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Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney causes ciliary elongation and cystogenesis, and cell-based proximity labeling proteomics and fluorescence microscopy show alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) are reduced in the cilia of DLG1-deficient cells compared to control cells. This phenotype is recapitulated in vivo and rescuable by re-expression of wild-type DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggest that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT., (© 2024. The Author(s).)

Alström syndrome (ALMS) is a very rare autosomal-recessive disorder, causing a broad range of clinical defects most notably retinal degeneration, type 2 diabetes, and truncal obesity. The ALMS1 gene encodes a complex and huge ∼0.5 MDa protein, which has hampered analysis in the past. The ALMS1 protein is localized to the centrioles and the basal body of cilia and is involved in signaling processes, for example, TGF-β signaling. However, the exact molecular function of ALMS1 at the basal body remains elusive and controversial. We recently demonstrated that protein complex analysis utilizing endogenously tagged cells provides an excellent tool to investigate protein interactions of ciliary proteins. Here, CRISPR/Cas9-mediated endogenously tagged ALMS1 cells were used for affinity-based protein complex analysis. Centrosomal and microtubule-associated proteins were identified, which are potential regulators of ALMS1 function, such as the centrosomal protein 70 kDa (CEP70). Candidate proteins were further investigated in ALMS1-deficient hTERT-RPE1 cells. Loss of ALMS1 led to shortened cilia with no change in structural protein localization, for example, acetylated and ɣ-tubulin, Centrin-3, or the novel interactor CEP70. Conversely, reduction of CEP70 resulted in decreased ALMS1 at the ciliary basal body. Complex analysis of CEP70 revealed domain-specific ALMS1 interaction involving the TPR-containing C-terminal (TRP-CT) fragment of CEP70. In addition to ALMS1, several ciliary proteins, including CEP135, were found to specifically bind to the TPR-CT domain. Data are available via ProteomeXchange with the identifier PXD046401. Protein interactors identified in this study provide candidate lists that help to understand ALMS1 and CEP70 function in cilia-related protein modification, cell death, and disease-related mechanisms., Competing Interests: Conflict of interest The authors declare no conflict of interest. The funders were not involved in the analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Biological complexity is achieved through elaborate interactions between relatively few individual components. Affinity purification (AP) has allowed these networks of protein-protein interactions that regulate key biological processes to be interrogated systematically. In order to perform these studies at the required scale, easily transfectable immortalized cell lines have typically been used. Gene-editing now affords the systematic creation of isogenic mouse models carrying endogenous tags for affinity proteomics. This may allow protein-protein interactions to be characterized in the appropriate tissue for a particular biological process or disease phenotype under physiological conditions, and for interaction landscapes to be compared across tissues. Here we demonstrate application to intraflagellar transport (IFT) proteins, which are WD40-domain-containing proteins that are essential for the formation and function of all types of cilia. We describe a method to generate mice with an endogenous C-terminal streptavidin/FLAG tag, using Ift80 as an example, and demonstrate the successful implementation of AP in this model. This method can easily be adapted for N- and C-terminal tagging of many other proteins in vivo., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Epilepsy-specific health-related quality of life (HRQOL) is an important outcome in youth with epilepsy (YWE). The PedsQL™ Epilepsy Module is the only caregiver-proxy and youth self-report epilepsy-specific HRQOL measure that can be used with youth 2-25 years. Multiple factors affect HRQOL, including epilepsy-specific characteristics, comorbid mental and behavioral health concerns, as well as sociodemographic factors. However, we have not yet examined the cumulative impact of these factors on epilepsy-specific HRQOL in YWE using the PedsQL™ Epilepsy module., Method: Youth with epilepsy (n = 281) and their caregivers completed questionnaires focused on sociodemographic factors (e.g., youth biological sex and age), mood/anxiety and behavior symptoms (i.e., Behavioral Assessment Scale for Children - Second Edition; BASC-2, Parent Rating Scale), epilepsy characteristics [e.g., seizure frequency, number of anti-seizure medications (ASMs), ASM side effects, and years since diagnosis], and the PedsQL™ Epilepsy module (subscales: Impact, Cognitive Functioning, Executive Functioning, Sleep, and Mood/Behavior)., Results: Hierarchical linear regressions were conducted to examine caregiver-proxy and youth self-reported factors that affect epilepsy-specific HRQOL. Results indicate the strongest key shared predictors of HRQOL in YWE, for both youth and caregiver informants, were mental and behavioral health symptoms. For instance, caregiver-proxy report of YWE HRQOL indicated BASC-2 Externalizing (p < 0.05), Behavioral Symptoms (p < 0.01), and Adaptive Skills (p < 0.001) explained 58 % of the variance in youth Cognitive Functioning HRQOL, while youth self-report of HRQOL indicated that BASC-2 Externalizing (p < 0.01), Behavioral Symptoms (p < 0.05), and Adaptive Skills (p < 0.001) contributed only 36 % of the variance in Cognitive Functioning HRQOL above and beyond the variance explained by sociodemographic and epilepsy-specific characteristics. Similar results were noted for Executive Functioning HRQOL domain, wherein caregiver-proxy report of YWE HRQOL indicated BASC-2 Internalizing (p < 0.01), Behavioral Symptoms (p < 0.001) and Adaptive Skills (p < 0.001) explained 65 % of variance in Executive Functioning, whereas youth self-report of Executive Functioning HRQOL indicated that caregiver-proxy BASC-2 Internalizing (p < 0.001) and Behavioral Symptoms (p < 0.01) explained 34 % of the variance in Executive Functioning HRQOL, above and beyond the variance explained by sociodemographic and epilepsy-specific characteristics. Unique mental and behavioral health predictors of YWE HRQOL were also found for both caregiver-proxy and youth self-report., Conclusions: Given the integral role of mental and behavioral health symptoms in epilepsy-specific HRQOL, it is critical to address mental and behavioral health symptoms preventatively and proactively to provide YWE with the most optimal health plan, including good seizure control, minimal ASM side effects, and the best possible HRQOL., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The intraflagellar transport (IFT) machinery is essential for cilia assembly, maintenance, and trans-localization of signaling proteins. The IFT machinery consists of two large multiprotein complexes, one of which is the IFT-B. TTC30A and TTC30B are integral components of this complex and were previously shown to have redundant functions in the context of IFT, preventing the disruption of IFT-B and, thus, having a severe ciliogenesis defect upon loss of one paralog. In this study, we re-analyzed the paralog-specific protein complexes and discovered a potential involvement of TTC30A or TTC30B in ciliary signaling. Specifically, we investigated a TTC30A-specific interaction with protein kinase A catalytic subunit α, a negative regulator of Sonic hedgehog (Shh) signaling. Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which, intriguingly, is also linked to a rare TTC30 variant. For an in-depth analysis of this unique interaction and the influence on Shh, TTC30A or B single- and double-knockout hTERT-RPE1 were employed, as well as rescue cells harboring wildtype TTC30 or the corresponding mutation. We could show that mutant TTC30A inhibits the ciliary localization of Smoothened. This observed effect is independent of Patched1 but associated with a distinct phosphorylated PKA substrate accumulation upon treatment with forskolin. This rather prominent phenotype was attenuated in mutant TTC30B. Mass spectrometry analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in protein complex patterns and identified an impaired TTC30A-IFT57 interaction as the possible link leading to synpolydactyly. We could observe no impact on cilia assembly, leading to the hypothesis that a slight decrease in IFT-B binding can be compensated, but mild phenotypes, like synpolydactyly, can be induced by subtle signaling changes. Our systematic approach revealed the paralog-specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA and the uptake of Smoothened into the cilium, resulting in a downregulation of Shh. This downregulation, combined with interactome alterations, suggests a potential mechanism of how mutant TTC30A is linked to synpolydactyly., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hoffmann, Bolz, Junger, Klose, Stehle, Ueffing, Boldt and Beyer.)

