexes, and average MD conformers represent the free state. The results are shown in Allosteric regulation is slowly surfacing as a common regulatory strategy in papain-like peptidases. Our understanding at the structural level has so far been restricted to the identity of allosteric sites and a limited number of effectors, whereas knowledge on how these effectors work has been limited due to the absence of obvious conformational change observable in X-ray structures. Herein we have made significant progress in our comprehension of this mode of action. We now have strong indications that allosteric regulation in cathepsin K, the primarily investigated papain-like peptidase from the allosteric perspective, proceeds via stabilization of conformational states with different accesibility of the S2 site, the primary specificity determinant, to the substrates. Conformational plasticity is an inherent property of cathepsin K and our data indicate that similar mechanisms also operate in other cysteine cathepsin peptidases. Altogether this analysis provides an important basis for experimental investigation of allostery in this family.www.rcsb.org). A complete list of entries used in this work is given in All coordinates used for analyses and simulations were retrieved from the RCSB Protein Data Bank  was constructed  Chimera  and the  Chimera . Polar h Chimera  was used Chimera , 45, 46)The same procedure was used to dock the substrate AGLKEDDA into the active sites of average MD conformers of cathepsin K derived from MD trajectories. Since structure averaging over MD trajectories results in distortion of flexible side chains, correct geometry of the receptors was first restored by energy minimization with NAMD . MinimizOther substrate molecules were docked into the active site of respective enzymes by the same procedure described in the previous section. The constructed substrates were Ala-Gly-Phe-Gly-Gly-His-His-Ala (AGFGGHHA) for cathepsins L and V, Ala-Ala-Val-Gly-Gly-Thr-His-Ala (AAVGGTHA) for cathepsin S and Ala-Gly-Leu-Lys-Ala-Ala-Ala-Ala (AGLKAAAA) for cathepsin F. The PDB entries used to retrieve the macromolecular coordinates were 1MHW, 1FH0, 2OP3 and 1M6D for cathepsins L, V, S and F, respectively. As above, docking was performed using the initial X-ray conformers and average structures calculated from MD trajectories.S1 FigThe conserved core is colored blue and conformationally variable regions discussed in the manuscript are colored dark yellow. The remaining part of the molecules is colored tan. PDB entries used in the representations are 1ATK for human cathepsin K, 1NB3 for human cathepsin H, 4YYQ for the fig protease ficin, 1YVB for falcipain-2, the major cysteine protease of Plasmodium falciparum, 3OIS for xyllelain from Xyllela fastidiosa, 4D59 for Cwp84 from Clostridium difficile, 1CSB for human cathepsin B, 1EF7 for human cathepsin X and 1K3B for dipeptidyl-peptidase I. The graphics were prepared with UCSF Chimera Software.(PDF)Click here for additional data file.S2 FigThe superpositions were constructed from the PDB entries shown individually in (PDF)Click here for additional data file.S3 FigNormal modes 1 through 5 (from top to bottom) calculated for an ensebmle of 55 non-reduntant cathepsin K structures. The representation contains only non-gapped positions in the sequence alignment (residues 3 through 215). The analysis was performed with the Bio3d package and the graphics prepared with UCSF Chimera Software.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.S2 Dataset(FA)Click here for additional data file.


CDKL5) gene has been associated with rare neurodevelopmental disorders characterized by the early onset of seizures and intellectual disability. The CDKL5 protein is widely expressed in most tissues and cells with both nuclear and cytoplasmic localization. In post-mitotic neurons CDKL5 is mainly involved in dendritic arborization, axon outgrowth, and spine formation while in proliferating cells its function is still largely unknown. Here, we report that CDKL5 localizes at the centrosome and at the midbody in proliferating cells. Acute inactivation of CDKL5 by RNA interference (RNAi) leads to multipolar spindle formation, cytokinesis failure and centrosome accumulation. At the molecular level, we observed that, among the several midbody components we analyzed, midbodies of CDKL5-depleted cells were devoid of HIPK2 and its cytokinesis target, the extrachromosomal histone H2B phosphorylated at S14. Of relevance, expression of the phosphomimetic mutant H2B-S14D, which is capable of overcoming cytokinesis failure in HIPK2-defective cells, was sufficient to rescue spindle multipolarity in CDKL5-depleted cells. Taken together, these results highlight a hitherto unknown role of CDKL5 in regulating faithful cell division by guaranteeing proper HIPK2/H2B functions at the midbody.The cyclin-dependent kinase-like 5 ( The subsequent identification of mutations in the CDKL5 gene in patients with the Hanefeld variant of Rett syndrome or early infantile epileptic encephalopathy (OMIM #300672) suggested the involvement of this gene in human brain functions2. In agreement, the two existing Cdkl5 knock-out mouse models are characterized by impaired learning and memory, autistic-like features, and motor deficits reconciling several aspects of the clinical spectrum present in patients with mutations in CDKL54.CDKL5 is a serine-threonine kinase that was identified through a transcriptional mapping study aimed at identifying disease causing genes in the Xp22 regionCDKL5/Cdkl5 gene is widely transcribed and the protein can be detected in most tissues and cells, both in the nucleus and in the cytoplasm6. However, CDKL5 expression reaches highest levels in the brain6 and because of the evident brain-associated functions, most studies have focused on the neuronal roles of CDKL5. In the brain CDKL5 expression is low at embryonic stages but a significant induction can be observed in the neuronal compartment in the first post-natal days supporting a role during neuronal maturation5. In post-mitotic neurons the levels and distribution of CDKL5 are regulated by neuronal activity indicating that the protein responds promptly to external stimuli8. Of relevance, neurons devoid of the kinase are characterized by defects in axon formation and outgrowth, dendritic arborization, spine morphology, and synaptic transmission, underscoring the importance of CDKL5 for brain development and functioning10.The 11 whereas CDKL5 inhibition, by RNAi or targeted gene disruption, was shown to increase bromodexoyuridine incorporation12. Although these data suggest the involvement of CDKL5 in cell proliferation, no information is available regarding the functions and the subcellular localization of the kinase during the cell cycle. In the current study we examined the localization of CDKL5 in interphase, mitosis, and cytokinesis of proliferating cells. Besides the typical nuclear punctuate staining of CDKL5 in interphase cells13, we also found CDKL5 to be localized at the centrosomes and at the midbody.While the functions of CDKL5 in post-mitotic neurons are under continuous investigation, its role in proliferating cells is still largely unknown. CDKL5 overexpression induces cell cycle arrest in neuroblastoma cells14. The centrosome serves as the main microtubule-organizing center that contributes to cell adhesion, motility, and polarity in interphase and to bipolar spindle formation and timely mitotic progression in mitosis16. During mitosis, the presence of two centrosomes per cell ensures the bipolar nature of the spindle and the equal segregation of chromosomes to two daughter cells. Quantitative or qualitative centrosome defects may lead to multipolar spindle formation and, eventually, loss of mitotic fidelity and acquisition of chromosome instability18.In animal cells, centrosomes form when a pair of orthogonally positioned centrioles assemble and organize a matrix of proteinaceous pericentriolar material around themselves. Centrioles act as the centrosome organizer and their duplication controls centrosome number. Like DNA, centrioles duplicate semi-conservatively exactly once per cell cycle19. Among the numerous midbody components, we have shown that HIPK2, an evolutionary conserved kinase whose large number of substrates includes the Rett syndrome associated factor MeCP220, localizes at the midbody and is required for faithful cytokinesis21. HIPK2 contributes to abscission, the last step of cell division, by phosphorylating extrachromosomal histone H2B at serine 14 (S14) at the midbody. In HIPK2-defective cells, expression of a phosphomimetic H2B-S14D mutant overcomes the cytokinesis failure21.The midbody is the narrow intercellular bridge containing bundles of microtubules derived from the mitotic spindle that connects the two daughter cells in cytokinesis. A complex network of components impacting on vesicle and membrane trafficking, cytoskeleton, chromosomes, cell cycle and lipid rafts affects midbody formation and cleavageBy biochemical and functional assays, we confirmed the presence of CDKL5 both at centrosomes and at the midbody and highlighted the involvement of CDKL5 in cell division through the regulation of HIPK2/H2B functions.13.To investigate the function(s) of the ubiquitously expressed CDKL5 in proliferating cells we started evaluating the subcellular localization of the kinase during the cell cycle. The distribution of endogenous CDKL5 was analyzed in HeLa cells by immunofluorescence (IF) during interphase, mitosis, and cytokinesis Fig. . We obseNext, through additional studies we validated the centrosomal and midbody localization of CDKL5 suggested by the above IF results.Regarding the centrosomal localization, we confirmed the overlapping staining of CDKL5 and γ-tubulin in different cells, such as SV40-transformed COS7 cells and human fibroblasts, MRC-5 Fig. . In the 22 in different cell types using two different anti-CDKL5 Abs  dynamics or to numeral