CDKL5) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual, and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD has indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3β, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3β activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3β activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3β. These compounds are very soluble in water but blood–brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces postsynaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity, and human neuropathology.Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 ( CDKL5)   cause CDCDKL5)  neurodevCDKL5) . CurrentCDKL5) . HemizygCDKL5) . These sCDKL5) . CDKL5 iCDKL5) . In otheCdkl5 rodents either enhanced long-term potentiation (LTP)   or did non (LTP)  in an agon (LTP)  and appeon (LTP) . This paCDKL5 is an X-linked gene encoding a 115 kDa serine-threonine kinase and member of the CMGC family that includes cyclin-dependent kinases (CDK), MAP-kinases, glycogen synthase kinases (GSK), and cyclin-dependent kinase-like (CDKL) (http://mbv.broadinstitute.org/) but significant homology to GSK3β (CDKL1-4 mRNA are expressed at much lower levels than CDKL5 in mouse and human brain (http://mouse.brain-map.org). Of these kinases, only GSK3α (e (CDKL) . It has to GSK3β . CDKL1-4ly GSK3α  and GSK3Cdkl5 knock-out mice have increased GSK3β activity, as evidenced by hypo-phosphorylation of GSK3β at S-9 and decreased β-catenin  were profiled using the GSK3β NanoBRET assay. NanoBRET is a cellular target engagement assay that relies upon bioluminescence resonance energy transfer (BRET) between a tracer with a red-shifted fluorophore attached and a protein of interest with NanoLuciferase appended. Test compounds, which compete with the tracer for binding to the ATP-binding site, are introduced in a dose-dependent manner and BRET plotted versus concentration, allowing calculation of a target engagement IC50 value , HW2-013 (B4), and LY-213 (B12)  . These rB1 is an SNS-032 analog, while B4 and B12 are analogs of AT-7519. SNS-032 and AT-7519 are published inhibitors of several members of the cyclin-dependent kinase (CDK) and cyclin-dependent kinase-like (CDKL) families  human as well as several nonhuman and mutant kinases , several CDKs  are potently inhibited (IC50 ≤ 100 nM) (colored green in the nested table in B1 (≤100 nM). Curves corresponding to the affinity data for CDKL5, GSK3, and several CDKs are included in B1 within the CDKL family was further profiled via thermal shift and enzymatic assays (B1 was modest (IC50 = 2.1–2.7 µM), and this compound did not inhibit CDKL1. We propose that only at higher concentrations  would inhibition of these kinases become significant, and, based on the data in B1. Importantly, these data support that when cells are dosed with B1 at ≤100 nM, no CDKL family member other than CDKL5 will be inhibited.For intained . All kinc assays . The higB4 and B12, which are analogs of AT-7519, also demonstrated good kinome-wide selectivity with S10(1 µM) scores of 0.02 and 0.01, respectively  or enzymatic (remaining kinases) assays. This orthogonal profiling of B4 confirmed high-affinity binding to CDKL5, GSK3α, and GSK3β  and inhibitory activity. Compounds B4 and B12 exhibited similar inhibition profiles and are potent inhibitors of CDKL5, GSK3α, and GSK3β. Despite its potency, compound B4 demonstrated suboptimal selectivity when compared with B1 and B12. This was determined based on the number of WT human kinases with PoC < 35 in the DiscoverX scanMAX panel when profiled at 1 µM: B1 = 12, B4 = 17, and B12 = 9.Binding and orthogonal assay results in combination with western blots confirmed that in cells B1 on CDKL5 activity   . Lapatincontrol) . B1 and t manner . B1 was B1) showed a significant reduction in pSer222 EB2/total EB2 at 500 nM concentration (B1) does not inhibit GSK3b (B4) was effective in reducing EB2 pSer222 at 500 nM but also inhibited GSK3β significantly at 500 nM with a trend of inhibition from 5 nM, indicating that this inhibitor affects GSK3b (B12) reduced pSer222 EB2 at 500 nM but also seemed to decrease β-catenin phosphorylation at this concentration. LY-213 (B12) showed comparable trends of inhibition for GSK3βand CDKL5 (B1) can inhibit CDKL5 activity without affecting GSK3 activity in neurons.Kinase domains of GSK3β and CDKL5 have high similarity. To test the cross-talk of these inhibitors with GSK3β, we measured phospho-β-catenin (Ser33/37/Thr41), which is directly phosphorylated by GSK3β . We measntration . There wntration . Phosphoit GSK3b . HW2-013ts GSK3b . Finallynd CDKL5 . There wnd CDKL5 . In suppnd CDKL5 . We concB1) could inhibit CDKL5 activity within intact brain tissue, we prepared acute hippocampal slices from P20–30 rats and incubated them with CAF-382 (B1) for 2 hr as described. As for primary cultures, CAF-382 (B1) reduced EB2 phosphorylation in a dose-dependent fashion (B1), also reduced EB2 phosphorylation in hippocampal slices (To determine whether CAF-382 ( fashion . Similarl slices .Cdkl5 in primary rodent brain cultures reduced glutamatergic excitatory synaptic transmission associated with loss of dendritic spines (B1) (10–100 nM) to hippocampal slices while measuring field excitatory postsynaptic potentials (fEPSPs) in the CA1 hippocampus. While varying the stimulation strength applied to presynaptic Schaffer-collateral pathway inputs, we were able to measure the input–output responsiveness through measuring the input (fiber volley size) versus output (fEPSP slope). The slope of this relationship was reduced in a dose–response relationship by CAF-382 (B1) within 30 min of application (B1) on glutamatergic synaptic transmission is primarily postsynaptic. Determination that amphiphysin-1, a presynaptic protein, is phosphorylated by CDKL5  (100 nM × 1 hr) had no effect on the paired-pulse ratio, confirming the acute effects of CAF-382 (B1) are restricted to the postsynapse. fEPSP slopes are mediated by AMPA-type glutamate receptors with NMDA-type glutamate receptors largely silent under these conditions. In order to visualize the impact of CAF-382 (B1) on NMDA-type glutamate receptor mediated responses, extracellular Mg2+ was removed from the recording medium (B1) (B1)-mediated CDKL5 inhibition is to selectively reduce AMPA-type glutamate receptor-mediated responses postsynaptically.Previous studies found that genetic knock-down of c spines . In thislication . In othelication  in a timlication . This suby CDKL5  suggeste changes . To inve changes . CAF-382g medium . Under tium (B1)  but was Cdkl5 in the mouse enhanced LTP at 7–13-week-old (wo) hippocampal Schaffer Collateral to CA1 synapses without alteration of the input–output relationship  (100 nM) for at least 30 min and, following stable baseline measurements of fEPSP slope, induced LTP via theta burst to Schaffer Collateral to CA1 synapses in the continuous presence of CAF-382 (B1). Compared to interleaved recordings from control slices, CAF-382 (B1) significantly reduced LTP at 60 min post theta-burst (B1) (100 nM) for only 1 hr prior to, but not during, recording. Compared to interleaved recordings from control slices, CAF-382 (B1) significantly reduced LTP at 30 min post pairing (B1) (A previous study found that germ-line knock-out of tionship . In anot synapse . In a rater ages . To clarta-burst . To inve pairing . The ind pairing . Since wing (B1) , and theing (B1)  removed B1 for use in vivo was unknown. To gauge whether B1 could be dosed in animals using a water-based formulation, we first analyzed its aqueous kinetic solubility (B1 demonstrated good solubility (196 µM), supporting that it readily dissolves in aqueous buffer without observed precipitation. Mouse liver microsomal stability data was also collected  after 30 min, and was not prone to non-NADPH-dependent enzymatic degradation. This stability is not true for other kinase inhibitor scaffolds, which are significantly degraded after just 30 min  and total system exposure (AUCinf) of 646 h*ng/mL . While CDKL5 is a key mediator of synaptic and network development and physiology, the precise role of this kinase as explored through genetic manipulations in rodent models remains unclear. This knowledge gap prevents a full understanding of the role of CDKL5 across development and has implications for determining and interpreting when protein and gene replacement strategies may be most efficacious. As CDKL5 kinase function is central to disease pathology , demonstrated potent activity in multiple assays, including target-based in vitro biochemical assays and functional assays utilizing both rodent neuronal cultures and brain slices. The results across all assays are consistent and align with the hypothesis that compound B1 inhibits CDKL5 and blocks phosphorylation of a known CDKL5 substrate, EB2 (50 ≤ 100 nM) inhibited (CDK9 ) noted in 10 [1 µM]) were calculated using the PoC values for only WT human kinases in the DiscoverX scanMAX panel. The S10 score expresses selectivity and corresponds with the percent of the kinases screened that bind with a PoC value <10. A lower S10 score indicates enhanced kinome-wide selectivity as a lower percentage of kinases bind with high affinity . Binding to a single kinase would generate an S10 score of 0.002, while binding to 10 kinases yields an S10 score of 0.025. WT human kinases in the scanMAX panel with PoC ≤ 20 (and GSK3β) for B1, with PoC < 10 for B4, and PoC < 35 for B12 are also included in The broad selectivity of compounds KinaseSeeker technology  format in duplicate. Curves with error bars showing standard deviation are included in 50 values generated are embedded in tables within A homogeneous competition binding assay for CDKL5 was executed at Luceome Biotechnologies, LLC  using their chnology . Brieflym value for ATP to generate dose–response (9-pt) curves for all kinases listed in https://www.eurofinsdiscoveryservices.com. Briefly, either full-length or a truncate of a human kinase was incubated with [gamma-33P]-ATP in the presence of buffer, salts (magnesium source), and kinase-specific additives (such as cyclins for CDKs), where necessary, in the presence of a known substrate. Scintillation counting after quenching and washing with phosphoric acid allowed plotting of the data versus concentration and calculation of an IC50 value. Inclusion of a positive control ensured assay performance.Radiometric enzymatic assays were executed at Eurofins DiscoverX Corporation (Fremont) at the KB1 was evaluated using an aliquot of a 10 mM DMSO stock solution dissolved in phosphate-buffered saline solution (PBS) at pH 7.4. This analysis was done by Analiza, Inc  as described previously  in the presence of 5× SyPRO orange dye (Invitrogen). Next, fluorescence was evaluated using a Real-Time PCR Mx3005p machine (Stratagene). A previously reported protocol was followed to run the Tm shift assays and assess melting temperatures  following a single intraperitoneal administration to CD1 mice . A dose of 2.29 mg/kg of the TFA salt of compound B1 in NMP/Solutol/PEG-400/normal saline  was prepared just before use. This dose was calibrated due to the compound being a salt form. Plasma was sampled 0.5, 1, 2, and 4 hr post-dose. Mean values from the two animals without dispersion are shown in The pharmacokinetics of compound B1 was evaluated by Pharmaron following a single intraperitoneal administration to CD1 mice . Two doses (2.29 mg/kg and 7.63 mg/kg) of the TFA salt of compound B1 in NMP/Solutol/PEG-400/normal saline  were prepared just before use. These doses were calibrated due to the compound being a salt form. The same dose as was used for Snapshot PK (2.29 mg/kg) was chosen to have a more comprehensive profile, including brain