Previous research has suggested that immunoglobulin E (IgE)-mediated events lead to several chronic diseases. We investigated the association between allergic conditions and age-related cataracts in the South Korean adult population.A cross-sectional study was performed using data obtained from 1,170 participants aged 40 years or older who were enrolled in the Korean National Health and Nutrition Examination Survey 2010. Multivariable logistic regression was used to examine the relationship between age-related cataracts and allergic conditions, including total serum IgE and allergen-specific serum IgE levels, after adjustment for potential confounders .P = 0.044). In particular, increased IgE levels were significantly associated with nuclear cataract . However, allergen-specific serum IgE levels did not differ significantly between groups. In the trend analysis, no significant relationship was observed between serum IgE and any type of age-related cataract.After adjusting for potential confounders, the odds ratio (OR) for age-related cataract was greater in participants with higher total serum IgE levels (OR = 1.37; Increased total serum IgE level is independently associated with age-related cataracts after adjustment for confounding factors. Cataract, defined as opacity of the crystalline lens, is the most common cause of visual loss worldwide . MoreoveImmunoglobulin E (IgE) is an antibody that binds to Fc  receptors, which are mostly found on the surface of mast cells . It playIt is well-known that anterior and posterior subcapsular cataracts are a common ocular complication of atopic dermatitis . HoweverIn the present study, we investigated whether increased serum IgE is associated with the prevalence of age-related cataract after adjustment for well-known risk factors . The study protocol was approved by the institutional review board of the Korean Center for Disease Control and Prevention (IRB Number: 2010-02CON-21-C). All participants signed forms consenting to the use of their health information in the study. The KNHANES 2010 was a nationwide, population-based, cross-sectional survey conducted by the Division of Chronic Disease Surveillance of the Korea Centers for Disease Control and Prevention [All analyses were based on the Korean National Health and Nutrition Examination 2010  to evaluate the participants’ cataract status . The staDermatophagoides farinae, cockroach, and dog allergen were also examined using anImmunoCAP100 kit  and a 1470 WIZARD gamma-Counter analyzer  [Demographic information on traditional cataract-related risk factors was obtained during the health interview. Specifically, each participant completed a questionnaire in which they provided information on age, smoking status, alcohol use, sun exposure, and medical history. Blood pressure was also measured by the health professionals. Sunlight exposure time was evaluated by a multiple-choice question with the following answer options: <2 hours, 2–5 hours, and >5 hours per day. Fasting blood samples of individual participants were collected to measure serum biomarkers . The blood samples were immediately refrigerated and transported to the Central Testing Institute in Seoul, South Korea. Levels of total serum IgE and antigen-specific IgE against Finland) . The uppFinland) .2 test  or the Wilcoxon rank-sum test (for continuous data). To assess the association between cataract and increased IgE levels and sensitization to specific antigens, we used multivariable logistic regression to estimate the respective odds ratio (OR). The estimated ORs were calculated in the following ways: crude OR, in which no adjustment was made for potential confounders; Model 1, wherein the data were adjusted for age, sex, body mass index [The characteristics of the participants in terms of cataract status were compared using the χss index , smokingss index , alcoholss index , and sunss index  . We considered P-values < 0.05 as statistically significant. All statistical analyses were completed using the SPSS Statistics 21.0 software .To avoid collinearity between the confounders , we selected systolic blood pressure and total cholesterol level as covariates. When calculating the correlation matrix of all covariates, there was no multi-collinearity problem .Ultimately, a total of 1,170 participants were included in this study. In P = 0.048) and Model 2 . In contrast, sensitization to specific antigens, including Dermatophagoides farinae, cockroach, and dog, was not significantly associated with age-related cataract either before or after adjustment. The possible effects of socioeconomic status on the association between IgE and age-related cataract were also examined. Additional adjustments for household income and education had no effect on the associations reported above, which showed an age-related cataract OR of 1.41 in Model 1 (P = 0.028) and 1.42 in Model 2 (P = 0.028) in subjects with an increased IgE level. Therefore, the effect of socioeconomic status on the association between IgE and age-related cataract was minimal.P = 0.001). Increased IgE remained a significant risk factor for nuclear cataract after adjustment for confounding factors in Model 1  and Model 2 . Sensitization to specific antigens was not a significant risk factor for any type of cataract.Multivariable logistic regression was performed in each cataract subtype group to examine the association of cataracts with both total and allergen-specific serum IgE levels. The OR results are listed in P for linear trend = 0.080). Subgroup analyses were also performed in each age group. As shown in To explore the more specific association between IgE levels and age-related cataracts, we carried out additional analyses involving the quartiles of IgE level. The ORs for each quartile of serum IgE concentration, adjusted for the variables in the fully adjusted model (Model 2), are shown in To our knowledge, this study was the first to evaluate the association between serum IgE levels and age-related cataracts in the general population. Our results demonstrated that study participants with increased total serum IgE levels had a significantly higher OR for age-related cataract after adjustment for confounding factors. In particular, increased IgE was significantly associated with nuclear cataract—the most common form of age-related cataract. However, neither of these findings was true of IgE sensitization to allergens in our regression models. The trend analysis of the ORs did not show a significant linear trend between the risk for age-related cataract and the serum total IgE quartiles. Since the analysis used data from a nationally representative sample of the population and was adjusted for a wide range of confounders, our results support the hypothesis that increased serum IgE levels are associated with age-related cataract.Recently, age-related cataract has been seen as a multifactorial disease that may be related not only to age and exposure to ultraviolet, but also to systemic conditions . PreviouThe possible association between IgE and cataract formation maybe mediated by the mast cell in several ways: the mast cell may aggravate the oxidative status of the lens proteins directly (by activating inflammatory cascades) or indirectly (by increasing vascular permeability). Furthermore, mast cells release several inflammatory mediators, such as cytokines, chemokines, histamine, proteoglycan, and mast cell protease, following IgE stimulation . These mIn the present study, the adjusted multivariable regression models revealed an independent relationship between increased IgE and nuclear cataracts. In our analyses, we adjusted for potential risk factors of age-related cataract; nonetheless, our results may have been influenced by various chronic illnesses associated with both cataract formation and increased IgE level. Recent studies have revealed that IgE-mediated events can lead to several chronic diseases, including not only allergic conditions associated with hypersensitivity, but also metabolic syndrome. In fact, increased serum IgE has been associated with diabetes, hypertension, atherosclerosis, and ischemic heart disease; all these conditions are also related to cataract formation . FurtherDermatophagoides farinae, cockroach, and dog, showed no significant association. The reason for this finding is unknown. Because sensitization to most of these allergens occurs in childhood, the progression of age-related cataracts in old-aged persons may not be closely associated with the process [Considering that increased IgE levels were associated with age-related cataracts, it was surprising that IgE sensitization to specific allergens, including  process . Accordi process . For exa process . Additio process . Overall process . TherefoThis study had certain limitations. Firstly, our analyses did not establish a causal relationship because of their cross-sectional nature. Moreover, our health data was based on a health interview survey taken on one occasion. Fasting plasma glucose, body mass index, blood pressure, and serum IgE may all differ depending on the time of measurement. Secondly, this was a single, Asia-specific study. Generally, the incidence and type of cataract are influenced by genetic background . TherefoIn conclusion, our findings demonstrated that increased total IgE levels significantly associated with age-related cataracts. Specifically, increased IgE was significantly related to nuclear cataracts. Due to the aforementioned limitations, further studies involving a larger sample size and other ethnicities are still needed. In addition, future studies should clarify more specifically the role of IgE in the development of age-related cataracts.S1 Table(DOCX)Click here for additional data file.