and structural centrosome alterationsi.e., each centrosome presents two spots of centrin-2; Fig. i.e., zero out of 140 spindles); we thus excluded loss of centriole cohesion or centrosome fragmentation as causative of the multipolar spindles. Furthermore, a significant increase of interphase cells with supernumerary centrioles (more than four per cell) was detected in siCDKL5 compared to siCtr cells  versus 59.07 ± 33.64 min in siCtr (n = 73); p < 0.001]. In these cells, we also observed a significantly longer cytokinesis time  and cytokinesis failure resulting in the formation of binucleated cells  in either siCtr or siCDKL5 cells during the imaging session , cytokinesis failure, and cell fusionlls Fig.  and S3. Altogether, these findings show that CDKL5 depletion leads to polyploidization and cytokinesis failure.i.e., Aurora B, PLK1, HIPK2) and microtubule-associated proteins 28 were assessed by IF in siCtr and siCDKL5 HeLa cells. Among the analyzed factors, we found only HIPK2 to be absent from the midbody in a high percentage of siCDKL5 cells compared with the siCtr cells . Indeed, the longer prometaphase time Fig.  and the 29, the main cause of Rett syndrome and an interactor and in vitro substrate of CDKL531. Whether a cross-talk between CDKL5 and MeCP2 exists at the centrosome still remains to be understood. Of note, the reduced MT nucleation capacity of centrosomes in MeCP2 deficient cells, but not in cells silenced for CDKL5, indicate that the two proteins have at least some separate functions in this organelle29.Conversely, we do not observe any apparent structural centrosome defects in CDKL5 deficient cells. However, this does not preclude the existence of centrosome-associated CDKL5 functions that may emerge from further studies. Interestingly, it is to note that the large group of proteins that have been functionally linked to the centrosome includes MeCP211. In agreement, in the developing mouse brain, genetic inactivation of Cdkl5 increases BrdU and Ki67 positivity12. Even if we observed an increased positivity for phosphorylated H3-S10 in our CDKL5-depleted cells, we did not find any increase in cell proliferation (data not shown), suggesting that the phospho-H3 positivity might be due to a prolonged cell division time. Our results, showing a role of CDKL5 in cell division, suggest that the influence of CDKL5 on cell proliferation might be linked to its effects on genome stability. This would be in agreement with the observation made in neuroblastoma patients, in which high expression of CDKL5 correlates with an increased overall survival11. However, the action of CDKL5 in tumorigenicity is definitively more complex since, at variance with neuroblastomas, in glioblastomas, CDKL5 was shown to be required for tumor cell survival and increased CDKL5 expression was found to be associated with reduced overall survival32. Future studies will be essential to investigate the role of CDKL5 in tumorigenesis.Previous loss- and gain-of-function studies in SH-SY5Y neuroblastoma cells suggested that CDKL5 inhibits cell proliferation33 and defects in neuronal progenitor proliferation and differentiation seem to be a convergence point in many neurodevelopmental disorders34. Of interest, defects in centrosomal proteins are known to impinge on neuronal migration and axon formation35. We thus speculate that the defects in radial neuronal migration and axon specification that have previously been associated with CDKL5 knock-down may be linked to a function of CDKL5 at this organelle9. Altogether, a further understanding of the precise role of CDKL5 in cell cycle progression and at the centrosome might be relevant to recognize the origin of some features of the human pathology associated with CDKL5.Although defects in CDKL5 have mainly been associated with a neurological disease we consider likely that its influence on cell cycle progression may be of relevance also in the pathology. In fact, it is well known that the precise control of the cell cycle critically regulates neurogenesis, neuronal differentiation and migrationProtocols and use of animals were approved by the Animal Ethics Committee of the University of Insubria and in accordance with the guidelines released by the Italian Ministry of Health. Adult mice were euthanized by cervical dislocation.36. pCAGGS-CDKL5-ires-GFP, expressing a siRNA resistant form of CDKL5, was cloned by inserting an EcoRI-EcoRV digested PCR product containing the murine CDKL5 cDNA (NP_0010795) into pCAGGS-ires-GFP digested with EcoRI and SmaI. siRNA resistant CDKL5 contains three nucleotide changes in the third position of codons 25–27 (ggagttgra =  > ggcggagtt). pBosH2B-GFP and pBosH2BS14D-GFP have been described in Rinaldo et al.21.pGFP-CDKL5, expressing the 107 kDa hCDKL5 isoform has been described elsewhere21); MgcRacGAP1 , MKLP1 ; PRC1 ; spastin , CREST centromere protein . HRP-conjugated goat anti-mouse or anti-rabbit secondary Abs for immunoblotting were purchased from Thermo Fisher Scientific. DAPI and secondary Alexa Fluor anti-rabbit, anti-mouse, anti-human, and anti-goat Abs for immunofluorescence were obtained from Life Technologies while FITC- or TRITC-conjugated Abs were from Jackson Immuno Research Lab.Antibodies (Abs) against the following proteins were used: β-actin , α-tubulin , β-tubulin , γ-tubulin , CDKL5 , AKT , Aurora kinase A , GFP , PLK-1 , phospho-H3-S10 , H2SB14-P ; centrin-2 , Cep170  and HIPK2  supplemented with FBS, L-glutamine, penicillin, and streptomycin as above; these cells were seeded on poly-D-lysine coated coverslips.Human cervix adenocarcinoma HeLa cells, the monkey SV-40-transformed kidney COS7 cells, and human fetal lung fibroblasts MRC-5 cells were maintained in DMEM  supplemented with 10% FBS, L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C with 5% COTM 2000 (Life Technologies) following the manufacturer’s protocol. Cells were collected or fixed 24 h post-transfection. For siRNA transfection, 20 nM siRNA oligonucleotides targeting CDKL5 or control GL2 siRNA targeting the luciferase gene  were transfected into HeLa or MRC-5 cells using LipofectamineTM RNAiMAX (Life Technologies).For transfection, cells were cultured in 6- or 12-well dishes and transfected with plasmids using LipofectamineFor CDKL5 rescue experiments, a siRNA resistant pCAGGS-CDKL5-ires-GFP vector was transfected 60 h after CDKL5 silencing and cells analyzed after another 24 h. For H2B-GFP or H2B-S14D expression, encoding vectors were transfected 24 h after CDKL5 silencing and cells were analyzed 24 and 48 h post-transfection.7 and plated on poly-L-lysine coated plates .Primary hippocampal cultures were prepared from embryonic day 17 (E17) CD1 mouse embryos, considering the day of the vaginal plug as E0, as described previously37. Briefly, E15.5 embryos were individually dissected in PBS and the neocortex was transferred in Dulbecco’s Modified Eagle Medium/F12 (DMEM) and dissociated by extensive enzymatic digestion with Papaine (Sigma-Aldrich). Cells were grown in medium containing DMEM/F12, 0.66% glucose, L-glutamine 1%, Pen/Strep 1%, 4 µg/mL of heparin, hormone mix with the addition of either 20 ng/mL epidermal growth factor (EGF) and 10 ng/mL basic fibroblast growth factor (bFGF). In such conditions, cells spontaneously formed neurospheres. Neurospheres were then dissociated into single-cell cultures and plated on matrigel-coated coverslips (BD Bioscience).Neurospheres were prepared as previously described29. Briefly, exponentially growing cells were treated with 10 μg/ml nocodazole and 5 μg/ml cytochalasin B (both from Sigma-Aldrich) for 90 min, followed by hypotonic lysis. Centrosomes were harvested by centrifugation onto a 20% Ficoll cushion and further purified by centrifugation through a discontinuous  sucrose gradient. Fractions of 0.3 ml were collected and analyzed by WB.Centrosome fractionation was performed as previously describedCells were lysed with lysis buffer  with addition of protease inhibitor cocktail (Sigma-Aldrich) and PhosSTOP (Roche Diagnostics). After 30 min on ice, the lysates were clarified by centrifugation and the supernatants collected. Samples were separated by either 10% or 8% SDS-PAGE, transferred to nitrocellulose membranes, and blocked in 5% non-fat milk in TBS  with 0.2% Tween-20 (T-TBS). Blots were incubated with primary Abs overnight at 4 °C, washed in T-TBS, and incubated with appropriate secondary Abs for 1 h at room temperature. After extensive washes, blots were developed with either West PICO Chemiluminescence kit (Pierce) or with ECL Prime Western Blotting Detection Reagent .2) with 0.5% Triton X-100 for 2 min and then fixed in 4% paraformaldehyde.Cells were grown on poly-L-lysine coated coverslips and fixed in ice-cold methanol or 2% formaldehyde, washed three times in phosphate buffered saline (PBS), permeabilized in 0.25% Triton X-100 in PBS for 10 min and then blocked in 5% bovine serum albumin (BSA) in PBS for 1 h before the required primary Abs were applied. For the staining of γ-tubulin and centrosome associated proteins, cells were permeabilized before fixation in PHEM  supplemented with protease- (Sigma-Aldrich) and phosphatase-inhibitors . Total cell extracts (TCE) from asynchronous and telophase enriched cells were analyzed in parallel.HeLa cells were enriched in telophase by treatment with nocodazole (100 ng/ml) for 4 h followed by mitotic shake-off. Nocodazole was washed out and the collected cells incubated for 80 min to reach telophase. Midbodies were isolated as described by Kuriyama 2. DIC images were acquired by using a DS-Qi1Mc camera. Image and video processing were performed with NIS-Elements AR 3.22.Cells were seeded in 8-well slides  and observed under an Eclipse Ti inverted microscope (Nikon) using a 40x objective . During the observation, cells were kept in a microscope stage incubator at 37 °C and 5% COt test or parametric ANOVA followed by Dunnet’s post hoc test was applied to determine the significance of quantitative experiments when the data distribution was normal. In other cases we used non-parametric Kruskal Wallis with Dunn’s post hoc test.For statistical analysis we assessed the normality distribution of data using the Shapiro-Wilk normality test: student’s Movie S1Movie S2Movie S3Supplementary information