penetrance, at this concentration. Since brain penetration was anticipated to be low, a higher concentration was also selected. Plasma and brain samples were collected 1 hr post-dose. Brain samples were homogenized at a ratio of 1:3 with PBS  and then final brain concentrations corrected. Bioanalytical assays were run using Prominence HPLC and AB Sciex Triple Quan 5500 LC/MS/MS instruments, and a HALO column . Brain/plasma concentration results are included in The brain/plasma concentration of compound Animals were housed in a controlled environment with 12 hr light/dark cycle. They were fed ad libitum and used in accordance with the Animals (Scientific Procedures) Act 1986 of the United Kingdom. Rat handling and housing was performed according to the regulations of the Animal (Scientific Procedures) Act 1986. Animal studies were approved by the Francis Crick Institute ethical committee and performed under U.K. Home Office project license (PPL P5E6B5A4B).2. Every 3–4 d, 20–30% of the maintenance media was refreshed. Between DIV14-16, neurons were treated with 5 nM, 50 nM, 500 nM, and 5 µM of the different compounds for 1 hr. The compounds were added directly to the media and the plates were placed at 37°C for the time of the treatment. DMSO was added to the well for the control condition.Primary cortical cultures were prepared from embryonic day (E) 18.5 embryos of Long–Evans rats as described . PregnanAfter treatment, neuronal cultures were lysed in 300 µL of 1× sample buffer (Invitrogen) containing 0.1 M DTT. Lysates were sonicated briefly twice and denatured at 70°C for 10 min. The samples were centrifuged at 13,300 rpm for 10 min and ran on NuPage 4–12% Bis-Tris polyacrylamide gels (Invitrogen). Proteins were transferred onto a Immobilon PVDF membrane (Millipore), which was then blocked in 4% milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 30 min. Primary antibodies were incubated at 4°C overnight, and HRP-conjugated secondary antibodies at room temperature (RT) for 2 hr. The following primary antibodies were used: rabbit anti-CDKL5 , rabbit anti-pS222 EB2 . Timed-pregnant Sprague–Dawley rats  gave birth in-house. All rodents were housed in micro-isolator cages with water and chow available ad libitum.All studies conformed to the requirements of the National Institutes of Health 2, 3 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM D-glucose, and bubbled with 95%/5% O2/CO2). Following removal of CA3, slices were recovered in a submersion-type chamber perfused with oxygenated artificial cerebral spinal fluid  at 28°C for at least 90 min and then submerged in a recording chamber perfused with recirculated ACSF. All electrophysiology was performed in the CA1 region at 28°C. Drugs were added to ACSF. Two twisted-tungsten bipolar-stimulating electrodes were offset in the CA1 to stimulate one or two independent Schaffer collateral-commissural pathways using a constant current source  with a fixed duration (20 µs), each at a rate of 0.033 Hz. fEPSPs were recorded from the stratum radiatum  region of CA1 using a borosilicate glass (WPI) microelectrode , amplified 1000× , and digitized  at 20 kHz using winLTP-version 2.4  for >30 min was allowed prior to measuring the effect of antagonists. Theta burst LTP was induced at test strength of 2 trains, separated by 20 s, of 10 trains of 4 stimuli at 100 Hz, separated by 200 ms. Potentiation was statistically compared over the last 5 min.As done previously , followision 2.4  to follosion 2.4 , Mg2+ wa2, 1.25 NaH2PO4, 1 MgSO4, 26 NaHCO3, 10 glucose, and 2 N-acetyl cysteine. Slices were incubated in either CAF-382 (B1) (100 nM) or vehicle (water) in modified ACSF for at least 1 hr prior to recording. Using infrared–differential interference contrast microscopy for visualization, whole-cell recordings (Axopatch 200B amplifier and pClamp software [Molecular Devices]) from CA1 pyramidal neurons (series resistance between 3 and 6 MΩ) were performed at 29–30°C with an intracellular solution containing (in mM) 130 Cs gluconate, 1 CsCl, 1 MgSO4, 10 HEPES, 1 EGTA, 4 MgATP, 0.4 MgGTP, spermine (0.01), and 2 QX-314, pH 7.3. AMPA-type EPSCs, evoked using a bipolar tungsten-stimulating electrode as above, at a holding potential of −65 mV were pharmacologically isolated using picrotoxin . EPSC peaks were evoked every 20 s. After establishing whole-cell access, a 3 min baseline recording at −65 mV was followed by LTP induction using a pairing protocol: cells were depolarized to 0 mV for 90 s and stimulated every 20 s then followed by delivery of 1 s × 100 Hz high-frequency stimulation. Evoked EPSC amplitudes were subsequently monitored at –65 mV. Potentiation was statistically compared over the last 5 min.For whole-cell, voltage-clamp electrophysiological recordings of excitatory postsynaptic currents (EPSCs), 350 μm horizontal hippocampal slices were prepared from rats at P 20–30 similar to previously . After 1Hippocampal slices were prepared and recovered as for electrophysiology with the addition of cuts to isolate CA1 from the remaining hippocampus; after recovery, the slices from a given rat were incubated in inhibitor or vehicle (water) in ACSF for 2 hr. Following this, slices were suspended in STE buffer, sonicated, boiled for 5 min, and then frozen until further use. To minimize the effects of slice preparation  and contData were analyzed via GraphPad Prism 9. Each assay was run in triplicate, and mean values are graphed in Data were analyzed using GraphPad Prism 9. Exact values of n and statistical methods are mentioned in the figure legends. Each concentration was compared to the control using a one-way ANOVA test. A p-value >0.05 was not considered statistically significant. Thresholds for significance were placed at *p≤0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001. All errors bars in the figures are SD where indicated. Nonsignificant statistics were not included.t-tests were used, where indicated and appropriate, for statistical comparisons for electrophysiological and biochemical using SigmaPlot 12.5 (Systat). Significance is reported at p<0.05 unless otherwise stated. All errors bars in the figures are standard error (SE) where indicated. Nonsignificant statistics were not included.Data are expressed as mean ± SEM with n = number of rats for a given treatment, unless otherwise stated. Data are plotted using SigmaPlot 12.5 . Mann–Whitney rank-sum, one-way, two-way, and repeated measures (RM) ANOVA (Holm–Sidak post hoc testing) and Student’s  This important study reports selective CDKL5 inhibitors that may be of high interest to investigate the role of this kinase in disease  and to address unsolved issues of inconsistency in the phenotypic characterization of CDKL5-deficient knockout mice. The evidence supporting the conclusions is convincing, with rigorous biochemical, in vitro and ex vivo assays. The work will be of interest to cell and medical biologists and epileptologists working in the fields of epilepsy and neural excitation. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Amy Andreotti as the Senior Editor.Thank you for submitting your article "Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Please further describe the screening procedure that led to the reported selective inhibitors .2) Please discuss potential limitations to using the reported inhibitors based on the seemingly limited brain penetration.3) Please revise figures, to clearly show statistically significant differences when detected, add legends, improve description in Figures' captions, etc.4) On the basis of the reviewers' comments. Please further discuss the statement "At issue, is whether observations of CDKL5 activity, either through genetic manipulations or interference with the kinase, are specific and not potentially subsumed by the activity of other kinases. Given the reduction of phosphorylation of the specific substrate, EB2, this is less likely here".Reviewer #1 (Recommendations for the authors):In the introduction, when stating: "Epilepsy in CDD does not respond to any antiseizure medications[11]; a newly approved therapy provides modest improvements[12]", the identity of this newly approved therapy should be revealed.Please indicate the source(s) of reactants used for synthesis.Even if some of the reported data were obtained through an outsource service  the details of the assay should be exhaustively described under the methods section to ensure reproducibility. Although a reference (ref. 43) has been included related to kinase inhibition selectivity, the authors are not from the same organization that has measured selectivity here, so I believe the details on the methodology should be made explicit.Regarding the preliminary PK study, I am curious about how tmax was estimated at 0.5 hours considering this is the earliest sampling point. Also, abbreviations used in Figure S10 should be defined and data should be presented with dispersion. How was the administered dose chosen? I am not sure about reporting Cmax and tmax with such a limited number of data points and no earlier sampling times.How were the doses used for the brain/plasma partitioning chosen?Details on the housing and feeding of the laboratory animals at The Francis Crick Institute should be provided.What is the total number of compounds included in the extensive libraries of SNS-032 and AT-7519 analogs?Whereas the S10 score is defined in the Figure 3 caption, it would be a good idea to include its definition in the main text, possibly in the Method section.Have the authors obtained data on the unbound fraction of B1?I am not convinced by the argument that, since B1 reduces the phosphorylation of the specific substrate EB2, interference with other kinases expressed in the brain might not be at play here. Please elaborate further.The authors indicate that brain penetration of B1 is rather low. Do they see this as an obstacle to using their inhibitor to further study the role of CDKL5 in CDD using in vivo assays? What about determining the kpuu, which is much more relevant pharmacologically speaking? . Haven´t the authors considered a hit-to-lead campaign to improve B1 PK profile?The potential utility of B1 could be further highlighted towards the concluding section of the manuscript.I suggest indicating the statistically significant differences in the means in Figure 6 graphically. Can the authors explain the apparent lack of a clear dose-response behavior?Seemingly, the authors have not observed their code to indicate different levels of statistical significance in Figure 7.What conclusion(s) can the authors draw from the similar microsomal stability observed with and without NADPH? Are the differences between 0 and 30 min statistically significant? Please provide a wider interpretation of the results.Reviewer #2 (Recommendations for the authors):I have several questions to clarify some descriptions.1. Selectivity scoreFigure 3 and the corresponding description of the selectivity score – the meaning of these is hard to understand. Do the dots in kinome tree diagrams represent 403 kinases? Which dot is CDKL5? Why were red circles drawn only on the CMGC family? The authors described that the S10 score is a way to express selectivity that corresponds with the percent of the kinases screened that bind with a PoC value <10 and indicated 0.017, 0.02, and 0.01 for B1, B4, and B12 respectively. But it is hard to interpret the significance of these values. Can you provide some scale or perspective and let us know how selective these values are in terms of the kinome? Why do some kinases have two IC50 (nM) values?2. Figure 7BPlease add legends for 3 traces.3. Lines 653-656"At issue, is whether observations of CDKL5 activity, either through genetic manipulations