To carry out a systematic literature review of the causes of preload loss of the abutment screws, of internal and external connection implants, tightened to different torque values and subjected to cyclic loading.in vitro studies in which internal and external connection implants were subjected to cyclic loads to determine the degree of loosening of the abutment screws after loading.A systematic search was conducted in PubMed, EMBASE, and Cochrane Library databases with reference to The reviewed studies tested distinct implant connections  subjected to diverse cycles (from 16667 to 1 million), with loads ranging from 0-400 Nw, using screws of different materials and designs that were tightened into torques between 20-45 Ncm, Accordingly after loading the percentage of torque loss ranges between 16.1% to 39%.Most of the studies indicate that the internal connection, together with the morse taper, best resists cyclic loading in terms of screw loosening in single-tooth implants. Key words:Dental Implants, Dental Implant-Abutment Design, Torque, In Vitro Techniques, Systematic Review. For many years now, the loss of natural teeth has created a need for tooth replacement for both aesthetic and functional reasons. In 1978, Brånemark and Albrektsson presented, at Harvard University, the results of their fifteen-year-long investigation concerning the integration of titanium in bones, at which time they coined the terms ‘osseointegration’ and ‘the implant-prosthetic complex’ .Initially, osseointegrated implants presented a high success rate (being 84% in the maxilla and 93% in the mandible during a 5-12 year observation period (4); however, soon after, relatively high levels of loosening of the screw abutment were observed (12.7% at 5 years) . This loThe first dental implants were comprised of an external connection system (0.7mm-high hexagon), with internal connection implants appearing later . In the The osseointegrated implant and the prosthetic abutment are joined together by a screw, and have therefore been called a screw joint .Abutment screw stability can be affected by preload, the effect of settling, and screw geometry ,14. PrelFrom a clinical point of view, it is thought that screw loosening is greater in an external connection than in an internal connection, where the incidence of loose screws is 38% in systems with an external hexogon ,16. HoweTorque loosening causes micromovements in the interface to appear that generate both mechanical problems  and biological problems. In the case of biological problems, microspaces that form within the interface permit the colonization of bacteria that can cause mucositis, peri-implantitis and finally implant loss, especially when the implant-prosthesis are subjected to cyclic loads .The clinician should be aware, when selecting the type of implant and torque to be applied, that the abutment screw can be influenced in terms of the biomechanical yield of the implant-prosthesis.in vitro studies related to the loosening of abutment fixation screws subjected to cyclic loads, in actual fact, there are only a few publications which compare the effect of the connection and the effect of the applied torque in the loss of preload in the presence of repeated occlusal loads. This work aims to review in a systematic way the existing literature regarding the conditioning factors of preload loss of the abutment fixation screws, in internal- and external-connection implants, tightened to varying torque values and subjected to cyclic loads.Despite the number of existing in vitro studies where dental implant units, with either internal or external connection, were subjected to cyclic loads to compare the degree of loosening of the abutment fixation screws using the measurement of counter torque.A literature search was conducted regarding articles, written in English, from 1995 to 2016 in relation to To do so, the PubMed, EMBASE, and Cochrane Library databases were searched with different search equations see , using kin vitro studies, in which implant units with internal and external connections had been subjected to cyclic loads, and the degree of the loosening of the abutment fixation screw was subsequently analysed using the measurement of counter torque.The following types of work were excluded from the search: those that did not specify the type of connection used, those whose objective was not to compare internal and external connections, where the implants were not subjected to cyclic loads, those that studied the behaviour of multiple prostheses or only bacterial filtration, and those where the analysis was based on finite elements. The search resulted in the identification of a total of 545 papers, after 70 studies were eliminated due to being duplicated. The abstracts of all 70 articles were reviewed, and 35 of these were selected and fully read. The other 35 articles were discarded since it was determined after reading the abstract that they did not comply with the objective of this work. Finally, 10 articles that fulfilled the criteria of inclusion were selected, as shown in the diagram . In addition, some authors used a thermo-cycler to reproduce the oral environmental conditions with greater accuracy ,20.et al.  and Gold-Tite®  , do impret al.  determin (et al.  concludeTherefore, the material and/or the design of the screws seem to have an influence on preload maintenance, in which a gold screws ,25 with -Settling effect and cyclical loadsThe main cause of screw loosening is the settling effect, which explains why all screws initially suffer from a preload loss of between 2% to 10% , withoutet al. (Siamos et al.  found thet al. ,12,20-24All of the works reviewed, except for the study by Tsuge and Hagiwara , showed Tsuge and Hagiwara  explain Although the reviewed works show internal validity and use a reproducible method, there is still a lack of a standardized methodology in the application of the cyclic loads to the implants. Park et al.  followedAs summary, in the reviewed literature, there seems to exist a certain level of consensus that establishes that the internal connection, together with the morse taper, is the type of connection that is the most resistant to cyclical fatigue in terms of screw loosening in single-tooth implants. However, screw loosening is a multifactorial event that depends, not only in the type of connection, but also in screw design and material, type and design of abutments, passive fit of the prosthetic elements and occlusal forces among others.


Ambra1 is linked to autophagy and neurodevelopment. Heterozygous Ambra1 deficiency induces autism-like behavior in a sexually dimorphic manner. Extraordinarily, autistic features are seen in female mice only, combined with stronger Ambra1 protein reduction in brain compared to males. However, significance of AMBRA1 for autistic phenotypes in humans and, apart from behavior, for other autism-typical features, namely early brain enlargement or increased seizure propensity, has remained unexplored. Here we show in two independent human samples that a single normal AMBRA1 genotype, the intronic SNP rs3802890-AA, is associated with autistic features in women, who also display lower AMBRA1 mRNA expression in peripheral blood mononuclear cells relative to female GG carriers. Located within a non-coding RNA, likely relevant for mRNA and protein interaction, rs3802890  may affect its stability through modification of folding, as predicted by in silico analysis. Searching for further autism-relevant characteristics in Ambra1+/− mice, we observe reduced interest of female but not male mutants regarding pheromone signals of the respective other gender in the social intellicage set-up. Moreover, altered pentylentetrazol-induced seizure propensity, an in vivo readout of neuronal excitation–inhibition dysbalance, becomes obvious exclusively in female mutants. Magnetic resonance imaging reveals mild prepubertal brain enlargement in both genders, uncoupling enhanced brain dimensions from the primarily female expression of all other autistic phenotypes investigated here. These data support a role of AMBRA1/Ambra1 partial loss-of-function genotypes for female autistic traits. Moreover, they suggest Ambra1 heterozygous mice as a novel multifaceted and construct-valid genetic mouse model for female autism. Causes likely converge at the synapse, as indicated by mutations of synaptic genes or by mutations causing quantitative alterations in synaptic protein expression, half-life or degradation, and are reflected by a virtually autism-pathognomonic neuronal excitation–inhibition dysbalance.15 Neuroligin-4 mutations, for instance, belong to the most common causes of monogenetic heritable autism.16 Construct-valid and face-valid mouse models of autism, building on monogenetic grounds, have helped in approaching the underlying common biology.18Autism spectrum disorders (ASD) are extremely heterogeneous neurodevelopmental conditions, affecting ~1% of the population. Typical, shared symptoms range from social communication and interaction deficits, including decreased attraction by and compromised reading of social signals, restricted interests, repetitive behaviors or pronounced routines, to reduced cognitive flexibility.3 Importantly, normal genetic variants, mainly single nucleotide polymorphisms (SNPs), likely contribute to the manifestation of autistic phenotypes. This is indicated by the results of genome-wide association (GWAS) and respective polygenic-risk studies on autism,20 but even more so by phenotype-based genetic association studies (PGAS), reporting an accumulation of ‘unfortunate’ normal variants, so-called pro-autistic genotypes, to be associated with increasing severity of autistic traits.23 In fact, phenotypical continua of autistic features from health to disease suggest underlying mechanisms of quantitative rather than qualitative nature.24 Genetic modifiers, like protective genes and environmental (co-)factors, mainly those acting during uterine and early postnatal development may also modulate autism severity.26The estimated heritability of autism approximates 90%. We note, however, that monogenetic forms including copy number variations altogether account for <20%, leaving ~80% of cases unexplained, which also enter the final common pathway of disease expression.27 Remarkably, little research has focused on the reasons for this disparity. Better understanding of this difference, however, could lead to major advancements in the prevention or treatment of ASD in both genders.28 We previously reported that an Ambra1 (activating molecule in Beclin1-regulated autophagy) partial loss-of-function genotype is associated with the autism-like behavior in female mice. The restriction to the female gender of autism generation by a defined genetic trait has thus far remained unique.29 Ambra1 is a positive regulator of a principal player in autophagosome formation, Beclin1. Importantly, autophagy has already been linked to autism in recent work.31 Moreover, Ambra1 is involved in other developmentally relevant processes in the nervous system and in neuronal function.33 Although homozygosity of the Ambra1 null mutation causes embryonic lethality, heterozygous mice with reduced Ambra1 expression appear completely normal at first view.33 Only upon comprehensive behavioral characterization, a striking autism-like phenotype of Ambra1+/− females emerges. This trait is quantifiable by the autism severity composite score, which even allows a behavior-based genotype predictability of >90%.34 As first mechanistic hint explaining the prominent gender difference, stronger reduction of Ambra1 protein in the cortex of Ambra1+/− females was found.29ASD has an overall gender distribution of ~4:1 males/females.AMBRA1 genotypes with autistic features has been described in humans, therefore still questioning the construct-validity of our mouse model.29 However, a recent GWAS on schizophrenia identified a genetic risk variation on chromosome 11 (11p11.2) in a region containing AMBRA1.35 Schizophrenia and ASD show considerable syndromic overlap, including deficits in social cognition and communication,36 and at least a subgroup of schizophrenia is also regarded as a disorder of the synapse.37Until now, no association of AMBRA1 genotypes would emerge in humans, thereby supporting the construct-validity of our Ambra1+/− mouse model. In addition, we aimed at defining potential further characteristics of ASD in Ambra1+/− mice, namely (1) decreased interest in social odors, as highly relevant social signals in mice, (2) increased