Newcastle disease (ND) and avian influenza subtype H9N2 (H9N2 AI) are two of the most important diseases of poultry, causing severe economic losses in the global poultry industry. Vaccination is an effective way to prevent and control the spread of ND virus (NDV) and H9N2 AI virus (AIV), but the antigenic differences between the current circulating strains and the vaccine strains might account for recent ND and H9N2 AI outbreaks in vaccinated poultry flocks.We developed an inactivated bivalent H9N2 and NDV vaccine based on the current prevalent strains of H9N2 AIV and NDV in China and evaluated its efficacy in chickens in this study.2 for NDV and 9.5 log2 for H9N2. Up until 15 wpv, HI titers were still detectable at a high level of over 6 log2. The immunized chickens showed no signs of disease after challenge at 3 wpv with the prevalent strains of NDV and H9N2 AIV isolated in 2012–2014. Moreover, viral shedding was completely inhibited in vaccinated chickens after challenge with H9N2 AIV and inhibited by at least 90% with NDV compared to the controls at 5dpc.The results indicated that the inactivated bivalent vaccine could induce a fast antibody response in vaccinated chickens. The hemagglutination inhibition (HI) titer in the sera increased rapidly, and the highest HI titer was observed at 4 weeks post-vaccination (wpv) with a mean titre of 8.6 logOur findings suggest that the inactivated NDV and H9N2 vaccine induces a fast and strong antibody response in vaccinated chickens and is efficacious in poultry against NDVs and H9N2 AIVs. Newcastle disease virus (NDV) and avian influenza virus (AIV) are two of the most important pathogens in poultry worldwide . NewcastIn China, the implementation of extensive vaccination programs in commercial poultry farms has reduced the number of epizootic outbreaks of ND in recent years. However, genotype VII NDV strains of class II have remained a constant threat to domestic poultry since the 2000s and most recent Chinese isolates belong to a sublineage of genotype VIId –9. The pOn the basis of clade classification, 69 genotypes were detected among H9N2 chicken viruses isolated in China from 1994 to 2013 . In contIn this study, we developed an inactivated H9N2 and NDV vaccine based on a H9N2 A/chicken/Hebei/G/2012 (G) virus and a ND Chicken/Shandong/aSG10/2010 (aSG10) attenuated virus that are closely related to the current endemic strains of H9N2 and ND virus in China and evaluated its efficacy in chickens.112RRQKR↓F117 and classified as genotype VII NDV, which showed high genetic variation with the genotype II LaSota vaccine.Based on phylogenetic analysis of the NDV F gene, three NDV isolates were selected as challenge strains in this study: Chicken/China/Hebei/01/2012 (HB12), Chicken/China/Shandong/01/2013 (SD13) and Chicken/China/Hebei/01/2014 (HB14) Table . All thrBased on phylogenetic analysis of the H9N2 AIV hemagglutinin (HA) gene, three H9N2 isolates were selected as challenge strains in the study: A/Chicken/Jiangsu/03/2012 (JS12), A/Chicken/Hebei/03/2013 (HB13), and A/Chicken/Shandong/03/2014 (SD14) Table . All thr50) values of the three NDV isolates were between 8.5 log10 EID50/0.1 mL and 9.3 log10 EID50/0.1 mL, and for the H9N2 viruses were between 7.5 log10 EID50/0.1 mL and 8.0 log10 EID50/0.1 mL.The pathogenicity of vaccine candidates and challenged strains was evaluated using the intracerebral pathogenicity index (ICPI) test in 1-day-old specific-pathogen-free (SPF) chickens and the intravenous pathogenicity index (IVPI) test in 6-week-old SPF chickens, the results are shown in Table To confirm the complete inactivation of two viruses, the formalin-treated viruses were performed three passages in 10-day-old embryonated SPF chicken eggs. All chicken embryos injected with formalin-treated viruses survived after 120 h, and no HA titer was detected.2 against NDV and 7.8 log2 against H9N2 antigen at 2 wpv. The highest HI titer was observed at 4 wpv, with a mean log2 HI titer of 8.6 for NDV and 9.5 for H9N2. Up until 15 wpv, HI titers were still detectable at a high level, with a mean HI titer of 6.7 log2 against NDV and 6.0 log2 against H9N2 , no NDV shedding was recorded in group B, and virus shedding was only detected in one of 10 birds in vaccinated groups A (log10 EID50 /0.1 mL < 1.00) and C (log10 EID50 /0.1 mL = 1.07), as shown in Table To evaluate the efficacy of the inactivated vaccine against virulent NDV, three genotype VII NDVs were selected for a challenge test. About 24 h post-challenge, all of the control birds showed the typical clinical signs of infection, including depression, ruffled feathers, greenish diarrhea, and trembling, and within 5 days post-challenge a 100% mortality rate was observed Fig. . Post-moTo evaluate the efficacy of the inactivated vaccine against prevalent H9N2 viruses, three selected viruses were used for the challenge test. When challenged with H9N2, no specific clinical signs were observed, with the exception of one bird from each of the control groups J and K, which exhibited signs of depression and death occurred at 6 days post-inoculation. Virus was detected in all of the chickens in the control groups at day 5. No virus shedding was recorded in any of the vaccinated groups Table .Table 3IIn China, virulent NDV genotype VII first emerged in the 2000s , 24, andVaccination is an effective way to prevent and control the spread of H9N2 AIVs, but the current vaccine used in China is prepared from isolates that were circulating in the early 1990s, and their protective efficacy has been decreasing in recent years. A HI test showed that the commercial vaccine strain SS/94 does not provide robust protection against emerging H9N2 AIVs in China . It is tAccording to previous phylogenetic analyses, most NDVs isolated in 2010–2014 belonged to genotype VII and most H9N2 AIVs belonged to subgroup III in the BJ/94-like clade, a predominant genotype. These strains showed high homology to each other and a lower level of homology to traditional vaccine strains in China. In this study, a new inactivated vaccine was developed based on the prevalent NDV and H9N2 AIV strains (NDV aSG10 and H9N2 G) and its efficacy was evaluated in chickens , 28.2 against both NDV and H9N2 AIV at 2 wpv. At 4 wpv, peak levels of HI antibodies were detected with a mean HI titer of 8.6 log2 against NDV and 9.5 log2 against H9N2. Antibody titers of more than 6.0 log2 were detected up until at least 15 wpv. The results suggested that this new inactivated vaccine was significantly more effective at generating HI antibody responses than the traditional vaccine strain.Evaluation of NDV or AIV vaccine immunogenicity is largely based on HI antibody titers. In this study, the HI antibody titers of all chickens vaccinated with the inactivated vaccine were detected at weekly intervals until 15 wpv by the HI test. The results indicated that the vaccine could induce a fast immunological reaction and the mean HI titers were over 6.9 log10 EID50/0.1 mL, compared with no mortality and a lower rate of viral shedding of 0–1.07 log10 EID50/0.1 mL in vaccinated groups. All of the unvaccinated chickens challenged with H9N2 shed virus, whereas no virus shedding was detected in vaccinated groups.We next evaluated whether vaccination of chickens with the inactivated NDV and H9N2 vaccine could reduce the disease rates of both endemic NDV and H9N2 viruses in China by challenge test. We challenged the birds via the injection route in order to the birds receive actual dose of challenge viruses. We also tried a natural route for NDV and H9N2 in our previous studies and the birds demanded higher HI antibodies to obtain good protection , 29. RedOur results showed that the inactivated NDV and H9N2 vaccine (strain NDV aSG10 and H9N2 G) induces a high and prolonged immune response in vaccinated chickens and good protection efficacy against prevalent viruses. This novel vaccine could therefore potentially be used in the poultry industry to prevent and control ND and H9N2 influenza in chickens in China.All SPF chicken eggs and chickens were purchased from Merial Vital Laboratory Animal Technology Co., Ltd . The chickens were kept in isolators at China Agricultural University throughout the experiment and the animal rearing facilities were approved by the Beijing Administration Committee of Laboratory Animals under the leadership of the Beijing Association for Science and Technology .The selected NDV candidate was strain Chicken/China/Shandong/aSG10/2010 (abbreviated as aSG10) of genotype VIId, which was an attenuated form of the virulent strain SG10 isolated from an outbreak of ND in chickens with an intracerebral pathogenicity index (ICPI) of 1.89 and a mean death time of 45 h . The selHA gene segments of 19 H9N2 viruses and F gene segments of 11 NDVs isolated in China from 2012 to 2014 were sequenced as described previously , 27. Mul50) was determined and calculated by the Reed–Muench method [Each virus was amplified in 10-day-old SPF embryonated chicken eggs, and the virus titer , then mixed with oil emulsion adjuvants Marcol 52  at a ratio of 1:2 (V/V).NDV strains aSG10 and H9N2 G were propagated in the allantoic cavities of 10-day-old SPF embryonated chicken eggs. Allantoic fluid was harvested 72–96 h after inoculation, and the virus titer in the allantoic fluid of 10Forty 3-week-old SPF While Leghorn chickens were divided into two groups and 20 for each. The birds in Group 1 were vaccinated with one single dose (0.25 mL/bird) of the inactivated vaccine via subcutaneous injection. Group 2 was the control. Blood was collected from each group (n = 10) at weekly intervals until 15 weeks post-vaccination (wpv). Serum was separated and stored at −20 °C until use. The antibody titers were determined by HI tests using 1% chicken red blood cells according to the OIE recommended protocol . The HI 5.0 EID50 for NDV (Intramuscular injection) and 106.0 EID50 for H9N2 (Intravenous injection) at 3 wpv. Chickens were monitored daily for morbidity and mortality for 14 days.SPF chickens were challenged with one of three LPAI H9N2 avian influenza viruses or one of three virulent NDV challenge strains three weeks after vaccination and observed for clinical disease and shedding for two weeks after challenge. A total of 95, 3-week-old, SPF chickens were randomly divided into 13 groups (A–M) and treated as shown in Table 50 determination in 10-day-old embryonated chicken eggs.To determine viral shedding from individual chickens, cloacal or throat swabs were obtained at 5 days post-challenge for virus isolation in eggs. Swab samples were suspended in 1.0 mL of PBS supplemented with penicillin-streptomycin  and then inoculated into 10-day-old embryonated chicken eggs (n = 3 eggs/sample) through the intra-allantoic route. The allantoic fluid was harvested at 5 days post-inoculation and checked for NDV and H9 AIV growth by a HA test using 1% chicken red blood cells. Positive samples were further titrated by EID