Ciliopathies are inherited disorders caused by defective cilia. Mutations affecting motile cilia usually cause the chronic muco-obstructive sinopulmonary disease primary ciliary dyskinesia (PCD) and are associated with laterality defects, while a broad spectrum of early developmental as well as degenerative syndromes arise from mutations affecting signalling of primary (non-motile) cilia. Cilia assembly and functioning requires intraflagellar transport (IFT) of cargos assisted by IFT-B and IFT-A adaptor complexes. Within IFT-B, the N-termini of partner proteins IFT74 and IFT81 govern tubulin transport to build the ciliary microtubular cytoskeleton. We detected a homozygous 3-kb intragenic IFT74 deletion removing the exon 2 initiation codon and 40 N-terminal amino acids in two affected siblings. Both had clinical features of PCD with bronchiectasis, but no laterality defects. They also had retinal dysplasia and abnormal bone growth, with a narrowed thorax and short ribs, shortened long bones and digits, and abnormal skull shape. This resembles short-rib thoracic dysplasia, a skeletal ciliopathy previously linked to IFT defects in primary cilia, not motile cilia. Ciliated nasal epithelial cells collected from affected individuals had reduced numbers of shortened motile cilia with disarranged microtubules, some misorientation of the basal feet, and disrupted cilia structural and IFT protein distributions. No full-length IFT74 was expressed, only truncated forms that were consistent with N-terminal deletion and inframe translation from downstream initiation codons. In affinity purification mass spectrometry, exon 2-deleted IFT74 initiated from the nearest inframe downstream methionine 41 still interacts as part of the IFT-B complex, but only with reduced interaction levels and not with all its usual IFT-B partners. We propose that this is a hypomorphic mutation with some residual protein function retained, which gives rise to a primary skeletal ciliopathy combined with defective motile cilia and PCD., (© The Author(s) 2023. Published by Oxford University Press.)