epileptic predisposition as in vivo readout of neuronal excitation–inhibition dysbalance and (3) early brain enlargement, as recognized in human autism. Indeed, we show here that also in humans, an AMBRA1 genotype, the intronic SNP rs3802890-AA, located in a long non-coding (lnc) RNA, is linked to autistic features and characterized by partial loss-of-function in females. Moreover, we demonstrate in Ambra1+/− mice prepubertal brain enlargement. Only in female mutants, we see loss of interest in sex pheromones and altered seizure propensity.The present study has therefore been designed to explore whether any autism-relevant phenotype association with normal In all experiments, the experimenters were unaware of genotypes (‘fully blinded’).38), recruited across Germany since 2005.40 Diagnosis is based on a comprehensive examination, lasting for at least 4 h . It is guided by the GRAS manual, which contains standardized interviews, psychopathology and neuropsychology testing. Moreover, careful study of all the medical discharge letters and charts of every single individual aids in assessing longitudinal aspects of the diagnosis as well.40 GRAS, complying with Helsinki Declaration, was approved by Ethics Committees of Georg-August-University, Göttingen, Germany, and participating centers. All study participants (European-Caucasian 95.6% other 1.8% unknown 2.6%) and, if applicable, legal representatives gave written informed consent. Of the 1105 successfully genotyped patients, 66.7% were male (N=737), 33.3% female (N=368), aged 39.46±12.58 years (range: 17–79). For genetic case–control analysis, healthy GRAS blood donors were employed,39 in total N=1258 (European-Caucasian 97.8% other 2% unknown 0.2%), 61.6% male (N=775), 38.4% female (N=483), aged 37.45±13.21 (range: 18–69) years. Voluntary blood donors widely fulfill health criteria according to the national guidelines for blood donation, ensured by a broad pre-donation screening process containing standardized questionnaires, interviews, hemoglobin, blood pressure, pulse and body temperature determinations.39The Göttingen Research Association for Schizophrenia (GRAS) data collection consists of >1200 deep-phenotyped patients, diagnosed with schizophrenia or schizoaffective disorder  years, N=1144 males, N=1215 females, from baseline examinations of Study of Health in Pomerania (SHIP), approved by the Ethics Committee, University Greifswald, and conducted in North-East Germany.41The general population sample comprises 42-based autism severity score (PAUSS)24 with slight modifications , higher score values  represent higher social support, that is, lower autistic features.For quantification of autistic phenotypes, we used the Positive and Negative Syndrome Scale (PANSS)ications , availab45 Markers were selected according to our SOP for PGAS23 using following selection criteria: (1) SNPs in Hardy–Weinberg equilibrium; (2) SNPs with minor allele frequency (MAF⩾0.2) allowing for statistical analyses; (3) SNPs not in high linkage disequilibrium (LD) with other selected SNPs (r2<0.8). Based hereon, only rs3802890-A/G remained for analysis.GRAS subjects were genotyped using semi-custom Axiom MyDesign Genotyping Array , based on a CEU  marker backbone, including 518 722 SNPs, plus custom marker-set of 102 537 SNPs. Genotyping was performed by Affymetrix on a GeneTitan platform with high quality .46 against 1000-Genomes (phase1v3) reference-panel using 869 224 genotyped SNPs.41 Rs3802890 was imputed with IQ=1.SHIP-0 subjects were genotyped using Affymetrix Genome-Wide SNP Array-6.0 (genotyping efficiency 98.6%). Imputation of genotypes was performed with software IMPUTE v0.5.047 and evaluated including detailed genome and transcriptome mapping. Expression of the rs3802890-containing RNA region was derived from latest largest collection of ESTs available at NIH.48 LncRNA matches were also established according to latest human lncRNA release at NCBI. RNA folding used RNAfold.49 For demonstrating rs3802890-A/G differences, thermodynamic ensemble structures drawing encoded base-pair probabilities were used. Protein binding regions were calculated using RNAanalyzer50 and CatRapid,51 coding potential was calculated using Genscan.52Genome sequences were established according to latest available releases . Iterative sensitive sequence comparisons were conducted, 1 μg of cDNA was applied using a mixture of oligo(dT)/hexamers, dNTPs, DTT and 200U SuperscriptIII . AMBRA1 RNA expression was measured using quantitative real-time PCR. The cDNA was diluted 1:25 in 10 μl reaction-mix, containing 5 μl of SYBR-green (Life Technologies) and 1pmol/primer:Peripheral blood mononuclear cells (PBMC) were isolated from morning blood, obtained via phlebotomy into CPDA-vials , applying standard Ficoll-Paque-Plus isolation procedure . Total RNA extraction was done using miRNeasy Mini-kit . For reverse transcriptionAMBRA1-Fw: 5′-GACCACCCAATTTACCCAGA-3′AMBRA1-Rv: 5′-GATCATCCTCTGGGCGTAGTA-3′GAPDH-Fw: 5′-CTGACTTCAACAGCGACACC-3′GAPDH-Rv: 5′-TGCTGTAGCCAAATTCGTTGT-3′AMBRA1 expression was calculated using the threshold-cycle method (LightCycler480 Software1.5.0SP3-Roche) and normalization to GAPDH. EST TCAAP2E6309 RNA expression was measured using traditional PCR. Extracted RNA, cDNA synthesized with oligo-dT primers with/without hexamers, or genomic DNA were used as template with the following primers:Technical triplicates were run on LightCycler480 . Relative EST TCAAP2E6309-Fw: 5′-GGCAGAGCAGAATGGATAGACA-3′EST TCAAP2E6309-Rv: 5′-AACGCCTGTTATCTGGGATCA-3′Investigations were carried out in agreement with guidelines for welfare of experimental animals, issued by the Federal Government of Germany and Max Planck Society, approved by local animal care and use committee .Ambra1 mutation in mice is caused by a truncated, non-functional Ambra1 protein via insertion of a gene-trapping vector into the murine Ambra1 gene.33Ambra1 wild-type  and heterozygous (Ambra1+/−) littermates of both genders with >99% C57BL/6 N background were used . Genotyping was performed as described.29 Males and females were kept in separate ventilated cabinets , group-housed, with woodchip bedding and nesting material, 12 h-light-dark cycle, 20–22 °C, food/water ad libitum.Ambra1+/+ and Ambra1+/− mice were separately group-housed in large type4 cages after weaning until age 8 weeks. After transponder implantation, they were put into intellicages , placed inside standard laboratory rodent cages  with floor covered by sawdust bedding.53 Each intellicage contains four housing shelters beneath the food hopper. Left and right of the intellicage, two social boxes are connected via plastic tubes, each equipped with two ring RFID-antennas to track individual mice. IntelliCage software records time spent in social boxes. Experiments are performed during the light phase. After habituation for 1 h to social boxes containing fresh bedding, mice undergo the pheromone-based social preference test: for 1 h they can freely choose between a social box with used bedding of C3H mice of opposite gender and another box with only fresh bedding. WT mice typically prefer used bedding containing pheromones.For pheromone-based social preference test, eff=55 ms, 12 differently phase-encoded echoes, 56 min measuring time) at isotropic spatial resolution of 100 μm. From these datasets, polygonal surface models of selected brain structures were generated by importing DICOM images into AMIRA . Structures of interest  were manually and semi-automatically labeled with segmentation editor on three-dimensional label fields .Mice were anesthetized (5% isoflurane), intubated and kept at 1.75% isoflurane/5% oxygen by active ventilation with constant respiratory frequency . Magnetic resonance imaging (MRI) was performed at 7 and 9.4T . Radiofrequency excitation and signal reception were accomplished with use of a birdcage resonator  and a four-channel phased-array surface-coil, respectively. T2-weighted MRI data were acquired with three-dimensional fast spin-echo MRI sequence . After injection, mice are observed for 30 min in their home cage.54 Response to PTZ injection is quantified: (1) hypoactivity: decrease in mobility until rest in crouched or prone position, abdomen at bottom; (2) partial clonus (PC): clonic seizure in face, head or forelimbs; (3) generalized clonus (GC): sudden loss of upright posture, whole-body clonus including all limbs and tail, rearing and autonomic signs; and (4) tonic-clonic seizure (TC): generalized seizure up to tonic hind-limb extension and death. Latencies to (2)–(4) are used to calculate individual seizure scores (ISS), where factors weight relative severity: ISS=1000/(0.2 × PC-latency+0.3 × GC-latency+0.5 × TC-latency).57Mouse groups were tested during the light phase at postnatal day 23 or at 13 months. Seizures were induced by single intraperitoneal injection of pentylentetrazol (PTZ) , STATA MP-v.13.1  and Prism4 . Statistical tests used are always given in figure legends. Data are presented as mean±s.e.m., statistical significance was set to P=0.05.Case-control analysis and test for deviation from Hardy–Weinberg equilibrium was performed using PLINK1.07.23—suitable SNP, AMBRA1-rs3802890-A/G, was available in our array. Case–control analysis  yielded comparable genotypic and allelic chi-square comparison . Thus, rs3802890 is not associated with the schizophrenia diagnosis.Only one directly genotyped and—according to our PGAS SOP24 as quantitative measure of autistic traits . Interestingly, when separating genders, the PAUSS association remains significant only for females and, likewise, most PAUSS sub-items show this trend in females but not males  with PAUSS in the discovery sample (GRAS) yielded a high negative Spearman rank correlation  versus male GG (N=33) carriers had higher expression levels , AA carriers of both genders  did not differ in their level , which was comparable to that of male GG carriers. Comparing both genders, AMBRA1 mRNA expression in PBMC of AA (risk SNP) relative to GG carriers is reduced in females but not males  brain dimensions in Ambra1+/− versus WT mice. Whole brain and hippocampus were enlarged in male and female mutants at postnatal day 23 (around puberty). Cerebellum was increased in female Ambra1+/− mice only  from brain dimensions.Brain enlargement has been described both in children and adults with ASD.ice only . Sizes omensions . We note10 Most likely, seizure predisposition reflects the autism-pathognomonic neuronal excitation–inhibition dysbalance.15 In our Ambra1+/− model, prepubertal female mutants displayed reduced response to PTZ, namely longer latency to the first whole-body seizure and decreased seizure score compared to female WT  carriers, may suggest an underlying autism-causing mechanism similar to that in heterozygous mice where Ambra1 reduction was stronger in female than male mutant cortex.29We previously reported in female 15 still remains unanswered. We may, however, speculate that reduced autophagy at synaptic terminals,63 likely more pronounced in Ambra1+/− females,29 influences synaptic protein turnover and function in a gender-specific manner. In fact, females may be particularly sensitive to reduced autophagy as suggested also by a recent paper reporting higher basal autophagy activity in the brain of neonatal female as compared to male rats.64 In this sense, AMBRA1/Ambra1 adds to the number of proteins shown to underlie sexually dimorphic effects on the brain.66The question of how AMBRA1/Ambra1 reduction may influence synaptic function, thereby causing the autism-pathognomonic neuronal excitation–inhibition dysbalance,in silico search for mechanisms, we saw that the lncRNA, covering the SNP region, shows highly diverse folding upon presence of G versus A allele. This pronounced structural effect may influence AMBRA1 mRNA and/or protein stability and will be subject for further investigation.In a first Ambra1+/− mouse model, we extend our earlier findings29 to crucial, additional autistic features, so far not systematically addressed in genetic models of autism, namely early brain enlargement, altered propensity towards epileptic seizures and reduced pheromone preference.Returning to the 8 Already upon first description of autism, increased size of the head was observed in affected children.67 The substrate underlying the enlarged brain has remained obscure, and the Ambra1+/− mouse model may now help to approach this question. Interestingly, we found Ambra1+/−-associated brain enlargement in both genders, thus uncoupled from the predominantly female behavioral phenotype. This finding may be important in connection with recent suggestions, based on genetically undefined autistic children, where brain volume overgrowth was linked to the emergence and severity of autistic social deficits, predicting a later autism diagnosis based on MRI deep-learning algorithms.7 This obvious discrepancy should stimulate further investigations considering gender, genetics and biological ASD subgroups.Brain enlargement is a consistently reported feature in human autism, both in adults and children.10 and mirror neuronal excitation/inhibition dysbalance.15 Also here, Ambra1+/− mice revealed a striking sexual dimorphism and may serve as future model to study and treat autism-associated epilepsy.Epileptic seizures are frequently observed, not only in syndromic autism, where they are often intractable,68 We note, however, that compared to humans, pheromone preference in mice represents a more prominent component of their social behavioral repertoire, thus more vulnerable to be disturbed in autism-like phenotypes of this species.Olfactory deficits in human autism have been reported, even though the literature is scarce, heterogeneous and inconsistent, likely reflecting subject selection and assessment biases or other methodological limitations, including statistical power issues.AMBRA1/Ambra1 gene for autistic traits across species. In humans, it will for instance be interesting to systematically screen ASD patients for AMBRA1 mutations, particularly female autists. Ambra1+/− mice may serve as a novel multilayered construct-valid genetic model of human autistic phenotypes.To conclude, our data suggest a fascinating sexual dimorphism regarding the role of the autosomal