Mucormycosis is a rare fungal infection but life-threatening, especially in lupus nephritis (LN). Mucormycosis may manifest as rhino-orbital-cerebral, pulmonary, cutaneous, gastrointestinal, renal, or disseminated forms.We report a case of a 52-year-old woman with cutaneous mucormycosis infection who was admitted because of LN.Histopathological analysis of the lesion confirmed the Rhizopus microspores from the family Mucoraceae.The mortality of mucormycosis remains unacceptably high. Our patient died at last even with standard therapy .It is difficult to diagnose because lacking of specific clinical features, it is necessary to identify the susceptible patients, and then make diagnosis rapidly through tissue biopsy. Despite the outcome is poor, aggressive surgical debridement and Amphotericin B/Posaconazole can be effective. Impaired immune function is common in patients with SLE. An impaired acute inflammatory, lymphopenia or neutropenia, the decreased number of T lymphocytes, and impaired T-helper cell activity make patients with SLE prone to infection.–6 Hypogammaglobulinemia and impaired complement function increase the vulnerability of these patients to infections. The use of glucocorticoids and immunosuppressive drugs constitutes a serious potential risk factor for infections.,5,8,9 It has been suggested that prolonged suppression of T-lymphocyte mediated immunity starts around the 21st day of administration of corticosteroids, and receiving intravenous pulses of corticosteroids increases the risk for opportunistic infections.,11Fever is very common in systemic lupus erythematosus (SLE) patients; infection (54.4%) and lupus disease activity (42.3%) were the most common causes of fever in a Chinese SLE cohort. There has been a marked increase in the incidence of opportunistic infections worldwide, such as fungal infections. Among fungal infections, those from Candida spp., Pneumocystis, and Cryptococcus neoformans are frequently reported in patients with SLE. Although mucormycosis is a rare fungal infection most commonly caused by Rhizopus and Mucor organisms, there is an even more pronounced increase with mucormycosis worldwide. Here, we describe a case of invasive cutaneous mucormycosis in a lupus nephritis (LN) patient.Bacteria are the most commonly implicated agents, followed by viruses and fungi.2The study was approved by the Ethics Review Committee of Beijing Anzhen Hospital, Capital Medical University, and implemented in accordance with the Declaration of Helsinki. A 52-year-old woman was presented with complaints of malar rash that had lasted for 18 days, edema that had lasted for 11 days, accompanied by fatigue, but no photosensitivity, no fever, no arthralgias, no oral ulcers, no gross hematuria, no oliguria. She visited at another hospital; urinalysis showed 3+ proteinuria; analysis of the urinary sediment revealed dysmorphic red blood cells (RBCs); and 24-hour urinary collection showed 7.66 g of protein. She was referred to our hospital for further examination. This patient had a history of hypertention for 9 years, a history of tuberculosis, and a history of previous myocardial infarction.Physical examination revealed blood pressure (BP) 160/100 mm Hg, butterfly facial erythema, normal sinus rhythm, presence of shifting dullness, and marked edema, especially in the face and extremities.9/L, platelet count was 71 × 109/L, absolute CD4+ T cell counts was 117 cells/mm3, serum complement component C3 was 0.24 g/L, component C4 was 0.08 g/L, ANA titers (1:1000) was elevated, anti-ds-DNA antibody was positive, and anticardiolipin antibody and anti-beta2-glycoprotein I antibody was negative. Transabdominal ultransonography revealed enlarged bilateral kidney volume and ascites.Laboratory assessment included urinalysis showed 3+ proteinuria, analysis of the urinary sediment revealed abundant dysmorphic RBCs per high-power field, and 24-hour urinary collection showed 9.4 g of protein. Blood biochemical tests revealed that serum creatinine was 247.9 μmol/L (94 μmol/L 18 days ago), serum albumin was 17.1 g/L, IgG was11.1 g/L, hemoglobin was 117 g/L, direct coombs’ test(+), white blood cells count was 6.13 × 10The histopathological diagnosis of LN was Class IV-G (A)+V See Fig. . GlomeruOn the basis of the clinical, serological manifestations, and histopathology of renal biopsy, SLE and LN were diagnosed, and the disease activity indices were higher. Immunosuppressive therapy was necessary for this patient to halt tissue injury as soon as possible. So, the patient was treated with intravenous pulses of methylprednisolone for a short period of time (0.5–0.8 g every other day for 3 times), then followed by the conventional glucocorticoid dose (methylprednisolone 40 mg/day).+ T cell counts decreased to 10 cells/mm3 and IgG decreased to 3.9 g/L rapidly, especially after intravenous pulses of methylprednisolone. This patient developed septicemia (blood cultures showed listeria monocytogenes) and fever, accompanied by chill and rigor. She was quickly treated with broad-spectrum antibiotics, including linezolid and meropenem. Fever lasted for a long time; the patient underwent oliguric and subsequently anuria 3 days after fever, and finally needed renal replacement therapy. Significantly, at the same time, the patient complained of a new symptom that is vulvar skin pain, gradually became ulcerated and indurated, then developed rapidly progressive tissue ischemic necrosis with a black eschar, accompanied by the pain of the involved lesions disappeared. The area of lesion gradually enlarged and adjacent tissue invasion increased  were used initially, but the patient could not tolerate the side effects, and then Posaconazole was used as salvage therapy. On the onset of surgical debridement and anti-mucormycosis drug therapy, there was a declined trend of the temperature, and a few fresh granulation tissues were seen in the involved vulvar lesion. Unfortunately, the area of the involved lesion was large and deep, and the patient died at last because of disease severity and lacking medical care costs.3 There are many risk factors that predispose to mucormycosis, such as diabetes or ketoacidosis, neutropenia or malignant tumor, organ or hematopoietic stem cell transplant (HSCT), and immunosuppressive therapy.,16 However, mucormycosis rarely appears in SLE but is an important cause of mortality.There were few reports of mucormycosis in SLE, especially seriously cutaneous mucormycosis. Mucormycosis is caused by fungi of Mucorales, which are widespread in the environment. Isolated renal mucormycosis is also identified, but rarely occurs. Vascular invasion resulting in thrombotic lesions and infarction is the most common feature of this infection. Cutaneous mucormycosis usually occurs in impaired skin. Penetrating trauma, dressings, and burns are the most common reasons. The typical presentation is erythema or induration of the skin, and further necrotic eschar. Cutaneous mucormycosis can not only be isolated cutaneous involvement locally, but also present invasive extension from cutaneous tissues to muscle, tendon, or bone.–20Because multiple systems or organs can be involved, the clinical features of mucormycosis present many forms, including rhinocerebral (39–66%), pulmonary (24%), central nervous system (9%), gastrointestinal (7%), and local cutaneous involvement (19%) forms, as indicated in a review of 929 reported cases. The gold standard for diagnosis remains pathologic findings of a tissue biopsy. Characteristic appearance of this fungi in histopathology is wide, ribbon-like, aseptate hyphae with right-angle branching  or restriction fragment length polymorphism (RFLP), which may serve as earlier diagnosis tools, are used for identification of Zygomycetes in tissues or in culture specimen.Clinical diversity of mucormycosis makes it difficult to diagnose. The diagnosis of mucormycosis mainly depends on conformation of the fungus in tissue by histopathology or culture.See Fig. .