or interference with the kinase, are specific and not potentially subsumed by the activity of other kinases. Given the reduction of phosphorylation of the specific substrate, EB2, this is less likely here."It is hard to understand the intent of these sentences.Reviewer #3 (Recommendations for the authors):The first part of the paper describing binding and orthogonal assays should be improved to render the read easier. For non-expert readers, it is impossible to really understand the specificity of the inhibitor, which is a central part of the manuscript. It is also not clear why the authors start with a panel of 25 inhibitors and test 20 in the rat neurons.While the work in general is well presented it could be improved by being updated and, in a few cases, more precise.Line 170: "epilepsy in CDD does not respond to any seizure medication" – that is a bit overstated.Line 182: I suggest citing Terzic and colleagues' work from 2021 showing that CDKL5 is required also during adulthood.Line 192-197: I suggest including De Rosa et al's work from 2023 suggesting a role of CDKL5 in GABAA-R mediated inhibition through the interaction with gephyrin.Line 590: the work by Kontaxi et al. has been published.Kinase assay: it is not clear whether full-length CDKL5, and which isoform, was used for the in vitro kinase assay. Furthermore, it is not clear why myelin basic protein was used as a substrate – does it contain the CDKL5 consensus site (which is by the way never mentioned in the paper)? Essential revisions:1) Please further describe the screening procedure that led to the reported selective inhibitors .We have modified the Results to include the size of the screened libraries based around AT-7519 and SNS-032. Descriptions of the NanoBRET, Eurofins DiscoverX scanMAX, Luceome CDKL5 binding, and Eurofins radiometric assays were also added to the Methods and Results. We have updated Figures 3 and S6. In the Methods section, we have added more clarification about the use of myelin basic protein as a substrate for CDKL5 and pertinent references. These additional details have further delineated our screening procedure for the reader.2) Please discuss potential limitations to using the reported inhibitors based on the seemingly limited brain penetration.We have added sentences to the Discussion to further detail potential limitations to using the reported inhibitors and highlighting in which situations their use is highly recommended. Specifically, these inhibitors will be useful for in vitro studies to study the role of CDKL5 pathophysiology with some limited uses in vivo.3) Please revise figures, to clearly show statistically significant differences when detected, add legends, improve description in Figures' captions, etc.With attention to the reviewers’ specific comments, we have added additional text and revised the figures and legends to address these important issues. More detail about statistics were added to the captions of Figures 6, 7, S4–S6 and S10.4) On the basis of the reviewers' comments. Please further discuss the statement "At issue, is whether observations of CDKL5 activity, either through genetic manipulations or interference with the kinase, are specific and not potentially subsumed by the activity of other kinases. Given the reduction of phosphorylation of the specific substrate, EB2, this is less likely here".We agree with the reviewers that this is not clear. We have deleted these sentences and added further clarification to the discussion. The more comprehensive NanoBRET profiling of CAF382 (B1) supports that at the concentrations we used the results generated are specific to CDKL5 inhibition and not due to inhibition of off-target kinases.Reviewer #1 (Recommendations for the authors):In the introduction, when stating: "Epilepsy in CDD does not respond to any antiseizure medications[11]; a newly approved therapy provides modest improvements[12]", the identity of this newly approved therapy should be revealed.This was added to the introduction.Please indicate the source(s) of reactants used for synthesis.We have added the most common commercial vendors of reactants to the first section of Supplementary Information. We have completed the Key Reagent Table.Even if some of the reported data were obtained through an outsource service  the details of the assay should be exhaustively described under the methods section to ensure reproducibility. Although a reference (ref. 43) has been included related to kinase inhibition selectivity, the authors are not from the same organization that has measured selectivity here, so I believe the details on the methodology should be made explicit.We have added more details to the Methods section for all assays, even those run externally. This should help ensure reproducibility.Regarding the preliminary PK study, I am curious about how tmax was estimated at 0.5 hours considering this is the earliest sampling point. Also, abbreviations used in Figure S10 should be defined and data should be presented with dispersion. How was the administered dose chosen? I am not sure about reporting Cmax and tmax with such a limited number of data points and no earlier sampling times.Tmax was defaulted to 0.5 hours because, as the reviewer stated, it was the earliest sampling point and also demonstrated the highest concentration (508 ng/mL after dosing at 2.29 mg/kg). All subsequent time points demonstrated concentrations of <320 ng/mL and decreased over time.We have made a note about this Tmax value as well as the Cmax value in the corresponding Methods section. The abbreviations used in Figure S10 are now all defined in the legend. The data included in Figure S10 was provided by Pharmaron as mean values only, without dispersion. We have indicated this in the figure legend as well as in the corresponding Methods section. We were aiming for a dose of 5 mg/kg but then had to adjust slightly given that the compound was prepared as a TFA salt. 5 mg/kg is a similar, but slightly lower dose than what has been used in mice for the parent compound of SNS-032 for cancer . We wanted to see if we could achieve efficacy at a slightly lower dose with our compound.How were the doses used for the brain/plasma partitioning chosen?We selected the same dose as was used for the Snapshot PK (2.29 mg/kg) to be able to have a more comprehensive profile of the compound at that dose. Since brain penetration was anticipated to be low, a higher concentration was also elected. Given the potency of B1 in vitro and to avoid potential toxicities caused by inhibition of off-targets, we chose to use lower concentrations than have been reported for SNS-032 (15 mg/kg[1]). Our goal is not to kill cancer cells, so we would like to find an efficacious dose without associated toxicity. Some of these details have been noted in the Analysis of Brain/Plasma Concentration Methods section.Details on the housing and feeding of the laboratory animals at The Francis Crick Institute should be provided.We added additional information to the Methods: “Animals were housed in a controlled environment with 12-hour light/dark cycle. They were fed ad libitum and used in accordance with the Animals (Scientific Procedures) Act 1986 of the United Kingdom. Protocols and procedures were approved by The Francis Crick Institute project license P5E6B5A4B."What is the total number of compounds included in the extensive libraries of SNS-032 and AT-7519 analogs?This detail has been added to the Results section. We profiled our extensive library of more than 50 SNS-032 and more than 100 AT-7519 analogs using the CDKL5 NanoBRET assay.Whereas the S10 score is defined in the Figure 3 caption, it would be a good idea to include its definition in the main text, possibly in the Method section.10 score is now defined in the Methods section as well, with details on how to calculate it.Thanks for this suggestion. The SHave the authors obtained data on the unbound fraction of B1?No, not at this time.I am not convinced by the argument that, since B1 reduces the phosphorylation of the specific substrate EB2, interference with other kinases expressed in the brain might not be at play here. Please elaborate further.We have rewritten this section to include additional data supporting that at the concentrations used (≤100 nM) that only CDKL5 is efficiently engaged by B1 in vitro. While this does not exclude the possibility that other kinases not included in the selectivity profiling are involved, the “unscreenable” kinome or other ATP competitive proteins would be required to bind B1 at ≤100 nM in order to influence the biology observed. While possible since we are screening ~80% of the kinome, this seems less likely.The authors indicate that brain penetration of B1 is rather low. Do they see this as an obstacle to using their inhibitor to further study the role of CDKL5 in CDD using in vivo assays? What about determining the kpuu, which is much more relevant pharmacologically speaking? . Haven´t the authors considered a hit-to-lead campaign to improve B1 PK profile?In the Discussion we have further qualified the use of B1 as a cellular tool to study the pathophysiology associated with CDKL5 dysfunction in vitro.50 of B1 in cell-free assays (Luceome KinaseSeeker and CDKL5 enzymatic assays) is nearly equivalent to its IC50 in the cell-based CDKL5 NanoBRET and Western blot assays: all between 1–10 nM. This supports that there is no shift in potency from biochemical to cell-based assays or permeability problems.There are other processes outside the brain , where CDKL5 inhibition via an inhibitor that does not cross the BBB would be of greater utility. Future in vivo studies using B1 via implantation of minipumps that are capable of stable dosing (and inhibition) over extended periods of time are still possible. In this instance, the effect of CDKL5 inhibition on behavior and seizure susceptibility could be directly studied. Furthermore, we have added text to indicate that a hit-to-lead campaign could furnish an analog better suited for in vivo studies. A hit-to-lead campaign would benefit from determining the kpuu for leads, as the reviewer suggests. The ICThe potential utility of B1 could be further highlighted towards the concluding section of the manuscript.We have highlighted in the last paragraph of the Discussion that B1 is one of the best available chemical probes for interrogating CDKL5 activity in cells and brain slices, especially across development.I suggest indicating the statistically significant differences in the means in Figure 6 graphically. Can the authors explain the apparent lack of a clear dose-response behavior?We have added this to Figure 6. While a dose-response relationship is clear with cultured neurons , it is not readily apparent in brain slices as presented in Figure 6. There are possible reasons. Both 45nM and 100 nM are highly effective in bringing pEB2 close to background levels, thus comparison between these doses may be problematic. Also, monolayer cultures have immediate access to inhibitors which may result in lower variability.Seemingly, the authors have not observed their code to indicate different levels of statistical significance in Figure 7.Figure 7 has been adjusted for clarity.What conclusion(s) can the authors draw from the similar microsomal stability observed with and without NADPH? Are the differences between 0 and 30 min statistically significant? Please provide a wider interpretation of the results.Additional relevance of running the assay in the presence and absence of NADPH has been added to the Methods section. The Results section was supplemented with some additional clarification that non-NADPH dependent enzymatic degradation was not observed when B1 was analyzed. The differences