Mechanosensing is essential for several physiological functions including touch and pain sensations, osmoregulation, and controlling the myogenic tone of resistance arteries. Understanding how mechanosensitive ion channels (MSCs) are gated can provide important information regarding these processes. We have previously demonstrated that during pathological conditions such as polycystic kidney disease, polycystin 2 (TRPP2) inhibits the activity of potassium-selective MSCs through a filamin A-mediated cytoskeletal effect, and renders tubular epithelial cells susceptible to apoptosis. However, the nature of this cytoskeletal inhibition remains poorly understood. In this study we use a combination of electrophysiology, structured illumination microscopy, and fluorescence recovery after photobleaching (FRAP) to examine the dynamic nature of the TRPP2-mediated cytoskeletal inhibition of the potassium-selective MSC TREK1. Our data indicate that this inhibition of MSC activity occurs through an accelerated cytoskeletal inhibition, and ultimately decreases the open probability of the TREK1 channel. These results shed light on a novel mode of regulation of MSCs gating, which may be at play in several physiological functions. The rapidity of this process suggests mechanosensitive ion channels (MSCs) are a central part of this effect. These channels are implicated in a series of essential physiological functions such as our senses of touch and hearing8, as well as the myogenic tone of resistance arteries9, baroreflex function10, and our ability to regulate hydromineral homeostasis13. Because of the importance of these functions, the activity of MSCs must be tightly regulated. One of the most important regulators of MSCs is the cytoskeleton22. Both the actin and tubulin cytoskeletons have been shown to modulate mechanosensory processes23. In epithelial cells, MSCs have been shown to be under tonic inhibition by both the F-actin and the tubulin cytoskeletons, and disassembly of these networks leads to significant increases in channel activity9. In neurons, on the other hand, the cytoskeleton appears to be necessary for the coupling between mechanical stimuli and channel gating. Indeed, rigidification of the cytoskeleton enhances the sensitivity of the mechanotransduction process, whereas cytoskeletal filament depolymerization uncouples mechanical stimuli from channel activation23. These modulatory effects can occur either via a direct interaction between the cytoskeleton and the channel subunits24, or by the cytoskeleton binding to the membrane near the channel and controlling lateral membrane tension or curvature25.The ability to perceive mechanical cues from our external or internal environments occurs through a process called mechanotransduction, during which mechanical stimuli are rapidly converted, often in microseconds26 that exerts a greater inhibitory effect on the open probability (Po) of MSCs9. This geometry can reduce the membrane radius of curvature and decrease the lateral tension in that domain, therefore effectively reducing channel Po9. We have previously reported that when TRPP2 separates from its binding partner polycystin 1, it can recruit FLNa to the plasma membrane, where the latter will indirectly reduce the activity of MSCs present in the membrane9. Importantly, this structural rearrangement only reduces the channels’ Po, not their absolute number in the membrane. This was demonstrated using the polymodal MSC TREK1, which can be activated by both mechanical stimuli and intracellular acidosis27. When TREK1 is co-expressed with TRPP2, its sensitivity to mechanical stimuli is significantly reduced18. However, its membrane expression and response to intracellular acidosis remain intact18. The nature of this cytoskeletal inhibition of channel activity, whether it is a static rigidification of the cell cytoskeleton or a dynamic recruitment of cytoskeletal components, remains poorly defined. Understanding the nature of the cytoskeletal modulatory effect is essential for a better understanding of how these channels contribute to health and disease. Here, we use a combination of electrophysiology, imaging with chimeric FLNa, structured illumination microscopy (SIM), and FRAP approaches to examine the nature of the inhibitory effect of FLNa and the F-actin cytoskeleton on TREK1, a potassium-selective MSC.In the case of the F-actin cytoskeleton, its effect on regulating channel activity can differ based on the geometrical arrangement of its filaments. Indeed, it has been proposed that in the presence of the actin crosslinker filamin A (FLNa), fibers are assembled into an orthogonal network18. Here we examined whether this inhibitory effect occurs through a dynamic process in which the F-actin cytoskeleton (CSK) not only inhibits channel activity, but does so at an accelerated rate. If this is the case, removal of the CSK would enhance channel activity, and if allowed to reform, the F-actin inhibitory effect would set in faster than in the absence of TRPP2. To examine this, we subjected the membrane patch during our cell-attached recordings, to repeated negative pressure pulses . To examine the localization of FLNa independently of the endogenous protein, we generated a stable cell line by expressing FLNa-RFP in the M2 cell background , we saw no difference in the recruitment of F-actin filaments to the subcortical cytoskeleton , painful mechanical stimuli are converted into depolarizing membrane potentials by non-selective cationic MSCs, which produce a depolarization large enough to activate voltage-gated ion channels at the action potential trigger zone31. This depolarizing effect can be blunted by the simultaneous activation of potassium-selective MSCs that tend to hyperpolarize the nerve terminal in response to mechanical stimuli. Thus mice lacking the TREK1 channel show a lower threshold to mechanical stimuli32. The contribution of these channels is also highlighted during inflammatory pain, in which inflammation causes a decrease in the expression of TREK1 and is associated with reduced thresholds to mechanical stimuli34. Alternatively, the expression level of these potassium-selective MSCs may be unaltered, but their gating by mechanical stimuli can be impaired. This has been demonstrated for epithelial cells of the proximal convoluted tubules which, during the cystogenesis that occurs as a result of polycystic disease, get compressed and undergo apoptosis. These cells normally express a functional mechanosensitive TREK2 ion channel which, during autosomal dominant polycystic kidney disease (ADPKD), becomes inhibited through a TRPP2-FLNa-dependent mechanism. It is therefore essential to better understand how the gating of MSCs can be modulated by accessory proteins such cytoskeletal elements.Mechanosensitive ion channels are a central part of several physiological processes, such as our senses of touch and pain20. We reasoned that if the cytoskeletal inhibition of TREK1 were a static phenomenon, then removing it by continuously stimulating the membrane with mechanical stimuli would prove to be difficult, and once the cytoskeleton was damaged, it would take a long time for it to reassemble and re-impose its inhibitory effect. However, if the inhibition were dynamic, we should be able to easily remove it and observe the time course at which it comes back. Our data demonstrates that within 10 seconds of stimulation at 2 Hz, we can remove most, if not all, of the inhibition caused by TRPP2 expression. Interestingly, the rate at which this inhibition returns is significantly faster in cells expressing TRPP2. This accelerated return of inhibition depends on the expression of FLNa in the cell, and is likely due to the membrane-targeting effect of TRPP2 on FLNa. Indeed, the subcellular localization of FLNa indicates a significant shift to the plasma membrane when TRPP2 is expressed. Filamin A changes the geometry of the F-actin network to a more orthogonal structure, thereby decreasing the directional dependence of fibers. This anisotropy can be measured by using super-resolution imaging of phalloidin-stained actin filaments. Our data indicate that A7 cells transfected with TRPP2 have a decrease in anisotropy compared to Mock-transfected cells. This decreased anisotropy is absent in TRPP2-transfected M2 cells, compared to Mock-transfected M2 cells.In this study, we examined the mechanisms through which TRPP2 can block the mechanical activation of TREK1. More specifically, we examined the dynamics of this inhibition and the contribution of the F-actin CSK to this inhibition. To examine the dynamic nature of the CSK inhibition, we designed an experimental approach derived from previously-published observations2 with the circle’s widest region on the membrane. It is therefore possible that the FRAP effect occurred only in a restricted area under the membrane and photobleaching a larger area diluted fluorescence recovery. Finally, (3) it is possible that the absence of a faster FRAP is accurate, and the F-actin filaments in Mock or TRPP2-transfected cells reassemble at the same rate, only that in TRPP2-expressing cells, this reassembly occurs with an orthogonal geometry, which has a more efficient inhibitory effect on MSC gating.We examined whether the accelerated recovery of inhibition was due to an increase in speed at which F-actin filaments are turned over. To test this hypothesis, we performed FRAP experiments on plasma membranes of Mock and TRPP2-transfected cells, and observed no difference in the two groups. The absence of a difference in FRAP time course between the two groups can be explained by several factors: (1) The cytoskeletal inhibition could be occurring within the microdomains where TREK1 is located instead of being present throughout the membrane. (2) Alternatively, the absence of a FRAP could be due to the large size of the field of view. Indeed, to efficiently photobleach a circular field, the minimum zone diameter had to be 6.53 μmBecause TREK1 is widely expressed in the nervous system, and has several mechanosensory functions, understanding how its gating is regulated may prove useful in the development of future therapeutic tools aimed at reducing its inhibition by the cytoskeleton.The fibroblast-like COS-7 cell line derived from African green monkey kidneys were used in addition to the human melanoma derived M2 and A7 cell lines. Cos-7 cells were maintained with Dulbeco’s Modified Eagle’s Growth Medium  supplemented with 10% Fetal Bovine Serum and 1% penicillin/streptomycin. M2 and A7 cells were maintained in Eagle’s Minimum Essential Medium  supplemented with 2% Newborn Calf Serum, 8% Fetal Bovine Serum, and 1% penicillin/streptomycin. A7 cells were additionally grown with 200 μg/mL of geneticin (Wisent) to maintain stable expression of Filamin A. One day prior to transfection, cells