[22] Rec,25 Azoles and echinocandin antifungal drugs are not effective against mucormycosis, Amphotericin B or its lipid formulations (LFAB) are recommended as the preferred therapy/first-line therapy for mucormycosis, and Posaconazole may be used as salvage therapy/second-line therapy.,15,24Effective management options for mucormycosis consist of early and accurate diagnosis, elimination of predisposing factors, and appropriate anti-mucormycosis therapy; more importantly, the critical therapy is aggressive surgical debridement that can directly improve outcomes. The outcome in patients with pulmonary mucormycosis is worse than for patients with rhino-orbital-cerebral involvement, with mortality rate about 87%. The mortality rate of generally disseminated mucormycosis is as high as 90% to 100%. Infection with Cunninghamella species and disseminated disease were independently associated with increased rates of death .According to a literature review, the mortality rate is 62% in patients with rhinocerebral infection.In our case, intrinsic defects in immune function and hypogammaglobulinemia and receiving intravenous methylprednisolone pulse therapy for control of LN were the risks for infection, especially opportunistic infections. This patient initially received micafungin as a preemptive treatment. When mucormycosis was diagnosed, the patient received standard therapy . Considered that mucormycosis is characterized by systemic clinical manifestations and significant functional impairment as described, suspected filamentous fungus was cultured in sputum/stools/nasal secretions, and the signs of chest and head images were obtained, but they were nonspecific. However, tissue biopsies except skin were not obtained because of the severity of the disease, and disseminated mucormycosis was highly suspected.4Mucormycosis is a rare fungal infection but life-threatening. The mortality with standard therapy remains unacceptably high. Disease may manifest as rhino-orbital-cerebral, pulmonary, cutaneous, gastrointestinal, renal, or disseminated forms. Despite the fact that it is difficult to diagnose because of lack of specific clinical features, it is necessary to make rapid investigations and biopsies to be performed and prevent delays in diagnosis.


Therefore, in the present paper, we studied patients with vestibular schwannoma (VS) undergoing trans-labyrinthine surgery (TLS). Our primary aim was to identify perilymph proteins in patients with VS on an individual level. Our second aim was to investigate the proteins identified at a functional level and our final aim was to search for biological markers for tumor-associated hearing loss and tumor diameter.Due to the surrounding bone, the human inner ear is relatively inaccessible and difficult to reach for cellular and molecular analyses. However, these types of investigations are needed to better understand the etiology, pathophysiology and progression of several inner ear disorders. Moreover, the fluid from the inner ear cannot be sampled for micro-chemical analyses from healthy individuals Sixteen patients underwent TLS for sporadic VS. Perilymph was aspirated through the round window before opening the labyrinth. One sample was contaminated and excluded resulting in 15 usable samples. Perilymph samples were analyzed with an online tandem LTQ-Orbitrap mass spectrometer. Data were analyzed with MaxQuant software to identify the total number of proteins and to quantify proteins in individual samples. Protein function was analyzed using the PANTHER Overrepresentation tool. Associations between perilymph protein content, clinical parameters, tumor-associated hearing loss and tumor diameter were assessed using Random Forest and Boruta. In total, 314 proteins were identified; 60 in all 15 patients and 130 proteins only once in 15 patients. Ninety-one proteins were detected in at least 12 out of 15 patients. Random Forest followed by Boruta analysis confirmed that alpha-2-HS-glycoprotein (P02765) was an independent variable for tumor-associated hearing loss. In addition, functional analysis showed that numerous processes were significantly increased in the perilymph. The top three enriched biological processes were: 1) secondary metabolic processes; 2) complement activation and 3) cell recognition.The proteome of perilymph in patients with vestibular schwannoma has an inter-individual stable section. However, even in a cohort with homogenous disease, the variation between individuals represented the majority of the detected proteins. Alpha-2-HS-glycoprotein, P02765, was shown to be an independent variable for tumor-associated hearing loss, a finding that needs to be verified in other studies. In pathway analysis perilymph had highly enriched functions, particularly in terms of increased immune and metabolic processes. Due to the surrounding bone, the human inner ear is relatively inaccessible and difficult to reach for cellular and molecular analyses. However, such investigations are needed in order to gain a better understanding of the etiology, pathophysiology and progression of several inner ear disorders. The inner ear harbors the membranous system, consisting of the cochlear and semicircular ducts with ampullas, the otolith organs (saccule and utricle) and the endolymphatic duct and sac. The labyrinth membrane separates the two extracellular inner ear fluids, referred to as perilymph and endolymph. Endolymph is rich in potassium, and in the cochlea, is also associated with an electric field potential  which is essential for hair cell transduction and hearing ,2.in vivo. However, the pioneers of proteomic analysis in perilymph, Arrer et al. . Fur. Fur19].To date, there have been no methods developed to predict hearing loss or tumor growth in patients with VS. This would be of significant value in the planning of clinical management. The present work used two estimates to study these clinically important variables. The difference in PTA between the affected and unaffected ear was used as an estimate of tumor-associated hearing loss and tumor diameter was considered as an estimate of tumor growth.The progression of hearing loss may depend on several factors such as the origin of the tumor along the vestibular nerve, the site of auditory nerve compression, the tumors tendency to infiltrate nerve axons and excretions from the tumor. The mechanisms are mostly unknown, therefore characterization of the perilymph proteome in VS patients may provide a valuable addition to understand the mechanisms of tumor-associated hearing loss. Recently studies of the proteome were applied to investigate various heat shock proteins, and they might be of importance for preservation of residual hearing after cochlear implantation . HoweverIn contrast to earlier mass spectrometry studies on perilymph ,19, the The lack of samples of perilymph from healthy individuals is an obstacle in mapping the pathological processes found in perilymph of VS patients, therefore an animal model is necessary. Compared with perilymph from patients having cochlear implants in a previous study shows the presence of alpha-2-HS-glycoprotein in many samples. The majhttp://pantherdb.org/.To understand the functions of the detected proteins, the use of pathway analysis, such as the PANTHER overrepresentation test, provides us with an informative presentation of protein-related activities in the inner ear. In the present study, we presented functional groups, including information relating to up- and down-regulation to present a clearer picture of their functions in perilymph. This method relies on the reported functions of the identified proteins in other tissues, and therefore the presented list of functional groups should be considered as an indicator of their possible roles in the inner ear. The procedure/data-base for this type of analysis is available at Investigating the biological process ontology in perilymph was probably the best method with which to understand the general function. In the top 10 enriched biological processes, three immune responses were present: complement activation, defense response to bacteria and B-cell–mediated immunity. Furthermore, three metabolic processes were identified: secondary metabolic processes, glycolysis and cholesterol metabolic processes. The other four processes identified were cell recognition, phagocytosis, blood coagulation and response to biotic stimulus . This inFrom the view of biological processes, alpha-2-HS-glycoprotein is part of the acute-phase response, an ancestor to response to stress and also part of neutrophil degranulation an ancestor to immune response , which wHence, our hypothesis is that alpha-2-HS-glycoprotein may be excreted from the VS into the extracellular fluid perilymph where it exerts pro inflammatory activity that contributes to sensorineural hearing loss. An alternative hypothesis is that factors derived from the VS influence the inner ear and induce the up-regulation of alpha-2-HS-glycoprotein in perilymph.The feasibility of using perilymph proteome as an approach for identifying biomarkers of VS is attractive. Our present study identified alpha-2-HS-glycoprotein as an independent factor for hearing loss and since it was part of the stable section of the proteome an interesting candidate as possible biomarker. However, further research is now needed to evaluate in an experimental model if alpha-2-HS-glycoprotein has a pathophysiological role in the hearing loss, and if it can be used as a biomarker to facilitate the decision-making process when choosing between “wait and scan” strategies or surgery.Using MS to analyze perilymph from 15 individual patients with VS, we found a stable section of the proteome consisting of 91 proteins which were detected in the majority of patients. The stable section was validated by comparison to earlier proteomic studies on perilymph and 89 proteins were confirmed. There was also a highly individual section of the proteome consisting of 184 proteins only detected in a minority of patients. This showed an important variation in the proteome between individuals, even in a cohort with a homogenous diagnosis.Furthermore, Random Forest and Boruta analysis showed that alpha-2-HS-glycoprotein, P02765, was an independent variable for tumor-associated hearing loss. This needs to be confirmed by future studies.Pathway analysis, using the PANTHER overrepresentation test, showed that perilymph exhibited numerous functions which were highly enriched compared to the level expected. Particularly noteworthy was the highly significant increase in the activity of immune and metabolic processes in the perilymph from patients with vestibular schwannoma.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.