between 0 and 30 min are statistically significant based on what we have observed in analyzing other published and unpublished kinase inhibitory scaffolds that significantly degrade after just 30 minutes. For example, after 30 min incubation with mouse liver microsomes, only 12.6% of a PIKfyve chemical probe and 40% of a CK2 chemical probe remained. We have added a relevant reference to support this in the Results section.Reviewer #2 (Recommendations for the authors):I have several questions to clarify some descriptions.1. Selectivity scoreFigure 3 and the corresponding description of the selectivity score – the meaning of these is hard to understand. Do the dots in kinome tree diagrams represent 403 kinases? Which dot is CDKL5? Why were red circles drawn only on the CMGC family? The authors described that the S10 score is a way to express selectivity that corresponds with the percent of the kinases screened that bind with a PoC value <10 and indicated 0.017, 0.02, and 0.01 for B1, B4, and B12 respectively. But it is hard to interpret the significance of these values. Can you provide some scale or perspective and let us know how selective these values are in terms of the kinome? Why do some kinases have two IC50 (nM) values?Thank you for drawing our attention to this. We have supplemented the Methods section that describes the DiscoverX scanMAX panel with details about how selectivity score is calculated and what it means. Some additional perspective and examples were added to further qualify the selectivity scores. To further address the concerns raised, we have also clarified in the legend that the dots represent kinases that bind with high affinity to the compound being assayed. Furthermore, we have labeled all red dots with the corresponding kinase. We have added a sentence to the discussion to highlight the important point about these compounds retaining CMGC family potency. Finally, the tables in Figure 3 have been reconfigured to have separate columns for biochemical (binding or enzymatic) and NanoBRET data. The legend has been updated to reflect this change as well.2. Figure 7BPlease add legends for 3 traces.Figure 7B and the legend has been modified for clarity.3. Lines 653-656"At issue, is whether observations of CDKL5 activity, either through genetic manipulations or interference with the kinase, are specific and not potentially subsumed by the activity of other kinases. Given the reduction of phosphorylation of the specific substrate, EB2, this is less likely here."It is hard to understand the intent of these sentences.We agree with the reviewer that this is not clear. This text has been deleted. The more comprehensive NanoBRET profiling of CAF-382 (B1) supports that at the concentrations we used that the results generated are specific to CDKL5 inhibition and not due to inhibition of offtarget kinases.Reviewer #3 (Recommendations for the authors):The first part of the paper describing binding and orthogonal assays should be improved to render the read easier. For non-expert readers, it is impossible to really understand the specificity of the inhibitor, which is a central part of the manuscript. It is also not clear why the authors start with a panel of 25 inhibitors and test 20 in the rat neurons.We have added a lot more detail to the descriptions of the orthogonal assays in the Methods and Results sections. This provides more context about what each assay is meant to evaluate and how to interpret the results. We tested all 25 putative CDKL5 lead compounds (Table S1) in rat neurons .While the work in general is well presented it could be improved by being updated and, in a few cases, more precise.Line 170: "epilepsy in CDD does not respond to any seizure medication" – that is a bit overstated.We have cited the literature that supports this statement. While I have not come across such an individual  and with our current clinical studies involving CDD (NIH/NINDS U01), all patients (>150) meet the ILAE definition of medically refractory (https://www.ilae.org/patient-care/drug-resistant-epilepsy), these have been reported so we have adjusted this statement and added those references.Line 182: I suggest citing Terzic and colleagues' work from 2021 showing that CDKL5 is required also during adulthood.Thank you. The reference to Terzic et al. have now been cited in this section.Line 192-197: I suggest including De Rosa et al's work from 2023 suggesting a role of CDKL5 in GABAA-R mediated inhibition through the interaction with gephyrin.Thank you. This is an important reference that has now been added.Line 590: the work by Kontaxi et al. has been published.The reference has been updated.Kinase assay: it is not clear whether full-length CDKL5, and which isoform, was used for the in vitro kinase assay. Furthermore, it is not clear why myelin basic protein was used as a substrate – does it contain the CDKL5 consensus site (which is by the way never mentioned in the paper)?We have added that we used full-length human CDKL5  to the Methods section. We added to the Methods that isoforms are homologous through the kinase domain.The basis for our selection of myelin basic protein as a substrate has been explained with references to support this choice. To execute the in vitro kinase assays, myelin basic protein  was employed as a substrate for recombinant CDKL5. Myelin basic protein is used as a substrate for multiple kinases, both serine/threonine and tyrosine kinases, to enable in vitro kinase assays due to the presence of multiple sites for phosphorylation. As such, we and others have used this protein as a kinase substrate for evaluating kinase activity. MBP does not contain a CDKL5 consensus site of RPXS/T*, and as such could be considered a less than ideal substrates to study CDKL5 activity, however for in vitro kinase assays MBP is still suitable as it can be phosphorylated by CDKL5. In addition, CDKL5 is known to phosphorylate substrates that do not contain a consensus motif[3].ReferencesWu, Y., et al., Cyclin-dependent kinase 7/9 inhibitor SNS-032 abrogates FIP1-like-1 platelet-derived growth factor receptor α and bcr-abl oncogene addiction in malignant hematologic cells. Clin Cancer Res, 2012. 18(7): p. 1966-78.SOX9 regulatory axis in epithelial cell death and kidney injury. Nat Commun, 2020. 11(1): p. 1924.Kim, J.Y., et al., A kinome-wide screen identifies a CDKL5-SOX9 signaling axis in rhabdomyolysis associated acute kidney injury. Am J Physiol Renal Physiol, 2020. 319(5): p. F920-F929.Kim, J.Y., et al., Involvement of the CDKL5-Kameshita, I., et al., TandeMBP: generation of a unique protein substrate for protein kinase assays. J Biochem, 2014. 156(3): p. 147-54.


Vanishing white matter (VWM) is a leukodystrophy that primarily manifests in young children. In this disease, the brain white matter is differentially affected in a predictable pattern with telencephalic brain areas being most severely affected, while others remain allegedly completely spared. Using high-resolution mass spectrometry-based proteomics, we investigated the proteome patterns of the white matter in the severely affected frontal lobe and normal appearing pons in VWM and control cases to identify molecular bases underlying regional vulnerability. By comparing VWM patients to controls, we identified disease-specific proteome patterns. We showed substantial changes in both the VWM frontal and pons white matter at the protein level. Side-by-side comparison of brain region-specific proteome patterns further revealed regional differences. We found that different cell types were affected in the VWM frontal white matter than in the pons. Gene ontology and pathway analyses identified involvement of region specific biological processes, of which pathways involved in cellular respiratory metabolism were overarching features. In the VWM frontal white matter, proteins involved in glycolysis/gluconeogenesis and metabolism of various amino acids were decreased compared to controls. By contrast, in the VWM pons white matter, we found a decrease in proteins involved in oxidative phosphorylation. Taken together, our data show that brain regions are affected in parallel in VWM, but to different degrees. We found region-specific involvement of different cell types and discovered that cellular respiratory metabolism is likely to be differentially affected across white matter regions in VWM. These region-specific changes help explain regional vulnerability to pathology in VWM.The online version contains supplementary material available at 10.1186/s40478-023-01599-6. Vanishing white matter  is one of the more prevalent leukodystrophies. It may present at any age, but primarily manifests in children . VWM is Typical patterns of VWM are dominated by degeneration of white matter structures with meagre reactive gliosis, lack of myelin, axonal abnormalities, and immature astrocytes and oligodendrocytes , 37, 41.It is important to elucidate how these differential regional vulnerabilities arise in order to understand VWM pathology. Here we aimed at gaining more insight into the molecular bases underlying selective regional vulnerability of the brain white matter in VWM. We performed a high-resolution mass spectrometry-based proteome analysis of 2 differently affected brain regions in VWM, that is, the white matter of the frontal lobe and the pons. Regional protein expression patterns were analyzed and compared to identify disease mechanisms that may contribute to regional vulnerability in VWM.This study focused on the white matter of the middle frontal gyrus and the mid-pons. Post mortem brain tissue from 4 genetically proven VWM patients was collected at the Amsterdam University Medical Centers location VU University Amsterdam . Patients were selected based on the severity of the white matter pathology . In addition, 4 controls obtained from the Netherlands Brain Bank were included. No confounding neuropathological or structural abnormalities were found in these controls. Tissue was obtained within 6 h post mortem. Informed consent was obtained in all cases. The study was approved by the Medical Ethical Committee of the Amsterdam University Medical Centers location VU University Amsterdam  and conducted according to the declaration of Helsinki. Demographic features of controls and patients are shown in Table 2O for 1 min. Next, tissue sections were stained using toluidine blue (1% w/v in sterile H2O) for 1 min at room temperature and washed 3 times in sterile H2O for 1 min. Afterwards, sections were dehydrated in 100% ethanol twice for 3 min and air-dried. Brain regions of interest were then microdissected using a Leica LMD6500 system (Leica Microsystems). A volume of 100mm3 of white matter tissue from the frontal lobe and the pons was collected into adhesive caps (Zeiss). Microdissected tissues were stored at -80℃ until further use.Fresh frozen tissue sections of 20µm thickness were mounted on polyethylene naphthalate-coated glass slides, fixed in 100% ethanol for 20 min, air-dried and rehydrated twice in sterile HMicrodissected tissue samples were incubated in lysis buffer phosphine, 10 mM chloroacetamide and 100 mM Tris-HCl in 50 mM ammonium bicarbonate) for 10 min at 99 °C with mixing at 750RPM. Tissue lysates were then sonicated with 20 mg protein extraction beads (Diagenode) using a Bioruptor Plus (Diagenode) for 20 cycles with on/off pulses of 30 s. Protein digestion was performed using Lys-C in a 1:100 enzyme-to-substrate ratio at 37 °C for 4 h. Afterwards, samples were diluted to a final concentration of 2 M guanidine hydrochloride followed by overnight digestion with trypsin at a 1:100 enzyme-to-substrate ratio at 37 °C. Digestion was quenched by lowering the pH below 2 using 10% formic acid. Peptide samples