were plated at ~50% confluency onto 35 mm plastic dishes. All constructs were transfected using FuGene 6 (Promega) according to manufacturer’s instructions using 2 μg of DNA. Cells co-transfected with two plasmids received 1 μg of each plasmid. One day following transfection, cells were plated onto 35 mm glass bottom dishes. All recordings were performed 48 hours after transfection.Cell-attached recordings were performed on transiently transfected COS7, M2, and A7 cells. The extracellular recording medium contained 155 mM KCl, 5 mM, EGTA, 3 mM MgCl2, and 10 mM HEPES . The pipette solution contained 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, and 10 mM HEPES . The pipette solution also contained 10 mM Tetraethyl-ammonium (TEA), 5 mM 4-Aminopyridine (4AP), and 10 μM glibenclamide to block contaminating potassium channels. After achieving a gigaseal in the cell-attached configuration and voltage clamping at 0 mV, membrane patches were stimulated with repeated 200 ms pressures pulses (−70 mmHg at 2 Hz) through the recording electrode/pipette setup using a high-speed pressure clamp device (ALA Scientific Instruments). After the 10 s stimulation, the membrane was allowed to rest for 2 s, 15 s, and then 30 s before subsequent pressure pulses were given. All recordings were performed with an Axon MultiClamp 700B amplifier (Molecular Devices) using non-coated fire polished glass pipettes (1.4–2.4 MΩ). Clampex 10.3 (Molecular Devices) was used for data acquisition. Data analysis and figure preparation was performed using Clampfit 10.3 (Molecular Devices), Microsoft Excel, and Prism 6 (GraphPad Software).2 area on the cell membrane. Images were captured with MetaMorph (Molecular Devices) before and for ~17 s after photobleaching to acquire fluorescence recovery at the cell membrane. Data was corrected for the overall bleaching of the background signal resulting from image acquisition. Fluorescence data was fit to an exponential function to extrapolate the recovery time constant using Prism 6 (GraphPad Software). The mobile fraction (MF) was calculated by taking the ratio of fluorescence signal that recovered post photobleaching  to the the total fluorescence signal that was photobleached (Fpre − Fpost).Fpre is the fluorescent signal pre-photobleaching; Fpost is the fluorescent signal immediately post-photobleaching; and Ffinal is the final fluorescent signal.FRAP experiments were performed on a spinning disk confocal microscope  using a 63x objective. A 100 ms laser pulse was used to photobleach a 6.53 μmThe S8 cell line was generated by stably transfecting M2 cells with a red fluorescent form of Filamin A . S8 cells transiently transfected with TRPP2 or a mock control were fixed with 4% paraformaldehyde and then treated with WGA-350 to label the plasma membrane. Images were captured at 63x on a spinning disk confocal microscope . The WGA-350 signal was used as a mask to determine the Filamin A-RFP signal at the membrane, which was then normalized to the overall Filamin A-RFP cell intensity. ImageJ  was used to perform data analysis.35. The cell anisotropy values were measured by analyzing 4μm thickness of actin staining just under the plasma membrane.Transiently transfected M2 and A7 cells were fixed with 4% paraformaldehyde and treated with phalloidin-488 (Cytoskeleton) to stain actin filaments. Super resolution images were collected using an OMX V4 microscope (Applied Precision/GE Deltavision) using a 100x objective. OMX and softWoRx softwares (Applied Precision/GE Deltavision) were used for image acquisition and structured illumination reconstruction, respectively. Analysis of SIM data was performed using ImageJ (NIH) and the plugin FibrilTool to calculate anisotropy, the property of being directionally dependent, amongst actin filamentsResults are represented as mean ± SEM. Statistical significance was tested using unpaired t-tests for comparison of means, or two-way ANOVA for comparisons of groups over time. Differences were considered significant for p < 0.05 (*).


Both spondylolysis and spondylolisthesis come in second place in the causes of pain among athletes. Treatment options include both conservative management and different operative methods. Athletes and adolescents are groups where the priority is to protect tissues from perioperative damage.We present our modification of the Buck’s, direct pars repair method, which we believe offers maximum protection of tissues. We used the modified surgical method in young, competitive athletes, in whom non-surgical treatment was not effective.Eight pars defects in five patients were treated using suggested method. All of them were young males (aged between 13 and 18 years), who practice soccer professionally. We use modified method of direct repair pars through the cannulated screw fixation, first proposed by Buck. Preoperative preparation consists of proper analysis of computer tomography images in multiplanar reconstruction mode: measuring screw length, measurement of inclination angle of the optimal screw trajectory in the frontal and sagittal plane. During the operation, the wire proper direction is performed by usage of the predetermined angles. Starting point for guide wire was also changed to the lower end of the facet. The fusion takes place with a screw of 3 mm diameter. After the operation patient need to use thoracolumbar spinal orthosis as a primary immobilization for 6 weeks and appropriate rehabilitation for another 6 weeks. We used these methods in eight pars fixations.All of the patients were painless in first week after surgery. All of them underwent total rehabilitation programme and returned to sport.Direct pars repair using Buck’s method with proposed modification, including adequate radiographic preparation, the use of a thin cannulated screw and changing the point of screw entry, allows precise and safe screw placement, regardless of the size of the bone at the defect site. Both spondylolysis and spondylolisthesis comes in second place in the causes of pain among athletes . This prDetermining the optimum path for screw insertion in preoperative planning in order to minimize the chances of improper entry of the implant.Maximizing the protection of soft tissues .Providing a safe margin of bone around the screw.Enabling an early return to full physical activity.Enabling proper screw placement without usage of computer tomography during surgery to avoid overdose of radiation in young athletes.To develop a method of:The indication for surgery was local pain in the lumbar region, coinciding with the place of occurrence of spondylolysis, which persists at least 3 months despite typical conservative treatment. The pain was present when typical moves were performed , only during a very intense physical activity. Activities of daily living did not trigger any discomfort. Each patient underwent magnetic resonance imaging as part of the diagnostic process, which has not revealed any changes that may be responsible for reported problems. We obtained written informed consent from parents of the minors in each case.Before surgery, standard computed tomography is performed. Then, in the multiplanar reconstruction modes, planes of maximum width of the pars interarticularis are determined Fig. . This alDuring the operation, the patient is positioned prone on a translucent operating table and a C-arm X-ray is used throughout the whole procedure. A marker wire is placed on the patient’s back and an anterio-posterior (AP) view is taken over the pars interarticularis, at the predetermined angle, and a line is drawn on the skin along the wire Figs. , 6.Fig. Another wire is bent at a right angle and placed on the patient’s back so that one arm is perpendicular to the long axis of the body and the other is in line with the pars as seen on the lateral view. A line is then drawn along this wire with a skin pencil. The intersection of the lines is the skin entry point Figs. , 8.Fig. Skin and fascia incisions are made. Since 1.1-mm wire is very flexible and is virtually impossible to keep it straight at the desired angle, a cannulated drill with the wire serving as stylet is used. When the tip of the drill crosses the defect, the wire stylet is advanced further. As the tip of the wire reaches the upper facet, the drill is removed and a cannulated screw is inserted over the wire. Drilling, wire insertion and screw advancement are performed under a radiologic oblique view.The patient is positioned prone on the translucent operating table. The pars defect is localized with the C-arm, in AP, lateral and oblique from 30 to 40 degrees planes, adapted to each operated pars, calculated on the basis of the computer tomography image.A skin incision approximately 1–1.5 cm long is made. The cut in the fascia should be slightly longer cephadally relative to the skin incision. This allows manoeuvring of the guide wire and drill. The guide wire, 1.1 mm diameter, is inserted through the skin incision. Unlike the original Buck method, where the screw was placed from the lower edge of the lamina, in our method the starting point is located at the bottom of the lower facet. Therefore, instead of the original method, it is moved slightly laterally. After the initial entry of the guide wire, it is positioned at the correct angle indicated by the computer tomography. Due to the high flexibility of the guide wire, transmission and maintenance of the proper trajectory can be improved with the use of a drill. After passing the drill and wire through the defect, the drill is withdrawn and the wire is left in place to drive the screw. Fusion is performed using a titanium, partially threaded, self-tapping, 3-mm-diameter screw. The length of the screw is predetermined from the computer tomography.All patients underwent postoperative radiological follow-up. Location of screws in both the frontal and sagittal plane was correct: screw ran through the previously predetermined trajectory without exceeding the bone border. Due to the age of patients control CT examination was done in two patients Fig. .Fig. 9PoFor 6 weeks after surgery, the patient uses a thoracolumbar spinal orthosis (TLSO). Flexion, hyperextension and rotation should be avoided. After the TLSO is discarded, the patient should perform isometric exercises of the abdominal muscles for 2 weeks. Over the next 2 weeks active exercises of the back and isometric exercises of the abdomen (without moving the lumbar spine) shall be performed and over the following 2 weeks active exercises of the back and aerobic exercise (e.g. cycling) should be done. So after 12 weeks the patient can return to normal physical activities, appropriate for the sport concerned. As in other articles about spondylolysis [We obtained approval for this study from an ethics committee of Poznan University of Medical Sciences. To date, eight pars defects in five patients were treated with the proposed method. All were males who professionally practice soccer. The age range was 13–18 years. In three cases, spondylolysis occurred unilaterally, on one level. In one case, spondylolysis had occurred on both sides on two levels. In one case, spondylolysis occurred on both sides on one level. Optimum screw location was achieved in all cases, i.e. passing from the base of the lower facet to the top of the upper facet without piercing the cortical bone. Pain levels in the first postoperative day were 2–3 on the visual analogue scale (VAS). All the patients were discharged on the first postoperative day. During the first postoperative week, the level of pain in all patients was 0 on VAS. There were no complications. All patients, after a 6 weeks period of immobilization in TLSO and 6-week rehabilitation, returned to sport at the previous level, while remaining completely free from pain and without functional deficits. Because the pain occurred only during intense physical activity, as it was mentioned at the beginning, ODI questionnaire was not applied, due to the lack of evaluation of this type of situation.Spondylolysis occurs in 6% of the population. The occurrence amongst athletes is much greater, averaging 15% , 5, 10. For patients who do not respond to conservative treatment, direct pars repair using Buck’s method with our modification, including adequate radiographic preparation, the use of a thin cannulated screw and changing the point of screw entry, allows precise and safe screw placement, regardless of the size of the bone at the defect site. Good functional results and a lack of complications support the clinical efficacy of our modifications.