Hearing loss is a major public health concern, affecting over 11 million people in the UK. While hearing aids are the most common clinical intervention for hearing loss, the majority of people that would benefit from using hearing aids do not take them up. Recent technological advances have led to a rapid increase of alternative listening devices to conventional hearing aids. These include hearing aids that can be customised using a smartphone, smartphone-based ‘hearing aid’ apps, personal sound amplification products and wireless hearing products. However, no systematic review has been published evaluating whether alternative listening devices are an effective management strategy for people with hearing loss.The objective of this systematic review is to assess whether alternative listening devices are an effective intervention for adults with hearing loss. Methods are reported according to the Preferred Reporting Items for Systematic reviews and Meta-analyses Protocols (PRISMA-P) 2015 checklist. Retrospective or prospective studies, randomised controlled trials, non-randomised controlled trials, and before-after comparison studies will be eligible for inclusion. We will include studies with adult participants (≥18 years) with a mild or moderate hearing loss. The intervention should be an alternative listening device to a conventional hearing aid (comparison). Studies will be restricted to outcomes associated with the consequences of hearing loss. We will search relevant databases to identify published, completed but unpublished and ongoing trials. The overall quality of included evidence will be evaluated using the GRADE system, and meta-analysis performed if appropriate.No ethical issues are foreseen. The findings will be reported at national and international conferences, primarily audiology, and ear, nose and throat, and in a peer-reviewed journal using the PRISMA guidelines.PROSPERO CRD4201502958. The protocol poses a clearly formulated research question identified and prioritised by key stakeholders in a recent James LindAlliance Priority Setting Partnership for mild-to-moderate hearing loss.The systematic review will be the first to pool together evidence assessing the effectiveness of alternative listening devices to conventional hearing aids.Studies assessing analogue  hearing aids will be excluded, as they are an obsolete technology.We will only include studies with adults (≥18 years), since audiological characteristics differ substantially between children and adults.Grey literature and dissertation abstracts will not be searched.Hearing loss affects over 11 million people in the UK (1 in 6 of the population), and is expected to rise to 15.5 million by 2030 given the ageing population profile.The most prevalent degree of hearing loss can be defined as mild-to-moderate, affecting 92% of all adults with hearing loss in the UK.Conventional hearing aids do not restore hearing but make sounds more audible through electroacoustic amplification. Hearing aids are regulated medical devices that deliver sound into the ear canal via air or bone conduction. Bone conduction hearing aids and bone anchored hearing aids (BAHAs) are for people that have a conductive hearing loss  and so are unable to wear air conduction hearing aids. Rather than sound from the hearing aid being delivered through the ear canal, sounds are sent through the skull directly to the inner ear. Depending on the technology they use to process sounds, hearing aids can be described as ‘analogue’ or ‘digital’, and can be worn behind-the-ear (BTE), in-the-ear (ITE) or receiver-in-the-canal (RIC). In the UK, hearing aids can be obtained and fitted free of charge from a qualified hearing healthcare professional trained in audiology via the National Health Service (NHS) or a registered hearing aid dispenser in the private sector.It may be taken for granted that hearing aids are an effective intervention for hearing loss. Indeed, a previous systematic review of literature published up to August 2004 suggested that hearing aids improve hearing-related quality of life, alleviating activity limitations and participation restrictions associated with hearing loss through improved communication. Nevertheless, the quality of the evidence was poor, with only two randomised controlled trials (RCTs) eligible to be included.13Recent advances in technology have also led to a rapid increase of innovative alternative devices that provide similar functionality  to conventional hearing aids.Alternative devices currently available include: (1) ‘made-for-smartphone’ hearing aids, which allow users to adjust and personalise their hearing aid settings with their smartphone; (2) smartphone apps (Android and Apple OS X), whereby a smartphone or tablet computer can act as a hearing aid when paired with wireless Bluetooth earphones or earbuds; (3) self-fitting personal sound amplification products (PSAPs) that provide an ‘off-the-shelf’ alternative to hearing aids; (4) wireless hearing products that enhance the use of hearing aids so that they can be connected to additional electronic devices such as mobile phones, MP3 players or televisions via frequency modulation systems or Bluetooth. Alternative listening devices may be particularly appealing to users in comparison to conventional hearing aids because of their convenience and accessibility, providing additional functionality, such as listening to music, video gaming or watching television.22To date, no systematic review has been published that specifically evaluates the quality of evidence concerning whether alternative listening devices are an effective intervention for people with hearing loss. This is important because a high-quality, impartial body of evidence is necessary to determine whether alternatives could be adopted and/or recommended to people with hearing loss. There is also a strong clinical need to determine the effectiveness of alternative listening devices to conventional hearing aids, particularly given that, in the UK, the majority of adults with hearing loss have difficulties accessing them. One significant barrier to access is that up to 50% of adults who have hearing loss are not referred onwards to hearing healthcare professional by their general practitioner.The primary objective of this study is to systematically review existing evidence that assesses whether alternative listening devices to conventional hearing aids are an effective intervention for adults with hearing loss. We define alternative devices as both standalone products and devices that provide additional features to a conventional hearing aid. Secondary objectives are to assess feasibility  of alternative listening devices. It is anticipated that this review will provide an evidence base to help inform future feasibility and effectiveness trials on alternative devices, an approach consistent with the Medical Research Council's (MRC's) guidelines for evaluating complex healthcare interventions.23The methods of the systematic review are reported according to the Preferred Reporting Items for Systematic reviews and Meta-analyses Protocols (PRISMA-P) 2015 checklist.The inclusion criteria are specified according to the Participant, Intervention, Comparison, Outcome and Setting (PICOS) study characteristics.Types of study: Retrospective or prospective studies, RCTs, non-RCTS, and before-after comparison studies will be eligible for inclusion. Articles reporting expert opinions, practice guidelines, case reports, case series, conference abstracts and book chapters will be excluded.Participants: We will include adults (≥18 years) with a mild or moderate hearing loss  or devices that provide additional features to a conventional hearing aid . Alternative listening devices should aim to improve hearing and communication outcomes in people with hearing loss through the amplification of external sound sources.Comparison: Comparisons can be inactive  or active . Conventional hearing aids are defined as a regulated medical device that delivers electroacoustic amplification via air or bone conduction, irrespective of where they are worn . Studies with analogue hearing aid instruments  will not be included. In 2000, the Modernising Hearing Aid Services programme was introduced, whereby all NHS patients are now provided digital hearing aids as standard. Analogue hearing aids are therefore an obsolete technology.Outcome measures: As this review is concerned primarily with the effectiveness of alternative listening devices, studies will be restricted to outcomes associated with the consequences of hearing loss. Primary outcomes will include one or more of the following: (1) behavioural measures of speech intelligibility ; (2) hearing-specific health-related quality of life, where participation is the key domain, measured using any self-report questionnaire .A systematic search strategy will be employed to identify articles that meet eligibility for inclusion. We will search CINAHL (via EBSCOhost), Cochrane Library, EMBASE (via Ovid SP), MEDLINE (via Ovid SP), PubMed, Scopus, Citations Indexes of Web of Science, ISRCTN Registry, ClinicalTrials.Gov and WHO International Clinical Trials Registry Platform (ICTRP) to identify published, completed but unpublished and ongoing trials. All database searches will be completed in 1 day and with no time, language, document type or publication status limitations.In addition, hand searching the past 6 months of publications from key audiology journals will be undertaken to ensure that any recently published articles are identified. Additional information will be identified manually through