were then desalted using C18 cartridges (Agilent Technologies) on the automated AssayMAP Bravo Platform (Agilent Technologies). Cartridges were first primed and equilibrated using 80% acetonitrile/0.1% formic acid and 0.1% formic acid, respectively. Peptides were then loaded and washed with 0.1% formic acid, and eluted with 80% acetonitrile/0.1% formic acid. Eluted peptides were dried using a SpeedVac centrifuge and stored at − 20 °C.6 ions) was acquired. Then the top 15 precursors were selected for MS2 analysis with higher-collision dissociation . Dynamic exclusion was set to 16 s. Precursor ions with unassigned charge state as well as charge state of 1 and 6 or higher were excluded from fragmentation.Mass spectrometry analysis was conducted on an Orbitrap Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific) coupled to an UltiMate 3000 RSLCnano System (Thermo Fisher Scientific). Peptides (1.5 µg/sample) were resuspended in 2% formic acid and loaded on a trap column  at a flow rate of 10 µL/min in 100% buffer A (0.1% formic acid). Peptides were separated on a self-packed analytical column. Elution was performed over a 175 min gradient at a flow rate off 300 nL/min with buffer B (80% acetonitrile/0.1% formic acid): 0–2.5 min 92% solvent A; 2.5– 57.5 min 8% solvent B; 157.5–161 min 35% solvent B; 161–165 min 100% solvent B; 165–175 min 92% solvent A. Data-dependent acquisition method was used to collect mass spectra. Briefly, first a full MS1 scan  and analyzed using MaxQuant (v1.6.6.0) . “Match 2-transformed. Only proteins detected in all replicates of at least one condition were considered for further analysis. Missing data were imputed using random draws from a left-shifted Gaussian distribution centered around a minimal mean value being the lowest observed expression value. The standard deviation was estimated as being the mean standard deviation of all detected proteins. Statistical significance was set to q-value < 0.05 with Benjamini-Hochberg’s (BH) adjustment for multiple comparison. Principal component analysis was conducted using FactoMineR R package [Statistical analysis was performed using the R statistical software (v1.3.1093). Differential protein expression analysis was analyzed using DEP R package . Peptide package . A Pearsq-value < 0.05 with BH adjustment for multiple comparison.Cell type enrichment analysis was performed using the EWCE R package based on a single nuclei RNA sequencing dataset of 5291 cells from brain white matter tissue of 3 controls . These cq-value < 0.05 with BH adjustment for multiple comparison. All proteins detected in our proteome analysis were set as background.Functional gene ontology overrepresentation analysis was conducted using PANTHER Classification System version 17.0) with a Fisher’s exact test , 35. The.0 with aTo gain insight into selective regional vulnerability in VWM, we conducted a high-resolution mass spectrometry-based proteome analysis on the white matter of the frontal lobe and the pons, the first representing a severely affected region and the latter being relatively spared. Four controls and 4 VWM patients with childhood-onset were included in the study Table . PatientProteome analysis identified an average of 22,053 peptides per sample. Missing values were imputed, mapping to a total of 3149 proteins across 14 samples . Amongst these, 288 were downregulated and 210 upregulated  and myelin proteolipid protein , as well as upregulation of the intermediate filament nestin  and vimentin   and oligodendrocytes , whereas proteins with increased expression were enriched for neurons  and OPCs  , of which 94 were downregulated and 130 upregulated  and MBP (p = 0.0095) expression, respectively. Proteins with higher expression were significantly enriched for immune cells , that is microglia and macrophages, and lymphocytes , whereas proteins with low expression were not significantly enriched for a particular cell type , suggesting strong difference in proteome patterns of the white matter in the frontal lobe compared to the pons   and OPCs   and oligodendrocytes   and lymphocytes , demonstrating involvement of immune cells in this specific region  cycle , 27, 32.We also found changes in mitochondrial oxidative phosphorylation. During oxidative phosphorylation oxidation/reduction reactions take place, followed by the release of energy in the form of adenosine triphosphate (ATP) , 14, 32.Some limitations apply to this work. A potential concern is the mismatch in ages between VWM patients and controls. Control brain tissue without confounding neuropathology, obtained at short post mortem time from young donors and children, is extremely rare. In particular, the long post mortem time at which the tissue is harvested elsewhere makes it unsuitable for selected experiments, including proteomics. It should, therefore, be noted that given the active myelination present in the brains of younger individuals, it is possible that certain proteome changes detected in this study may in part be caused by aging.Furthermore, our proteome analysis was focused on regional changes in the abundance of proteins that are relevant to VWM pathogenesis. It would be of interest to further explore other white matter disorders in general and leukodystrophies in particular to gain insight into the possible commonalities and differences between different diseases at the protein level.In conclusion, our novel proteomic data shed light on the pathophysiology of VWM in different ways. They show that relatively or allegedly completely spared brain regions are affected in VWM at a molecular level, in line with previous observations concerning the cortex. The data also pinpoint the basic cellular molecular pathways and components that are differentially affected in the VWM brain. Further studies are warranted to explore how this information translates to a cellular functional level.Below is the link to the electronic supplementary material.Supplemental Table 1 List of all unique proteins detected and quantified across the white matter of the frontal lobe and pons in control and VWM casesSupplementary Material 1: Supplemental Table 2 List of significantly differentially expressed proteins in the white matter of the frontal lobe and pons in VWM compared to controlSupplementary Material 2: Supplemental Table 3 Results of gene ontology and pathway analysis of significantly differentially expressed proteins in VWM frontal and pons white matterSupplementary Material 3: Supplemental Table 4 Comparison of proteins with differential expression in the frontal and pons white matter in VWMSupplementary Material 4: Supplemental Table 5 Results of gene ontology and pathway analysis of all 650 protein found significantly differentially expressed across the frontal and pons white matter in VWMSupplementary Material 5: Supplemental Fig. 1 Prototypic MRI of a 32-year-old VWM patient with age of onset at 7 years. (a-b) T2-weighted images show (a) a normal pons and (b) a severe cerebral white matter disease with diffuse signal abnormality and atrophy. (c) FLAIR image shows that the cerebral white matter is largely rarefied. Red arrows indicate in (a) the normal pons and in (b-c) the affected cerebral white matterSupplementary Material 6: Supplemental Fig. 2 Principal component analyses of all white matter in the frontal lobe and pons in control and VWM cases. Analysis of all samples reveal separation of VWM frontal white matter samples from all other samples in the first component, explaining 29.5% of the variability. The second component separates control pons white matter samples from control frontal as well as pons white matter samples. This accounts for 17.0% of the variability. PC1 principal component 1, PC2 principal component 2, Ctrl control, VWM vanishing white matterSupplementary Material 7: Supplemental Fig. 3 EWCE analysis of proteins found altered in the same direction in both the frontal and pons white matter in VWM (n = 40). Analysis of (a) upregulated and (b) downregulated proteins. * q-value < 0.05Supplementary Material 8:


GJB2 variants in the Chinese population of the Dongfeng Tongji cohort and to analyze the features of the hearing phenotype. Methods: We used data from 9910 participants in the Dongfeng Tongji cohort in 2013 and selected nine GJB2 variants. Pure tone audiometry was employed to measure hearing. Differences in genotype and allele frequencies were analyzed via chi-squared test or Fisher’s exact test. Results: Of the 9910 participants, 5742 had hearing loss. The genotype frequency of the GJB2 variant c.109G>A was statistically significantly distributed between the normal and impaired hearing groups, but not for the variant c.235delC. A higher frequency of the c.109G>A homozygous genotype was found in the hearing loss group (0.5%) than in the normal hearing group (0.1%). Patients with c.109G>A and c.235delC homozygous mutations exhibited varying degrees of hearing loss, mainly presenting sloping and flat audiogram shapes. Conclusions: A significant difference was found in the genotype frequency of the GJB2 variant c.109G>A between the case and control groups, but not in that of the variant c.235delC. Different degrees of hearing loss and various audiogram shapes were observed in patients with c.109G>A and c.235delC homozygous mutations.Background: This study aimed to describe the distribution of the genotype and allele frequencies of  GJB2) gene (OMIM: 121011), which encodes the gap junction protein connexin 26 (Cx26) on chromosome 13q12.11 [Hearing loss is a common sensory disorder affecting more than 1.5 billion people worldwide, including 430 million with moderate or severe hearing loss . Heredit13q12.11 ,9,10. GJB2 was first described in 1997 [GJB2 have been reported [GJB2 vary widely [GJB2, is also common among deaf patients in East Asia [GJB2 mutations in affected individuals [GJB2 variants show diverse phenotypes, ranging from mild to profound [GJB2 mutation, auditory phenotypes show great variability [GJB2 mutations. Since hearing loss related to  in 1997 ,12, morereported ,12. In dast Asia . The c.1ast Asia . Later, ividuals ,15,16. Cividuals , and theividuals ,17. Simiprofound ,18, whicprofound ,19. Patiprofound ,19. Howeiability ,20. TherGJB2 mutations in hearing loss and their genetic heterogeneity and phenotypic diversity, it is important to analyze the frequency distribution and genotype–phenotype correlation of GJB2 mutations in hearing loss patients for genetic diagnosis and counseling. This study aimed to: (1) analyze the frequencies of genotypes and alleles of GJB2 mutations in the Chinese population from the Dongfeng Tongji (DFTJ) cohort and (2) investigate audiogram shapes in patients with hearing loss with regard to different genotypes of c.109G>A and c.235delC mutations in the GJB2 gene.Given the dominant role of The DFTJ cohort is a prospective study launched in September 2008 in Shiyan City, Hubei Province, China, based on retired employees of Dongfeng Motor Corporation . The basGJB2 mutations and audiometry. After those participants missing the age variable data (n = 2) were removed, 9910 participants were used for the final analysis.After the exclusion of 57 participants with invalid audiometric data and 1544 participants who lacked the genotype and failed quality control, this study included 9912 participants with complete data on nine 0.5–4 kHz) greater than 25 decibels of hearing level (dB HL) in the better ear [0.5–4 kHz ≤ 40 dB HL), moderate (40 dB HL < PTA0.5–4 kHz ≤ 60 dB HL), and severe–profound (PTA0.5–4 kHz > 60 dB HL). In a special soundproof room at Dongfeng General Hospital, participants underwent pure tone tests administered by professional audiologists using a calibrated pure tone audiometer  after an otological examination. Air conduction thresholds at 0.5, 1, 2, 4, and 8 kHz were recorded for each ear and coded as a maximum when the maximum was unresponsive . Hearingtter ear . The sevBased on Liu’s  classifiGJB2 variants or single nucleotide polymorphisms were selected as follows: c.11G>A (rs111033222), c.79G>A (rs2274084), c.