Sea ice is an important component of the global climate system and a key indicator of climate change. A decreasing trend in Arctic sea-ice concentration is evident in recent years, whereas Antarctic sea-ice concentration exhibits a generally increasing trend. Various studies have investigated the underlying causes of the observed trends for each region, but possible linkages between the regional trends have not been studied. Here, we hypothesize that the opposite trends in Arctic and Antarctic sea-ice concentration may be linked, at least partially, through interdecadal variability of the Pacific Decadal Oscillation (PDO) and the Atlantic Multidecadal Oscillation (AMO). Although evaluation of this hypothesis is constrained by the limitations of the sea-ice cover record, preliminary statistical analyses of one short-term and two long-term time series of observed and reanalysis sea-ice concentrations data suggest the possibility of the hypothesized linkages. For all three data sets, the leading mode of variability of global sea-ice concentration is positively correlated with the AMO and negatively correlated with the PDO. Two wave trains related to the PDO and the AMO appear to produce anomalous surface-air temperature and low-level wind fields in the two polar regions that contribute to the opposite changes in sea-ice concentration. Sea ice, an important component of the polar climate system, is a prominent indicator of global climate change123432819In contrast to the declining trends in Arctic sea ice, Antarctic sea-ice cover has exhibited an overall increasing trend in the past few decades323242526272830313231The associations found in previous studies, whether for the Arctic or Antarctic, between trends in sea-ice cover and low-frequency global climate oscillations such as the AMO, PDO and IPO suggest a possible connection between the opposing trends in sea-ice cover in the two polar regions. Most previous studies, however, have examined separately either the decreasing Arctic or the increasing Antarctic sea ice cover, rather than a more comprehensive examination of global sea-ice cover that concurrently considers trends in the Arctic and Antarctic. We hypothesize that the opposing trends in sea-ice cover in the two polar regions are, in part, connected via low-frequency global modes of climate variability, and provide an initial evaluation of this hypothesis. We focus on two indices of climate variability, the AMO and PDO, which capture atmosphere-ocean anomalies in the Atlantic and Pacific, respectively. Statistical analysis is used to assess the degree of association between time series of the AMO and PDO indices and sea-ice concentration. We employ both short-term remotely-sensed observations of sea-ice concentration and century-long estimates obtained from observational datasets and atmospheric reanalyses.We first focus on the concurrent temporal trends in high-resolution, remotely-sensed Arctic and Antarctic sea-ice concentrations obtained from the National Snow and Ice Data Center (NSIDC) for the period 1979 through 2013. Empirical orthogonal function (EOF) analyses are performed to jointly identify for the two polar regions (50°–90°N and 50°–90°S) the prevailing spatial patterns of the variability of seasonal mean sea-ice concentration for MAM (March-May), JJA (June-August), SON (September-November) and DJF (December-February). The sea-ice concentration time series were not detrended or filtered prior to the EOF analysis because of the short length of the NSIDC observational record, especially in comparison to the low-frequency PDO and AMO climate anomalies. The percentages of the total variance explained by the first EOF mode (EOF1) range from 16% for DJF to 22% for MAM. The EOF1 spatial patterns  show negThe time coefficients, or principal components (PC), of EOF1 show an overall increasing trend (significant at the 99% confidence level) for all four seasons , with coThe PC1 time series is compared to the AMO and PDO time series, both of which display significant  overall trends, positive for AMO and negative for PDO , althougThe analyses presented above lend support for the proposed connectivity between the two polar regions of the trends in sea-ice concentration. The short length of the NSIDC sea-ice concentration time series is problematic, however. To further explore the proposed hypothesis, our second set of analyses focuses on long-term estimates of sea-ice concentration obtained from the Twentieth Century Reanalysis (V2) for the period 1856 through 2011The results of the analysis of the longer sea-ice concentration datasets also support a connection between the opposing trends in sea-ice concentrations in the Arctic and Antarctic and a relationship with the AMO and PDO. As for the NSIDC time series, EOF analysis was applied to the two time series. For brevity, only the results for SON, when the variance explained by EOF1 is largest, are discussed and displayed. For the Twentieth Century Reanalysis, EOF1 for SON  explainsThe spatial patterns of EOF1 for the HadISST sea-ice concentration also display opposite anomalies for the two polar areas (negative for the Arctic and positive for the Antarctic) , althougLastly, we return to the NSIDC dataset to explore possible mechanisms for the global sea-ice trends. Sea-surface temperature (SST), 200-hPa geopotential height, and 2-m air temperature anomalies separately are regressed on PC1 obtained from the NSIDC time series of sea-ice concentrations. Here we only show the regression maps for SON  since th29We hypothesized that sea ice loss in the Arctic and net sea ice gain in the Antarctic during recent decades are connected via global climate oscillations, namely the AMO and PDO, and provided an initial assessment of the proposed hypothesis. Although this assessment is constrained by the limitations of the observational record of sea-ice concentrations, the congruent findings from statistical analysis of three time series of sea-ice concentration of varying length and resolution  point to a possible teleconnection between the opposite changing trends in sea-ice coverage in the two polar regions. These findings are consistent with those from previous studies that focused separately on either the north or the south polar regions. For example, several studies suggested that the changes in the SST over the tropical Pacific2324252627283132331619http://nsidc.org/data/NSIDC-0051). The sea-ice concentrations are derived from the Nimbus-7 Scanning Multichannel Microwave Radiometer (SMMR), the Special Sensor Microwave/Imagers (SSM/I) on the F8, F11, and F13 satellites, and the Special Sensor Microwave Imager/Sounder (SSMI/S) on the F17 satellite of the Defense Meteorological Satellite Program (DMSP). The NSIDC dataset, available daily from 26 October 1978 to the present, has a grid cell resolution of 25 × 25 km in a polar stereographic projection. Sea-ice concentration, defined as the areal percentage covered with ice, is generated using the National Aeronautics and Space Administration (NASA) team algorithm developed by the Oceans and Ice Branch, Laboratory for Hydrospheric Processes at NASA Goddard Space Flight Center (GSFC). More details are available from the NSIDC web site. Although the NSIDC dataset has been utilized widely to understand the variability of sea ice in Arctic and Antarctic, it has some limitations that may affect the interpretation of the results from the current study. First, and most important to our analysis, the NSIDC dataset is of relatively short length, especially in comparison to the low-frequency modes of climate variability. Second, snow and surface flooding on sea ice and seasonal variations in the physical characteristics of sea ice may influence the accuracy of the sea-ice dataThree data sets of sea-ice concentration were used in this study. The first dataset was obtained from the National Snow and Ice Data Center (NSIDC) in Boulder, Colorado (The NSIDC observed sea-ice data were supplemented by long-term sea-ice data from the Hadley Centre Sea Ice and Sea Surface Temperature (HadISST) data sethttp://www.atmos.washington.edu/~mantua/abst.PDO.html) and the monthly AMO indiceshttp://www.esrl.noaa.gov/psd/data/correlation/amon.us.data).The large-scale atmospheric circulations were derived using data from the National Centers for Environmental Prediction (NCEP)-Department of Energy (DOE) global reanalysisThe empirical orthogonal function (EOF) technique was used to reveal the dominant patterns of the variability of the seasonal mean sea-ice concentrations in the two polar regions. The EOF analysis can produce a set of modes that consist of spatial patterns (EOFs) and corresponding time series [principal components (PCs)]. For each mode, its EOF and PC are orthogonal to the EOFs and PCs of all other modes. Each mode has a corresponding eigenvalue that depicts the percentage of variance explained by the mode. In this study the EOF technique was utilized to identify the prevailing spatial patterns and time variations of seasonal-mean sea-ice concentration for MAM (March-May), JJA (June-August JJA), SON (September-November SON), and DJF (December-February DJF). Finally, regression analysis was used to explain the large-scale circulation anomalies associated with the leading EOF patterns. Specifically, a large-scale circulation variable  was regressed to the leading PC time series to explore the origin of the leading EOF patterns. This latter analysis was limited to only the higher resolution NSIDC sea-ice concentration data set.How to cite this article: Yu, L. et al. Possible connections of the opposite trends in Arctic and Antarctic sea-ice cover. Sci. Rep.7, 45804; doi: 10.1038/srep45804 