snowballing of the reference lists from included studies, as well as screening of related articles by shortlisted authors, to identify any relevant articles that may not have been returned by the initial database searches. Searches will be repeated 1 month prior to submission for publication to ensure that any newly published studies are included. Contact with study authors will be permitted to ascertain whether any studies are ongoing. Manual searches and personal author contact will continue up to the end of the data collection phase.At the end of study selection process, search strategies for each database will be reported in a reproducible and replicable way, and a PRISMA flow diagram will be presented.The search terms were collected based on free text and controlled vocabularies , Excerpta Medica Tree (EMTREE) and CINHAL Headings), expert opinion, literature review and checking the test search results.We will use the following search strategy for PubMed, which will be adapted for other databases:(‘Hearing Loss’[MeSH] OR Hypoacus*[tiab] OR ‘Hearing Loss’[tiab] OR ‘Hearing Losses’[tiab] OR ‘Hearing Impairment’[tiab] OR ‘Hearing Impairments’[tiab] OR Deaf*[tiab] OR ‘Hearing Problem’[tiab] OR ‘Hearing Problems’[tiab]) AND  AND (Prosthes*[tiab] OR Device*[tiab] OR Aid[tiab] OR Aids[tiab] OR Product[tiab] OR Products[tiab])) OR ‘Ear Mold’[tiab] OR ‘Ear Molds’[tiab] OR Conventional[tiab] OR Unaided[tiab] OR ‘No Treatment’[tiab] OR ‘Control Group’[tiab] OR ‘Usual Care’[tiab] OR ‘Waiting List’[tiab] OR Waitlist[tiab] OR ‘Treatment as Usual’[tiab] OR ‘Usual Treatment’[tiab] OR Routine[tiab] OR ‘Care as Usual’[tiab]) AND  AND .http://www.covidence.org/). All identified articles will be recorded electronically, and can be tracked throughout the data screening and extraction process. Reasons for exclusion will be noted and included articles allocated a unique study ID code so that the record can be linked to the corresponding full text and data collection sheet.DWM will be responsible for data management using Covidence online software . Where necessary, study authors will also be contacted for additional information to resolve questions concerning eligibility. Discrepancies will be adjudicated by MAF.We plan to use a standardised data collection form constructed via Covidence. Detailed guidance notes will be devised by DWM prior to starting the review. The guidance notes and data collection form will be piloted by DWM and ABB, and revised if necessary before the review to ensure consistency. Data collection will be conducted by DWM and ABB independently, but in duplicate for every record included. Study authors will be contacted to resolve any uncertainties. MAF and JX will sample a subset (∼10%) of data collection sheets for each author to confirm consistency of approach. All authors will resolve any disagreement through discussion.The data collection form will include a list of fields given in DWM and ABB will independently assess risk of bias of each included study with the Cochrane Risk of Bias Tool, which rates the studies as ‘high risk’, ‘low risk’ or ‘unclear risk’ in the following six domains: sequence generation, allocation concealment, blinding of participants and outcome assessment, incomplete outcome, selective reporting and other bias. The overall quality of the evidence will be evaluated using the GRADE system.For binary data, we will calculate risk ratio with 95% CI where possible. For continuous data, we will calculate mean difference (MD) with 95% CI where the studies use the same outcome measures; otherwise, we will instead use standardised MD with 95% CI. If summary effects are reported, we will analyse these using generic inverse variance and report the effect estimates with its 95% CI.In the absence of meta-analysis, primary and secondary outcomes will be assessed at the individual study level, with the main study findings examined through narrative synthesis.2 statistic. If I2 is >50%, we will explore potential statistical, clinical or methodological causes for heterogeneity through subgroup analysis. We will not pool studies if I2 exceeds 60%,Heterogeneity across studies will be examined using IFunnel plots will be used to estimate the influence of unpublished papers on the overall effects .If heterogeneity is identified, subgroup analyses will assess the impact of participant age, gender, degree of hearing loss and the presence of comorbidity. Age will be defined as older adults (≥55 years) and younger adults (<55 years) since people with age-related hearing loss are typically 55 years and older, which is also the age eligibility threshold adopted by the services specification developed by the UK Department of Health.http://www.prisma-statement.org/; DWM and MAF). A public and patient involvement representative will be involved in disseminating the outcomes of the review through print and events aimed at non-specialist audiences.No ethical issues are foreseen. The finding will be reported at national and international conferences and meetings, primarily audiology and ENT (DWM), and in a peer-reviewed journal using the PRISMA guidelines (


We found a severe reduction of c-Fos expression in V1 of Cdkl5−/y mutants, suggesting circuit hypoactivity. Glutamatergic presynaptic structures were increased, but postsynaptic PSD-95 and Homer were significantly downregulated in CDKL5 mutants. Interneurons expressing parvalbumin, but not other types of interneuron, had a higher density in mutant V1, and were hyperconnected with pyramidal neurons. Finally, the developmental trajectory of pavalbumin-containing cells was also affected in Cdkl5−/y mice, as revealed by fainter appearance perineuronal nets at the closure of the critical period (CP). The present data reveal an overall disruption of V1 cellular and synaptic organization that may cause a shift in the excitation/inhibition balance likely to underlie the visual deficits characteristic of CDKL5 disorder. Moreover, ablation of CDKL5 is likely to tamper with the mechanisms underlying experience-dependent refinement of cortical circuits during the CP of development.Cyclin-dependent kinase-like 5 (CDKL5) mutations are found in severe neurodevelopmental disorders, including the Hanefeld variant of Rett syndrome . CDKL5 loss-of-function murine models recapitulate pathological signs of the human disease, such as visual attention deficits and reduced visual acuity. Here we investigated the cellular and synaptic substrates of visual defects by studying the organization of the primary visual cortex (V1) of Cdkl5 De novo mutations of the cyclin-dependent kinase-like 5 (CDKL5) gene are responsible for the Hanefeld variant of Rett syndrome (RTT), also referred to as CDKL5 disorder. This is an X-linked neurodevelopmental condition with a broad range of deficits including stereotypical hand movements, deficient language acquisition and in some cases respiratory dysregulation. Differently from classic RTT, patients affected by CDKL5 disorder exhibit early onset (in the first months of life) epilepsy, severe hypotonia, characteristic sideways glance, abnormal eye tracking and severe visual impairment  mouse  in specific brain circuits  and the Bioethics Committee of the University of Torino, Italy. Animal suffering was minimized, as was the number of animals used. Mice for testing were produced by crossing Cdkl5For most immunolabeling experiments, animals were anesthetized with an intraperitoneal injection of Avertin (Sigma-Aldrich) and transcardially perfused with ice-cold 4% formaldehyde in 0.1 M phosphate buffer (PB). The brains were then dissected and kept in the same fixative solution overnight at 4°C. Afterwards, brains were cryoprotected by immersion in 10%, 20%, and 30% sucrose solutions, cut into 30 μm sections with a cryostat and stored at −20°C in a solution containing 30% ethylene glycol and 25% glycerol until use. Cryosections were subsequently processed free-floating for immunohistochemistry or peri-neuronal nets (PNNs) visualization  with 0.01% H2O2 for 3 min. Sections were mounted on gelatin-coated glass slides, air dried, dehydrated in ethanol, cleared in xylene, mounted in DPX mounting media  and coverslipped. Control sections in which the primary antiserum was omitted were completely unstained (not shown). Sections were observed with a light microscope  equipped with a CCD camera . Images of V1  were captured with a 10× objective from 4 to 6 animals per genotype using at least two corresponding coronal brain sections.For c-Fos detection, after a blocking step in phosphate-buffered saline (PBS) containing 10% normal goat serum (NGS) and 0.05% Triton X-100, sections were incubated overnight at room temperature with rabbit anti-c-Fos , diluted in PBS with 3% NGS and 0.05% Triton X-100. Sections were then rinsed, incubated for 1 h with goat anti-rabbit biotinylated secondary antibody , and transferred to a biotin-avidin complex containing solution . The immunoreactivity was visualized by incubating the sections in a solution containing 3,3For immunofluorescence, sections were kept in PBS solution containing 0.05% Triton X-100 and 10% normal donkey serum (NDS) for 1 h followed by an overnight incubation at 25°C with the appropriate primary antibodies . Subsequently, sections were incubated for 1 h at RT with streptavidin Texas Red . After several PBS rinses, the sections were mounted on gelatin-coated glass slides and observed with a confocal microscope.For the detection of PSD-95 and gephyrin immunoreactivity, mice were anesthetized intraperitoneally using Avertin (Sigma-Aldrich) and decapitated. The brains were rapidly excised and