*84T>C (rs3751385), c.109G>A (rs72474224), c.235delC (rs80338943), c.341A>G (rs2274083), c.368C>A (rs111033188), c.571T>C (rs397516878), and c.608T>C (rs76838169). These variants were genotyped in a genome-wide association study scan of the DFTJ cohort using Affymetrix Genome-Wide Human SNP Array 6.0 chips and Illumina Infinium Omni Zhong Hua-8 Chips  [p value of Hardy–Weinberg equilibrium < 10−5. There were 703,302 SNPs in the Illumina dataset and 549,196 in the Affymetrix dataset. Genotypes of markers in the Illumina and Affymetrix datasets were imputed separately using the 1000 Genomes Project ALL Phase 3 Integrated Release Version 5 haplotypes as a reference panel. Detailed descriptions of the genotyping and quality control processes are available elsewhere [For the purpose of genetic analysis related to hearing loss, nine common GJB2 mutations in the Chinese population were selected. Nine , China) . In brielsewhere ,27.p-value of less than 0.05. All statistical analyses were carried out using R software .The categorical variables were expressed as numbers with percentages, and the continuous variables as means with standard deviations. Comparisons of differences in genotype and allele frequencies between groups were conducted using the chi-squared test or Fisher’s exact test. The threshold for significance was set at a two-sided GJB2 gene in the cohort and between the hearing-impaired and normal groups. Based on the variant annotation in the ClinVar database , two variants are reported as pathogenic variants (c.109G>A and c.235delC); five are benign or likely benign variants ; one is uncertain (c.571T>C); and the last has a conflicting interpretation of pathogenicity (c.11G>A). We found a significant difference  in the genotype frequency distribution of c.109G>A between the case and control groups (p = 0.369). Likewise, no statistical difference was found for the frequency distribution of c.109G>A alleles across hearing loss severity groups (p = 0.274)  .p = 0.661)  .Moreover, among the five benign variants, we found statistically significant differences in the genotype and allele frequencies of c.*84T>C between case and control groups . The allGJB2 gene and analyzed the audiogram phenotypic profiles of two pathogenic variants for the hearing loss group. A significant difference in genotype frequencies between cases and controls was observed for the variants c.*84T>C and c.109G>A, while the other seven variants (including c.235delC) were not statistically different between cases and controls. Homozygous mutations in c.109G>A and c.235delC showed different degrees of hearing loss and various shapes of audiogram, mainly sloping and flat. This study was a large-scale population analysis of GJB2-related variants , and contained objective pure tone audiometric measurements that could effectively characterize the hearing phenotype.In this study of 9910 participants from the DFTJ cohort, we described an overview of the genotype and allele frequency distributions of nine variants with the GJB2 were statistically significantly distributed between cases and controls. The frequency of homozygous genotype in the hearing impaired group was 0.5%, lower than that reported in other Chinese populations in a recent study [GJB2 gene [CDH23 coexisting with the c.109G>A homozygous mutation in one patient [Consistent with our expectations, we found that the genotype frequencies of the c.109G>A variant in nt study , where cJB2 gene . In agreJB2 gene ,28, we o patient . Therefo patient ,17,28. HGJB2-associated variant in Chinese patients with hearing loss [GJB2 gene causes a frameshift mutation that prematurely terminates translation and produces a non-functional gap junction protein [The c.235delC variant has been reported to be the most prevalent ing loss ,12,29,30 protein ,32. Pati protein ,33,34. HGJB2 gene and has been identified as a benign variant. Technological advances in sequencing and many other assays have facilitated the elucidation of the non-coding genome [We found that the genotype and allele frequencies of c.*84T>C were statistically different between the normal and impaired hearing groups. The c.*84T>C is located in the 3′UTR region of the g genome . Many nog genome . The geng genome . We obseg genome .GJB2-related hearing loss were generally residual, sloping, or flat, but similar to those observed in hearing impaired patients without the GJB2 mutation, suggesting a possible poor correlation between the type of GJB2 mutation and audiogram shape [In agreement with a previous study , the audam shape .The limitations of this study should also be considered. First, the participants were from a single cohort and might not be representative of the general Chinese population, which limited the generalizability of the results. Second, other pathogenic variants in GJB2 were not included in the study. Finally, this study also did not consider the case of compound heterozygotes in the GJB2 gene or in combination with other genes, which requires further research.GJB2 variants c.*84T>C and c.109G>A between cases and controls in a Chinese population in the large-scale DFTJ cohort, but not for the c.235delC variant. Patients with c.109G>A and c.235delC homozygous mutations exhibited diverse hearing loss severities and audiogram shapes. These findings could provide evidence for genetic diagnosis and counseling for hearing loss.To sum up, we observed a statistically significant distribution of genotype frequencies of the


Beauveria bassiana can colonize plants resulting in growth promotion and protection against phytopathogenic microorganisms. However, physiological changes in potato plants (Solanum tuberosum) during this interaction are poorly understood. In the present work, gas chromatography–mass spectrometry and high-performance liquid chromatography were used to analyze sterol, fatty acid, and phenolic acid concentrations in potato plants inoculated with B. bassiana conidia in soil. We showed an increase in amounts of stigmasterol, minor sterol compounds, and some hydroxy fatty acids in leaves after the fungal treatment. Moreover, levels of hydroxycinnamic acids, especially chlorogenic acid, were elevated in roots following the B. bassiana inoculation. We propose that these changes could have been caused by oxidative reactions, and the alterations may have resulted in growth-stimulatory and protective effects of B. bassiana on the plants.The entomopathogenic endophytic fungus  Beauveria and Metarhizium are promising candidates for improving plant health and yield [Arabidopsis by Beauveria led to reprogramming of metabolic pathways related to resistance to environmental stresses and phytopathogenic microorganisms. Proietti et al. [B. bassiana. Entomopathogenic fungi are able to alter the biochemical composition of plant tissues [Entomopathogenic endophytic fungi such as nd yield ,2,3. Trand yield ,2. Growtnd yield ,5,6,7. F tissues  and the  tissues .Beauveria or Metarhizium) with potato Solanum tuberosum plants. Rios-Moreno et al. [Metarhizium brunneum to colonize potato tissues and produce destruxin under laboratory conditions after spraying the plants with conidial suspensions. Krell et al. [M. brunneum is capable of endophytic colonization of potato plants, thus increasing dry biomass, nitrogen, and phosphorus contents of the plants, as well as improving water use efficiency. Changes in these parameters proved to be mediated by different nutrient conditions, and these authors stated that M. brunneum can mitigate nutrient deficits in the soil, further improving plant productivity [Beauveria bassiana or Metarhizium robertsii was found to alter pigment ratios, peroxidase activity, levels of thiobarbituric acid reactive substances, and proline in various tissues of potato [Beauveria and Metarhizium propagules in the soil of potato fields (102–104 CFU/g), the occurrence of Metarhizium in plants without surface sterilization may reach 50%, but the colonization of internal potato tissues occurs rarely [B. bassiana or M. robertsii reduced the incidence of rhizoctoniosis (Rhizoctonia solani), changed immune-activity parameters, and improved crop quality under field conditions [B. bassiana (strain Sar-31) significantly decreased Rhizoctonia disease and improved the tuber yield [Several important effects have been found during interaction of entomopathogenic fungi . To ourChaetomium–Miscanthus sinensis system [Bacillus subtilis can themselves produce chlorogenic acid and its derivatives [Interactions of plants with fungi may change the amounts of phenolic compounds . These cs system . Some enivatives . This acivatives ,33 and nivatives .B. bassiana under laboratory conditions.We have previously observed growth stimulation, increases in levels of antioxidant enzymes, and enhanced resistance to phytopathogens in potato plants exposed to entomopathogenic fungi ,15. TherB. bassiana at 14 days postinoculation was 45% for roots, 75% for stems, and 10% for leaves (B. bassiana-colonized tissues were found in the control treatment group. Cultures reisolated from plants showed 100% identity of the secreted-lipase gene fragment (445 bp) toward each other and to the paternal strain Sar-31 .Plants inoculated with B. bassiana colonization, the total amount of sterols in leaves increased 1.3-fold, but the change was not statistically significant . Nevertheless, a significant 1.5-fold increase in a major compound (stigmasterol) was registered in response to the fungal inoculation  and significant  upregulation of a minor compound  were registered. There were no significant changes in the amounts of sterols in roots in response to B. bassiana treatment , that of C15:0 14Me by 1.2-fold , and the amount of C16:0 2-OH by 1.6-fold . There were no significant effects of B. bassiana on any fatty acid amounts in roots  and 17 saturated ones (C9–C28) were detected in potato leaves and roots . The totrol ones A,B. In pin roots C,D. PCA in roots , indicatB. bassiana . In particular, the amount of chlorogenic acid rose by 1.5-fold  and the amount of caffeic acid by 1.1-fold (p = 0.40) as compared to the control.In extracts of roots, three hydroxycinnamic acids were found: chlorogenic (5-O-caffeoylquinic) acid, caffeic acid, and an unidentified hydroxycinnamic acid . The totB. bassiana treatment  acid and cryptochlorogenic (4-O-caffeoylquinic) acid . No signreatment B.B. bassiana to colonize potato and to increase plant biomass is consistent with our previous studies [The ability of  studies . In the  studies ,5,35,36; studies ,12. FurtB. bassiana (Arabidopsis in response to infection with biotrophic (Pseudomonas syringae) and necrotrophic (Botrytis cinerea) pathogens. Higher expression of sterol desaturase has also been detected after infection of Arabidopsis with biotrophic and hemibiotrophic fungi Golovinomyces cichoracearum and Phytophthora infestans [B. bassiana colonization, as evidenced by upregulation of malondialdehyde and antioxidant enzymes [B. bassiana, Metarhizium brunneum, or Trichoderma harzianum was observed by Gupta et al. [B. bassiana strains induce genes involved in oxidative stress processes in Arabidopsis thaliana, also in agreement with results on “Solanaceae plant–entomopathogenic fungus B. bassiana” systems [B. bassiana. Notably, the increased concentration of the stigmasterol in Arabidopsis after P. syringae infection did not lead to the downregulation of