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Caseinolytic protease from Listeria exploits two paths of proteolytic stimulation: heterooligomerization and partial inhibitor binding both enhance ClpX chaperone affinity. Listeria monocytogenes expresses two isoforms (LmClpP1 and LmClpP2). LmClpPs assemble into either a LmClpP2 homocomplex or a LmClpP1/2 heterooligomeric complex. The heterocomplex in association with the chaperone ClpX, exhibits a boost in proteolytic activity for unknown reasons. Here, we use a combined chemical and biochemical strategy to unravel two activation principles of LmClpPs. First, determination of apparent affinity constants revealed a 7-fold elevated binding affinity between the LmClpP1/2 heterocomplex and ClpX, compared to homooligomeric LmClpP2. This tighter interaction favors the formation of the proteolytically active complex between LmClpX and LmClpP1/2 and thereby accelerating the overall turnover. Second, screening a diverse library of fluorescent labeled peptides and proteins with various ClpP mutants allowed the individual analysis of substrate preferences for both isoforms within the heterocomplex. In addition to Leu and Met, LmClpP2 preferred a long aliphatic chain (2-Aoc) in the P1 position for cleavage. Strikingly, design and synthesis of a corresponding 2-Aoc chloromethyl ketone inhibitor resulted in stimulation of proteolysis by 160% when LmClpP2 was partially alkylated on 20% of the active sites. Determination of apparent affinity constants also revealed an elevated complex stability between partially modified LmClpP2 and the cognate chaperone LmClpX. Thus, the stimulation of proteolysis through enhanced binding to the chaperone seems to be a characteristic feature of LmClpPs.Caseinolytic proteases (ClpP) are important for recognition and controlled degradation of damaged proteins. While the majority of bacterial organisms utilize only a single ClpP,  Each subunit carries an active Ser-His-Asp catalytic triad that is essential for activity.6 ClpP by itself lacks proteolytic activity but is able to digest small peptides that diffuse into the barrel via axial pores.8 The first specific inhibitors reported for ClpP include the beta-lactones, which exhibit an irreversible mode of action and, depending on their chemical structure, can cause either retention of the tetradecameric state or deoligomerization of ClpP.10 More detailed insights into inhibitor-mediated complex disassembly were provided through a new generation of covalent phenyl esters and relevant modeling studies. These studies suggested that steric clash of the inhibitor within the active site triad triggers a rearrangement at the heptamer interface, causing dissociation of the ClpP tetradecamer into two heptameric rings.11 Recently, the first reversible ClpP inhibitors were reported, which distort the active site catalytic triad through structural rearrangements.12 However, this inactive state of the ClpP peptidase could be reversed through formation of the ClpXP complex, highlighting the power of conformational control within this dynamic system. ClpXP interaction is mainly mediated by ClpX-loops which bind into hydrophobic clefts located at the ClpP axial surface.13ATP-dependent proteolysis represents an important mechanism for removal of misfolded or ribosome-stalled proteins under stress conditions. In prokaryotes AAA+ chaperones  recognize and unfold substrate proteins by ATP consumption and direct the linear peptide chain into a proteolytic barrel of caseinolytic protease P (ClpP).Mycobacterium tuberculosis and Listeria monocytogenes encode two ClpP isoforms (ClpP1 and ClpP2).18 While L. monocytogenes ClpP2 (LmClpP2) resembles related enzymes in other bacteria such as Escherichia coli and Staphylococcus aureus, LmClpP1 shares only 44% sequence identity with E. coli ClpP, forms predominantly inactive heptamers, lacks a conserved N-terminal chaperone binding motif and exhibits a truncated catalytic triad in which Asp172 is replaced by an Asn residue.15 Mutational studies and in-depth X-ray crystallographic analysis revealed that this Asn residue is responsible for a conformational selection of the inactive heptameric state.15 Accordingly, mutation of this Asn to an Asp induced tetradecamer formation and increased catalytic activity of LmClpP1.15Interestingly, some bacterial strains such as 14 Importantly, it was found that LmClpP1 is only active when complexed with LmClpP2, which forces LmClpP1 into an active conformational state.15 The molecular reason for this surprising finding could be explained by a crystal structure of the LmClpP1/2 heterocomplex.16 All active sites within the heterocomplex, including Asn of LmClpP1, were aligned in an active conformation, which demonstrated that heterocomplex formation regulates LmClpP1 activity. While the heterocomplex was less active in peptidase assays, a 10-fold increase in proteolytic activity over the homocomplex was observed when in the presence of LmClpX. From a functional perspective, this implies that the cell produces a hyperactive enzyme during stress conditions, when misfolded proteins must be removed efficiently. Indeed, quantitative real-time PCR showed increased clpP1 and clpP2 expression under heat stress.16 So far, no systematic analysis of LmClpP1 and LmClpP2 peptidolytic and proteolytic cleavage specificities has been performed, leaving their role in substrate recognition unresolved. Structural studies and beta-lactone inhibitor screenings showed subtle differences in the P1 binding pockets and revealed a preference of LmClpP2 for medium to long aliphatic and aromatic side chains. LmClpP1, on the other hand, did not bind inhibitors specifically and only bound the natural product vibralactone.15Negative stain EM images of mixed LmClpP1 and LmClpP2 indicated the formation of heterocomplexes composed of two homoheptameric rings.M. tuberculosis (MtClpP1 and MtClpP2) also assemble into a similar heterocomplex.19 However, many mechanistic and functional differences compared to the L. monocytogenes heterocomplex have been reported. For example, MtClpP1 and MtClpP2 are both tetradecamers, which show only weak proteolytic activity on their own in the presence of chaperones.17 Peptidolytic activity is only enabled through MtClpP1/2 heterocomplex formation and requires the presence of activating peptides.20 Proteolytic substrate turnover is facilitated in association with the chaperones MtClpX or MtClpC1.21 Moreover, peptide substrate screenings with MtClpP1 revealed a cleavage preference after Met, Leu, Phe and Ala residues, while MtClpP2 was largely inactive.22 Although structural information remains elusive, mutational studies within the hydrophobic clefts of MtClpPs showed that chaperones only bind the heterocomplex via MtClpP2.23 The stoichiometry of the L. monocytogenes complexes remains unknown. However, as tetradecameric LmClpP1(N172D) is inactive in proteolysis assays with LmClpX, the LmClpP1/2 heterocomplex does not seem to bind LmClpX via the ClpP1 ring.16Interestingly, ClpP1 and ClpP2 from Here, we utilize peptide libraries to identify LmClpP1 and LmClpP2 cleavage specificities. While these preferences were largely abrogated in the proteolytic complexes, both isoforms retained a certain degree of individual specificity in protein digests. Biochemical analyses were used to dissect the multiple steps of proteolysis and revealed that the elevated activity of the heterocomplex stems from a 7-fold increased binding affinity for the LmClpX chaperone. Surprisingly, stimulation of proteolysis was also observed when a customized chloromethyl ketone-based (CMK) inhibitor partially modified the active sites of homotetradecameric LmClpP2. A closer mechanistic inspection of this inhibition mode revealed an increased affinity for the chaperone LmClpX to be the fundamental activation principle.16 Specifically, LmClpP1 exhibited a smaller and more defined cleft, restricting accessibility to smaller substrate side chains, while LmClpP2 resembles ClpPs from other organisms, e.g. E. coli, and provides access to larger substituents.24 In light of this divergence, we tested a previously established synthetic library of 172 fluorogenic 7-amino-4-carbamoylmethylcoumarin (ACC)-tagged peptide substrates containing various natural and unnatural amino acid substituents at the P1, P2 and P3 sites  as well as the active site mutants LmClpP1/2(S98A) (LmClpP1wt/2m) and LmClpP1(S98A)/2 (LmClpP1m/2wt) in order to unravel cleavage preferences of the individual heterocomplexes  and nicotinate phosphoribosyltransferase (LmPncB), were fused to a ClpX SsrA recognition tag and the purified recombinant proteins were subjected to LmClpXP protease assays  coupled to a nano-HPLC using established procedures.25 Peptide fragments were sequenced and analyzed via the Protein|Clpper software . The algorithm calculates log2 scores S by dividing the occurrence of an amino acid A at cleavage site position P by the natural occurrence of this amino acid in the respective protein. Thus, log2(S) values larger than 0 indicate that the respective residue is enriched at a given position, whereas log2(S) values smaller than 0 reflect depletion.25 Overall, a sufficient sequence coverage comprising 4668 unique peptides and 11 508 peptide-to-spectrum matches were obtained, which led to the analysis of 22 925 cleavage reactions   were strongly depleted in the protease screening. In addition, Protein|Clpper analysis of the digests showed much lower overall specificity at the P2, P3 and prime sites than at the P1 site. Nevertheless, Pro appears to be a crucial cleavage blocker if present at P2 or P1′ sites, which is likely attributable to its rigidity and locking the protein backbone in a kinked conformation. However, Pro