cut into coronal slabs that were fixed by immersion in ice-cold 4% formaldehyde in 0.1 M PB for 30 min. After fixation, the tissue slabs were rinsed in PB, cryoprotected via immersion in ascending sucrose solutions , cut into 20 μm sections with a cryostat, mounted on gelatin-coated slides and stored for a maximum of 1 month at −20°C  and expressed as puncta/μm2 (neuropil density) or puncta/μm (somata/dendritic density).Synaptic puncta in the neuropil were analyzed from 5 to 6 mice per genotype on five serial optical sections (0.5 μm Z-step size) that were acquired from layers 2 to 5 of the V1 cortex with a laser scanning confocal microscope  using a 100× objective  and the pinhole set at 1 Airy unit. Synaptic puncta were identified if present in at least two consecutive optical sections. PV+ PNNs was estimated in at least five animals per genotype. Confocal images spanning from the pial surface to the white matter of V1 were captured in at least two corresponding coronal brain sections with a 40× objective (1.0 NA) using a 1-μm Z-step. Digital boxes in width (230.34 μm) spanning from the pial surface to the white matter  were superimposed at matched locations on each coronal section of the V1 cortex, divided into 10 equally sized sampling areas , and immunopositive cells were manually counted in each bin . To compare the distribution of frequencies relative to WFA staining intensity categories, we used χ2-test. All values are presented as mean ± SEM, and n indicates the number of mice.If the data sets passed normality test and equal variance test, we performed Student’s −/y male mice. As shown in Figure +/y, 1167 ± 56.3 cells/mm2; Cdkl5−/y, 683.1 ± 39.2; p < 0.05; n = 6 for each group) in male mutants compared with WT littermates  and 2 (VGluT2), which are markers of cortico-cortical and thalamo-cortical glutamatergic axon terminals, respectively  INs, the regular-spiking calretinin+ (CR) INs, and the burst-spiking somatostatin+ (SOM) INs. Moreover, since CR+ cells can be further subdivided by the differential expression of SOM  in Cdkl5−/y mice compared to WT littermates . Figures . Separat+ cells, we also evaluated the number of PV+ INs relative to the total number of neurons identified with NeuN labelling. As shown in Figures + cells was unaffected by Cdkl5 deletion . However, the analysis of layer distribution revealed a specific increase of NeuN+ cell density in layer V of mutant mice . Reciprocally, the number of c-Fos+ cells also expressing PV was also reduced  boutons decorating the cell body of layer V pyramidal neurons in mutant mice compared to WT animals . Together, these data reveal that the lack of CDKL5 leads to excessive glutamatergic connectivity onto PV+ cells in V1 that in turns make more synapses with corticofugal layer V pyramidal neurons.We then investigated the synaptic output of PV+ cells, we next evaluated the density and pattern of expression of chondroitin sulfate proteoglycans (CSPGs) visualized with WFA labeling. CSPGs condense around the cell body and major dendrites of PV+ INs forming extracellular matrix specialized structures called PNNs . However, the density of cells surrounded by WFA+PNNs in V1 of mutant mice showed an increase compared to WT , consistent with the augmented density of PV+ INs , at the transition toward the opening of the CP , and at the end of the CP  , null mice displayed a higher number of PV+ INs at the closure of the CP  in the visual cortex .Given the increased density of PV+ PNNs before the opening and at the end of the CP. Interestingly, we found no difference in the percentage of PV+ INs surrounded by PNNs irrespective of the developmental stage and genotype , such developmental maturation of PNNs was completely abolished in Cdkl5−/y mice . These data indicate that the lack of CDKL5 leads to profound alterations in the establishment of PV+ IN assembly, together with defective formation of PNNs at the end of the CP.Since the mature configuration of PNNs coincides with the end of the CP in V1 . However, the density of postsynaptic gephyrin clusters was similar in Cdkl5 KO and control mice. One possible explanation for this discrepancy is that in the absence of CDKL5 PV+, INs establish supernumerary inhibitory synapses with the cell body of pyramidal neurons, where gephyrin is undetectable (Patrizi et al., + cells and their connections or whether it reflects a more general rearrangement of GABAergic synapses.In addition to changes of glutamatergic synapses, we found an increased number of GABAergic axonal boutons in Cdkl5 mutants. This observation is consistent with the increase in the number of PV+ cells were the only type of inhibitory IN modified after ablation of Cdkl5. The density of these cells and their synapses was increased in layers II–III and V of mutant V1, and glutamatergic contacts onto PV+ dendrites were also increased. These changes are likely to contribute considerably to the visual deficits reported in Cdkl5 KO mice, because PV+ cells are involved in mediating feedback and feedforward inhibition and are crucial for the generation of network oscillations underlying perception (Atallah et al., + INs is needed for equalizing E/I ratio of pyramidal cells following increased activity (Xue et al., + dendrites represent critical regulators of PV+ cell activity (Kameda et al., + cells observed in V1 of Cdkl5 KOs is, therefore, expected to cause a major perturbation of cortical activity, resulting in hypoactivation of projection neurons. Interestingly, several lines of evidences support the concept that dysfunctions of GABAergic circuits specifically involving PV+ cells might play a prominent role in RTT syndrome (Chao et al., + INs in the somatosensory cortex of MeCP2 KO mice (Tomassy et al., + circuitry in the V1 of MeCP2 mutants, albeit with no changes in the density of PV+ INs.PV+ INs, as reported in previous studies (Mainardi et al., + expressing PV+ cells is further reduced in the KO mice, suggesting that, hypoactivation of these INs might be a compensatory mechanism to counteract the increased GABAergic drive and/or the strong impairment of glutamatergic transmission. Remarkably, c-Fos levels were also diminished in somatosensory cortex (Figures + (Sceniak et al., + inhibitory connectivity (Durand et al., The reduced expression of c-Fos in V1 of Cdkl5 KO mice (Figure  Figures , suggest Figures , indicat+ INs in Cdkl5 mutants are presently unclear. However, CDKL5 was reported to be expressed at high levels in GABAergic INs (Rusconi et al., + expression (Gonchar et al., + INs in Cdkl5 KO mice is already detectable at the closure of the CP, suggesting that CDKL5 loss may affect visual stimuli-driven refinement of cortical circuits during postnatal development. In line with this hypothesis, Ueno et al. (+ cells in the visual cortex of dark reared mice.The mechanisms responsible for the increased density of PVo et al.  revealed−/y mice (Amendola et al., + cells and PNNs during the CP. This hypothesis is supported by the maturation sequence of PNNs in Cdkl5 KO mice, in which PNNs intensity appears strongly attenuated at the end of CP, but becomes comparable to WT in adulthood. The same altered visual stimuli-driven refinement of cortex might be responsible for the increase in the number of PV+ synapses on pyramidal cells, which is observed in adult mice only. According to this interpretation, the excessive PV+ cells in the mutant mice would need a longer time to establish their connections onto excitatory cells, resulting in a surplus of PV+ synapses detectable only in adult mice.The structure and function of PNNs play a crucial role in physiological and pathological conditions and, conceivably, tools enabling the manipulation of PNNs may have to some extent a potential for the development of new therapies aimed to normalize neuronal plasticity in neurodevelopmental disorders (Carulli et al., + INs disrupting CP mechanisms were found in V1 of MeCP2 null mice (Krishnan et al., + cells completely abolished CP plasticity (He et al., + circuitry is accelerated, leading to a premature opening and closure of CP of plasticity in V1. Importantly, in MeCP2 mutants the accelerated maturation of the PV+ network was not associated with an increase in the density of PV+ INs, as we found in Cdkl5 mutant mice, but rather with increased levels of PV expression (Krishnan et al., + INs and pyramidal cells in V1 associated with an analysis of the CP using a monocular deprivation paradigm will help defining the impact of Cdkl5 on cortical function and plasticity.These data suggest that the CP might be prolonged in Cdkl5 mutants. Accordingly, PSD-95 KO mice exhibit lifelong ODP (Huang et al., in vivo that is likely to underlie the severe visual impairments associated with CDKL5 pathology. Furthermore, we unveiled an unknown crucial role of CDKL5 in the organization of cortical inhibitory transmission. Future studies are needed to pinpoint the molecular machinery responsible for CDKL5-mediated E/I balance functionality. Moreover, our study further supports the idea that RTT variants may actually share common pathogenic mechanisms, involving disruption of the E/I ratio and an enhancement of GABAergic transmission mediated by PV+ INs (Dani et al., In conclusion, our study points to a primary role of CDKL5 in the correct formation and maintenance of the E/I balance in V1 RP, MS-P and MG conceived and designed the study. RP, EC and AG performed the experiments. RP, AG, MS-P and MG analyzed the data. EA, CG, RP, AG, MS-P and MG provided reagents, materials and analysis tools. RP, MS-P and MG wrote 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.