β-sitosterol [B. bassiana.We observed enhancement of stigmasterol production in leaves of potato plants inoculated with bassiana . Stigmasnfestans ,40. Grienfestans  report t enzymes ,15. An ia et al. . Raad eta et al.  showed t systems ,41. Pose systems  demonstrtosterol , consistP. syringae but not the necrotrophic pathogen B. cinerea. Those authors suggested that stigmasterol can interfere with a specific plant defense pathway  or act on microorganisms directly by enhancing their growth. The behavior of B. bassiana in a plant is biotrophic according to the classification of Spanu and Kamper [Interestingly, Griebel and Zeier  found thd Kamper  because d Kamper . It is pB. bassiana. Previous works showed changes in the profile of fatty acids caused by arbuscular mycorrhiza. For instance, Cao et al. [Poncirus trifoliata plants by Piriformospora indica. Lida et al. [B. bassiana. We detected statistically significant quantitative changes only for the third type of fatty acids in their broad spectrum: C15:0, C15:0 14Me, and C16:0 2-OH  was also detected by Homayoonzadeh et al. [B. bassiana or fungal biosynthesis of some compounds; this issue should be elucidated in future studies. It is well known that chlorogenic acid is an antioxidant; therefore, this phenomenon may be associated with a response of potato to oxidative processes. Mei et al. [2O2 and malondialdehyde in apple leaves treated with an herbicide inducing oxidative stress. Moreover, exogenous chlorogenic acid modulates peroxidase and catalase production and the expression regulation of genes related to the antioxidant system and phenolic compound metabolism [We observed an elevated amount of hydroxycinnamic acids, especially chlorogenic acid, in potato roots after culation . It was h et al. . Research et al.  hypothesi et al.  demonstrtabolism . Those aPyrus betulaefolia, and a high concentration of the acid can inhibit it [B. bassiana. Chlorogenic acid has antifungal properties, in particular, the inhibition of spore germination, a reduction in mycelial growth, or cell lysis, which have been documented for Sclerotinia sclerotiorum, Fusarium solani, Verticillium dahliae, Botrytis cinerea, Cercospora sojina [Phytophthora infestans [Phlyctaena vagabunda [F. solani and Candida albicans [B. bassiana treatment is in agreement with diminished incidence of diseases caused by phytopathogenic fungi [Chlorogenic acid plays an important role in processes of plant growth and organogenesis of shoots and roots and also affects fruit ripening ,50,51. Thibit it ,53. Thera sojina , Phytophnfestans , and Phlagabunda . This acalbicans ,56. Morealbicans . Thus, tic fungi .Beauveria bassiana s.s. (strain Sar-31) from the collection of microorganisms at the Institute of Systematics and Ecology of Animals (the Siberian Branch of the Russian Academy of Sciences) was used. The strain was isolated from Calliptamus italicus  in Western Siberia (53°41′ N 78°02′ E) in 2001. The fungal species was identified on the basis of the sequence of a translation elongation factor region (GenBank accession No. MZ564259). The fungus was grown in Petri dishes (90 mm) with 1/4 Sabouraud dextrose agar supplemented with 0.25% of yeast extract (1/4 SDAY [v/v). The concentration of conidia was determined using a hemocytometer. The germination of conidia on 1/4 SDAY was 98% after 16 h of incubation (n = 100).An isolate of 1/4 SDAY ) at 26 °Solanum tuberosum variety Agata) were used in the experiments. Tubers (diameter 3–4 cm) were treated with 7% sodium hypochlorite (2 min) and then 70% ethanol, followed by three rinses in sterile distilled water [Metarhizium and Beauveria by plating on CTAB medium [Potato plants  in the 0.03% (v/v) Tween 80 solution 3 days after planting. Control plants were treated with a conidia-free aqueous Tween 80 solution.Plants were grown at 22 °C and 40–60% relative humidity with a 16 h/8 h photoperiod (light/dark) using full-spectrum Flora phytolamps . We used soil drenching with the conidial suspension around seedlings  were used for the determination of lipid and hydroxycinnamic acid amounts.Fourteen days after fungal treatment, plant roots and leaves were collected, lyophilized in individual packs, and stored at −20 °C. In the 2SO4 in MeOH at 80 °C for 3 h [The dry plant material was crushed in a ball mill and extracted with a mixture of chloroform and methanol by the Folch method ,72 in an for 3 h ,74 and tv/v) [The plant material was crushed in the ball mill, and phenolic acids were extracted with a mixture of methanol and 2% aqueous acetic acid (2:3 v/v) . The matGC/MS analysis was performed with a 6890 N gas chromatograph and a 5975 N mass-selective detector . Separation was carried out on an HP-5MS capillary column , at an injector temperature of 280 °C, ion source temperature of 230 °C, quadrupole temperature of 150 °C, and a flow rate of a carrier gas (helium) of 1 mL/min. A temperature gradient was implemented as follows: the temperature was initially held at 50 °C for 2 min and then raised to 280 °C at a rate of 10 °C/min. The ChemStation software, the NIST 14 MS spectral library, and literature data ,77,78 wev/v) trifluoroacetic acid in an H2O gradient; the methanol percentage in the gradient was 2–100% (minutes 0–20) and 100% methanol (minutes 20–25); the flow rate was 0.8 mL/min; the injection volume was 2 μL; and the detection was performed simultaneously at four wavelengths: 254, 280, 320, and 360 nm. Chlorogenic acid (Sigma-Aldrich) was used for the quantification of hydroxycinnamic acids. Identification of cryptochlorogenic and caffeic acids was carried out on the basis of UV spectra and retention times of peaks [HPLC analysis was performed on an Agilent LC 1100 chromatograph  equipped with a quaternary pump, an autosampler, and a diode array detector. The chromatographic conditions were as follows: a ZORBAX Eclipse XBD-C8 column  (Agilent Technologies); a mobile phase consisting of methanol/0.1%  of fatty acid composition was performed using a variance–covariance matrix. The Past v.3 software [Data were checked for normality by the Shapiro–Wilk e’s test . Normallsoftware  was utilB. bassiana. The main observed effects were increases in the amount of stigmasterol in leaves, hydroxy fatty acid (C16:0 2-OH) in leaves, and hydroxycinnamic acids in roots. Most probably, these changes are associated with oxidative reactions in potato plants during colonization by this B. bassiana strain (Sar-31), in agreement with our previous results [B. bassiana on the chemical composition of potato tubers because it may affect human health.This is the first report on changes in the lipid and hydroxycinnamic acid profiles of potato plants after colonization by  results ,15. At t


Current knowledge and further development in the field of otoprotection,” presents a selection of different papers approaching this important topic. A diverse range of approaches to tackling this topic is presented, from everyday usable solutions to clinical applications such as intraoperative monitoring or imaging to highly technical laboratory approaches. This spectrum depicts the wide approach taken by the community to this topic.Over the next 30 years, up to 2.5 billion people will experience various degrees of hearing loss. Approximately 700 million people will be dependent on technical rehabilitation, like different hearing implant solutions. Thus, hearing impairment will become a medical and economic burden in the coming decades. Given this context, the emphasis on different otoprotective efforts is understandable and necessary. This special issue, “Fischer et al., smart watches can play an important role in monitoring personal noise exposure and provide a widely available and cost-effective measure for otoprotection. Additionally, generating fundamental knowledge of the pathophysiological mechanisms underlying noise-induced hearing loss is a further milestone in understanding this unique burden. The development of therapeutic and protective options is aided by an improved understanding of noise-induced hearing loss. Cederholm et al. contributed by utilizing a type III intermediate filament peripherin gene knockout mouse model in a noise-induced hearing setup. The authors demonstrated a clear association between disruption of OHC-type II SGN sensory input, near elimination of MOC efferent-mediated contralateral suppression at moderate to high sound levels, and decreased otoprotection in noise-induced hearing loss.Nowadays, noise-induced hearing loss, resulting from increased daily noise exposure, is a major cause of acquired hearing loss. Awareness of individual excessive noise exposure is an important tool to protect the population from noise-induced hearing loss. As elaborated by Nacher-Soler et al. elaborated on experimental molecular therapeutic access. A robust temporal NOX3 downregulation could be demonstrated by intracochlear delivery of NOX3-siRNAs. Various alternative therapeutic approaches have been evaluated so far. Growth factors have been shown to have a positive effect on the inner ear. This otoprotective branch has been further investigated by Bieniussa et al.. In a special progressive motor neuropathy mouse model, the deterioration of the auditory nerve and the outer hair cells could be diminished by the application of PEGylation insulin-like growth factor. Insulin-like growth factor PEGylation is a modification that prolongs the factor's half-life while maintaining full activity. Aside from studying the direct pharmacological effect of otoprotective agents, another area of focus was drug delivery to the inner ear. In the included review by Barbara et al., the large field of nanoparticles and their applicability in inner ear drug delivery was included. Nanoparticles have been proven to be safe, with no significant decrease in cell viability or signs of ototoxicity. This drug application form includes encapsulation, polymerization, surface functionalization, and enhanced drug release. Dexamethasone and antioxidants represent the most frequently used drugs in the evaluated studies.Aside from such specific approaches, the field of otoprotection includes a variety of therapeutic approaches, ranging from growth factors to modulations of reactive oxygen species-generating enzymes. These otoprotective ideas show the breadth of creativity used in the development of various treatment modes. Reactive oxygen species, like NOX3, have an implicated role in acquired sensorineuronal hearing loss. Unfortunately, no specific pharmacological inhibitor is available so far. In a novel attempt, Wimmer et al. evaluated electrode impedance variability as a potential biomarker for residual hearing. In 42 CI patients, the group associated a 1 kΩ increase with a 4.4 dB deterioration of residual hearing.In addition to drug identification and application strategies, the broad field of otoprotection extends to daily clinical routines, ranging from CI electrode insertion surveillance technologies to improved MRI technologies. Due to the expanding indication criteria for cochlea implantation candidacy, the procedure is now performed with significant residual hearing. Monitoring of residual hearing during the electrode insertion process is another otoprotective focus. This special topic issue includes a variety of publications reflecting the breadth and diversity of otoprotective research.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Colorectal self-expanding metal stent insertion is a useful procedure to treat acute colorectal obstruction caused by advanced colorectal cancer; however, guidewire perforation has been reported to occur as a result of inadequate endoscopic visibilityVideo 1 Stent insertion to treat a colonic stricture using a small-caliber tapered transparent hood.The patient was an 84-year-old woman with acute colorectal obstruction due to advanced colorectal cancer of the sigmoid colon. We inserted a colonic stent to decompress the obstruction. To maintain endoscopic visibility, a transparent hood was placed on the endoscope tip, and endoscopy was performed using an electrolyte-free gel; however, due to the severity of the stricture, endoscopic visualization with the standard hood was restricted . The taUse of the small-caliber tapered transparent hood and gel is a reliable method to maintain adequate endoscopic vision and ensure safe colonic stent insertion.Endoscopy_UCTN_Code_TTT_1AQ_2AF