at P3 increases the probability of being processed in the case of LmClpP2.Having identified substrate preferences of LmClpP1 and LmClpP2 at the peptide level, we investigated if this specificity holds true for cleavage of proteins as well. Therefore, two cognate e assays . SsrA-taP1m/2wt) . Althoug9 did not inhibit LmClpP1, thus failing to lend any insights into the heterocomplex mechanism.15 However, with the peptide screening data in hand, we were able to design customized LmClpP inhibitors in order to analyze the role of each isomers in more detail. A tripeptide scaffold with a C-terminal chloromethyl ketone (CMK) group was used to irreversibly bind the active site. Although peptide–CMK inhibitors are less selective and exhibit limited cell-permeability for in situ applications, the main advantages of these compounds are their stability upon enzyme active site binding as well as their customizability. We thus synthesized two inhibitors that incorporated the best residues from the P1, P2 and P3 library screening. Hence, 2-Aoc and Leu were selected for P1, while P2 and P3 sites contained hArg and 3,4-dichlorophenylalanine, respectively, yielding Leu-CMK and Aoc-CMK inhibitors -hArg-COOH dipeptide was synthesized on 2-chlorotrityl chloride resin and coupled with NH2-Leu-CMK (or NH2-2-Aoc-CMK). In addition, we included E2 and D3 in our study as representative beta-lactone inhibitors, as well as AV170, a recently introduced ClpP inhibitor with an electrophilic phenyl ester moiety  was measured by the hydrolysis of Ac-Ala-hArg-Leu-ACC, a substrate that showed optimal turnover by both isomers. Inhibitors were used in three concentrations, 100, 10 and 1 μM to estimate their potency  complexes were quite different. Here, only the CMKs showed any inhibitory effect, and Leu-CMK exhibited significantly higher potency compared to the 2-Aoc analog. Leu-CMK completely abolished LmClpP1 activity in the heterocomplex at 1 μM, reflecting its preference for small residues at the P1 site. With the first potent LmClpP1 inhibitors in hand, we commenced with mechanistic studies of LmClpXP proteolysis.The peptidolytic activity of LmClpP2, LmClpP1 potency  and S2†.wt/2wt, LmClpP1wt/2m and LmClpP1m/2wt were reconstituted with LmClpX and proteolysis of GFP-SsrA was monitored by a decrease in fluorescence signal according to previously established protocols.27 Inhibitors were added at three concentrations  to estimate their effects on enzymatic activity. Phenyl ester AV170 turned out to be the most effective inhibitor against each complex, except LmClpXP1wt/2m, highlighting the restricted binding site preferences of the LmClpP1 isoform  while higher concentrations decreased, and finally abolished, complex activity at 50 μM. Intact-protein MS analysis of the most activated species revealed 20% LmClpXP2 complex occupancy of the inhibitor. This suggests that incomplete inhibitor binding to the LmClpP2 tetradecamer stimulates proteolysis in association with LmClpX, but not peptidolysis, when in the absence of chaperone (see results above). The degree of modification reached 52% at 100 μM, which was sufficient to fully abolish proteolytic activity  which contributed to a model of functional communication between ClpX and ClpP during substrate processing.28 Here, we show that the mechanism of proteolytic stimulation extends beyond complete active site binding and is largely affected by the degree of modification, inhibitor structure as well as the ClpP isoform investigated.Third, LmClpXP2 assembly is in equilibrium with individual LmClpP2 and LmClpX complexes. This equilibrium precedes all subsequent steps required for protein turnover, including ClpX-mediated substrate binding, unfolding and translocation into the proteolytic chamber . A shifttivation . A similAoc-CMK. However, it should be noted that all heterocomplex constructs, including those that contain an active-site mutated ring, are about 10-fold more active compared to the LmClpXP2 homocomplex for yet unknown reasons.16 An effect on LmClpX ATPase activity by heterooligomerization has already been excluded in previous studies.16 In order to understand the basis for the enhanced activity, we dissected the mechanism and investigated key steps relevant for proteolysis.Proteolytic studies of the three heterocomplexes did not reveal pronounced activation upon partial modification with Aoc-CMK inhibition, we focused on the interaction between LmClpP1/2 and LmClpX, and determined their affinity. Proteolytic assays were performed with varying LmClpP1/2 concentrations, and apparent affinity constants were calculated as outlined above ,30 which are small-molecule chaperone mimics that bind to the same hydrophobic pockets and thereby, like ClpX, induce pore opening and conformational changes of ClpP to the activated state.33 Thus, binding to chaperones, or mimics thereof,34 represents the first known method of ClpP activation. Surprisingly, we identify here additional mechanisms of activation of LmClpPs, based on their affinity for chaperones. Key to this analysis was the fact that ClpP and ClpX are in a dynamic equilibrium for the formation of the proteolytically-competent ClpXP complex, which is the critical first step for proteolysis (27Protein degradation is tightly controlled by the cell in order to prevent unwanted proteolytic damage. Thus, major proteases such as ClpP and DegP are only activated for proteolysis in association with cognate chaperones and oligomerization, respectively.teolysis .27S. aureus homolog,25 prefers substrates with a long aliphatic side chain at the P1 site. As ClpP exhibits hydrolytic activity of small substrates that access the proteolytic chamber by diffusion, it is possible that it may have specialized functions beyond the cleavage of proteins. Although the preference for aliphatic amino acids such as Leu and Met, similar to other ClpPs, was retained in proteolytic studies, the overall selectivity observed for peptidase activity was largely abrogated.25 However, a striking feature of LmClpP1 is its additional preference for Gln at the P1 site. An additional feature of LmClpP1/2 is the accelerated proteolytic rate when associated with LmClpX. We show that the reason for this second principle of ClpP activation is a higher affinity between LmClpP1/2 and LmClpX, which shifts the equilibrium to the active LmClpXP1/2 heterocomplex. As the apical sites of LmClpP1 and LmClpP2 do not significantly change in the homo- and heterocomplexes, structural differences in the loop regions, which could not be resolved so far, may be responsible for a tighter interaction. Importantly, LmClpP2 is prone to a third mechanism of activation based on partial active site binding by irreversible inhibitors. While modification of 20% of all catalytic serines by a CMK inhibitor increased proteolysis by 160%, alkylation of approximately 50% of the sites resulted in full inhibition. Again, the reason for this proteolytic stimulation was an increase in binding affinity of the chaperone for partially CMK-modified LmClpP2, which shifted the equilibrium to the active proteolytic complex. Thus, modulating the affinity between ClpP and ClpX seems to be a unifying principle for proteolytic fine-tuning. Interestingly, the activation heavily depended on the inhibitor used. While Aoc-CMK induced the strongest effect, a CMK inhibitor containing Leu at the P1 site was only marginally stimulatory, suggesting that binding of long aliphatic chains into the S1 pocket could be a crucial parameter. Similarly, the long aliphatic beta-lactone inhibitor D3 also stimulated proteolysis, although to a much lesser extent, which suggests that this effect is more general and can also be observed by lactone active-site acylation in addition to CMK alkylation. On the contrary, aromatically-decorated lactone E2, a known disruptor of the ClpP tetradecamer,10 inhibited turnover, which emphasizes the importance of the ligand introduced into the S1 pocket. While a slight activation by Aoc-CMK was also observed for SaClpP, the more pronounced effects in LmClpP2 suggest a more functionally relevant role in this system. Based on this data, the LmClpP2 active site and substrate pocket seem to control the fate of proteolytic activity. Partial binding of long aliphatic ligands or heterooligomerization with LmClpP1 induce proteolysis via tighter binding to ClpX, which may be necessary under certain stress conditions. For instance, heat stress was previously shown to induce the expression of clpP1 and clpP2, resulting in an increased number of heterocomplexes, which might result in elevated proteolytic rates for removal of misfolded proteins.16 In addition, it is intriguing to speculate that metabolites containing long aliphatic chains may act as native stimulants. While the existence of such metabolites has to be investigated in future studies, it is important to note that activating peptides have previously been reported for heterooligomeric MtClpPs.19 In fact, these small molecules bind to active sites and thereby enhance activity.20We initiated our studies by inspecting both LmClpP isoforms and discovered that the LmClpP2 peptidase, similar to its Listeria system, which is already unique through its unusual expression of two ClpP isoforms. Due to the fundamental relevance of ClpP for cell homeostasis under stress conditions, both activation pathways ensure a boost in ClpP activity when needed, while remaining tightly regulated by ClpX interactions in order to prevent uncontrolled damage.In total, our in-depth analysis of LmClpP1/2 activity revealed two principles of proteolytic stimulation that both rely on elevated affinity between LmClpPs and LmClpX. This phenomenon appears to be special to the