BackgroundFractures of the skull base occur in 3-30% of head injury presentations to the emergency department. Overall, 9-40% of the cases have temporal bone fractures (TBFs). This fracture may disrupt the intervening structures causing edema, hematoma, bleeding, hearing loss, dizziness, cerebrospinal fluid otorrhea, and facial nerve paralysis. This study aims to evaluate the type of TBF, its correlation with hearing loss, and the outcomes of hearing loss.MethodologyA prospective observational study was done among 50 patients who presented to the emergency department following trauma with clinical features and CT of the temporal bone suggestive of TBF. A complete evaluation of the patients was done, and patients were managed as per the departmental protocol. The patients were followed up for six months and monitored for otological symptoms. Periodic assessment of hearing loss by pure tone audiometry (PTA) was performed at the end of one week, one month, and six months.ResultsThe most common type of fracture in our study was longitudinal TBF (72%), followed by transverse TBF (20%) and mixed TBF (8%). According to the newer classification, otic capsule-sparing fracture was more common than otic capsule-violating fracture. Most patients presented with conductive hearing loss (60%) following the TBF. On follow-up, there was a statistically significant improvement in hearing loss at the end of six months.ConclusionsOur study found that in most cases hearing loss improved over time. Patients with conductive hearing loss showed maximum improvement in comparison to patients with sensorineural and mixed hearing loss. A significant lateral force to the cranium can cause complex injuries of the petrous region resulting in temporal bone fractures (TBFs). These injuries can result from a motor vehicle accident (MVA), high-impact sports, or, less frequently, from trivial falls. The incidence of TBF varies greatly. In polytrauma cases, the incidence has been reported to be 32 per 1,000 cases [As the temporal bone houses several important structures, TBFs can be complicated by facial nerve palsy, cochlear injury, vestibular organ injury, and injury to the carotid artery/jugular vein. Thus, each case of head injury/polytrauma needs to be evaluated for trauma to the temporal bone as this may be frequently overlooked at the initial point of contact in the emergency department (ED). This may be critical in determining the acute as well as long-term management plan.This study aimed to determine the incidence, clinical presentations, types, and outcomes of hearing loss in different types of TBFs.This prospective observational study was conducted in a tertiary care hospital of Mahadevappa Rampure Medical College (MRMC) in the Department of Otorhinolaryngology. This study was conducted over 18 months from March 2021 to August 2022. Ethics approval  was granted by the MRMC Institutional Ethics Committee before recruitment. All participants provided written informed consent.Patients and data collectionPatients irrespective of their age group with a high-resolution computed tomography (HRCT)-diagnosed/confirmed TBF were included in this study. All included patients were either directly referred to our department from the ED or were subsequently referred from the Department of Neurosurgery or Oral and Maxillofacial Surgery. Patients with a history of any hearing loss and neurological insults  were excluded from the study.Case history was noted detailing the specifics of the trauma  and clinical symptomatology. A detailed clinical evaluation was performed including a general physical, otological, and neurological examination including House Brackman’s staging for facial nerve palsy if relevant. The demographic details were also collected.In HRCT, serial 1 mm thick sections, 120 kV, 140 mA, 1 mm interval, 0.8 mm nominal single collimation width and 1.6 mm total beam collimation width, and 0.562:1 pitch were obtained in both axial and coronal planes. Axial images were obtained parallel to the orbitomeatal plane. Coronal sections were obtained at a scanning angle parallel to the vertical ramus of the mandible.Traditional TBF classification system was followed in this study, namely, longitudinal, transverse, and mixed fractures, based on the orientation of the fracture line relative to the long axis of the petrous pyramid ,4. An alWe developed an algorithm for the management of all patients with TBFs to standardize and deliver a consistent line of care Figure .OutcomesThe primary outcome assessed was hearing loss. For up to the initial six months, patients were followed up and monitored for otological symptoms. Hearing loss was assessed by performing a pure tone audiogram (PTA) at the end of the first week, first month, and sixth month. Secondary outcomes included the clinical presentation and any complications such as tympanic membrane perforation, hemotympanum, facial nerve palsy, or cerebrospinal fluid (CSF) otorrhea.Statistical analysisData was analyzed using SPSS version 25.0  software. For quantitative data analysis of descriptive statistics, mean and standard deviation were calculated initially. Independent-sample t-test was used to compare the mean values between two variables for statistical significance. For qualitative data analysis, the chi-square test and Fisher exact probability tests were applied for statistical significance. P-values ≤0.05 were considered statistically significant for all comparisons.A total of 50 patients met the inclusion criteria of HRCT-diagnosed TBF. The age range of the included patients was 12-65 years, and the mean age was 32 (±15) years. There was a male preponderance (80%) in our cohort with a male-to-female ratio of 4:1. MVAs were the most common mode of injury seen in 31 patients (62%)  were OCS fractures while eight (16%) were OCV fractures  patients. Reduced hearing was the second most common symptom at presentation seen in 24 (48%) patients, followed by otalgia in 18 36%) patients, and watery ear discharge in 4 (8%) patients. Other rarer symptoms included giddiness in 8 (16%) and tinnitus in three (6%) patients. Facial palsy was present in two patients at presentation , sensorineural hearing loss (p = 0.017), and mixed hearing loss p = 0.025) with time  patients, 10 (20%) had transverse TBF, and four (8%) had mixed TBF. As per otic capsule involvement, 42 (84%) had OCS fractures while eight (16%) had OCV fractures. Studies by Prasad et al.  and PadmAll our patients were followed up for six months and were evaluated clinically. In the study conducted by Maradi et al. , tympaniIn our study, all four (8%) cases of CSF leaks had resolved with conservative management in about a week. CSF leaks generally heal in two weeks. CSF leaks that persist longer than 10-14 days most likely require surgical repair . In our Conductive hearing loss is the most common type of hearing loss in TBF. This could possibly be due to the presence of blood in the EAC, ossicular disruption, or tympanic membrane perforation. The high rate of hemotympanum and tympanic membrane perforations may be the cause of this high ratio of hearing loss. Among the 17 patients with hearing loss, eight had conductive hearing loss, five had sensorineural hearing loss, and five had mixed hearing. A similar distribution of the type of hearing loss was seen in a study by Abhishek et al. . In anotThe longitudinal fracture traverses the EAC and produces hemotympanum, typically with conductive hearing loss. conductive hearing loss can be due to the hemotympanum itself behind the tympanic membrane, or from tympanic membrane perforation or ossicular chain disruption. Transverse fractures, on the other hand, can compromise the otic capsule and cause profound sensorineural hearing loss. It is possible to sustain direct injuries that disturb the membranous labyrinth or concussions . SensoriLimitations of the study include a small sample size. In the future, a multi-institutional prospective study would yield better quality results. The data derived from such studies may be applicable at the national level to develop a standardized operating protocol for all patients with TBF. A longer follow-up period and more frequent assessment of hearing would have provided a much better picture of the increase/decrease of hearing. The absence of evidence of pre-trauma hearing loss also added to the limitations as our study population included patients more than 60 years of age and may have had some age-related sensorineural hearing loss.TBFs occur due to high-intensity trauma resulting in a wide range of otological manifestations. Our study found that among patients with TBFs, conductive hearing loss was more common than sensorineural hearing loss and mixed hearing loss. Our follow-up showed that in most cases hearing loss improved over time. Patients with conductive hearing loss showed significantly better improvement in comparison to patients with sensorineural and mixed hearing loss. Early intervention in the form of surgery is required in a few cases to avoid worsening of hearing loss and poor quality of life.This study showed that a majority of TBF-associated hearing loss improves over time with conservative management. However, in all cases, neuroimaging plays a crucial role in determining whether active surgical management may be required in some cases for better outcomes.Further studies are required with a bigger sample size and longer follow-up periods. Studies comparing the two classification systems in terms of the degree, severity, and long-term prognosis of each type of hearing loss are also required.


Metabolites, “Resting Metabolic Rate of Individuals”, contains five original articles and one narrative review, which can be organized into four topics: (i) estimation of RMR using predictive equations; (ii) association between RMR and circulating cardiometabolic risk factors; (iii) effects of two different interventions on RMR; and (iv) RMR in persons with narcolepsy.The resting metabolic rate (RMR) represents the energy required to sustain normal body functions and homeostasis in an awake individual under ambient thermoneutral conditions and during rest. The RMR is the most relevant 24-h energy expenditure (EE) component. In fact, the RMR  commonly accounts for 60–70% of the total 24-h EE, making it a potential indicator of future weight (re)gain. This special issue of Regarding the estimation of RMR, Maury-Sintjago et al.  observedTwo cross-sectional studies reported the associations between RMR and cardiometabolic risk factors in middle-aged  and younTwo other studies were published which investigated the effects of exercise   and of wLastly, a review article was included in this Special Issue. Dhafar et al.  conducteThe present Special Issue summarizes the progress on the topic of RMR and human health in different populations, which will be of interest from both clinical and research perspectives. In addition, it highlights the current limitations of certain research areas and the need for more studies to be carried out in order to advance scientific knowledge.