Leber congenital amaurosis (LCA) is a group of inherited retinal diseases characterized by early-onset, rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy, we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localized at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient in lebercilin exhibited early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, adeno-associated virus-based LCA5 gene augmentation partially restored the bulge region, preserved OS axoneme structure and membrane disc formation, and resulted in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.

Establishment and maintenance of the primary cilium as a signaling-competent organelle requires a high degree of fine tuning, which is at least in part achieved by a variety of post-translational modifications. One such modification is ubiquitination. The small and highly conserved ubiquitin protein possesses a unique versatility in regulating protein function via its ability to build mono and polyubiquitin chains onto target proteins. We aimed to take an unbiased approach to generate a comprehensive blueprint of the ciliary ubiquitinome by deploying a multi-proteomics approach using both ciliary-targeted ubiquitin affinity proteomics, as well as ubiquitin-binding domain-based proximity labelling in two different mammalian cell lines. This resulted in the identification of several key proteins involved in signaling, cytoskeletal remodeling and membrane and protein trafficking. Interestingly, using two different approaches in IMCD3 and RPE1 cells, respectively, we uncovered several novel mechanisms that regulate cilia function. In our IMCD3 proximity labeling cell line model, we found a highly enriched group of ESCRT-dependent clathrin-mediated endocytosis-related proteins, suggesting an important and novel role for this pathway in the regulation of ciliary homeostasis and function. In contrast, in RPE1 cells we found that several structural components of caveolae (CAV1, CAVIN1, and EHD2) were highly enriched in our cilia affinity proteomics screen. Consistently, the presence of caveolae at the ciliary pocket and ubiquitination of CAV1 specifically, were found likely to play a role in the regulation of ciliary length in these cells. Cilia length measurements demonstrated increased ciliary length in RPE1 cells stably expressing a ubiquitination impaired CAV1 mutant protein. Furthermore, live cell imaging in the same cells revealed decreased CAV1 protein turnover at the cilium as the possible cause for this phenotype. In conclusion, we have generated a comprehensive list of cilia-specific proteins that are subject to regulation via ubiquitination which can serve to further our understanding of cilia biology in health and disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Aslanyan, Doornbos, Diwan, Anvarian, Beyer, Junger, van Beersum, Russell, Ueffing, Ludwig, Boldt, Pedersen and Roepman.)

Mutations in PDE6D impair the function of its cognate protein, phosphodiesterase 6D (PDE6D), in prenylated protein trafficking towards the ciliary membrane, causing the human ciliopathy Joubert Syndrome (JBTS22) and retinal degeneration in mice. In this study, we purified the prenylated cargo of PDE6D by affinity proteomics to gain insight into PDE6D-associated disease mechanisms. By this approach, we have identified a specific set of PDE6D-interacting proteins that are involved in photoreceptor integrity, GTPase activity, nuclear import, or ubiquitination. Among these interacting proteins, we identified novel ciliary cargo proteins of PDE6D, including FAM219A, serine/threonine-protein kinase NIM1 (NIM1K), and ubiquitin-like protein 3 (UBL3). We show that NIM1K and UBL3 localize inside the cilium in a prenylation-dependent manner. Furthermore, UBL3 also localizes in vesicle-like structures around the base of the cilium. Through affinity proteomics of UBL3, we confirmed its strong interaction with PDE6D and its association with proteins that regulate small extracellular vesicles (sEVs) and ciliogenesis. Moreover, we show that UBL3 localizes in specific photoreceptor cilium compartments in a prenylation-dependent manner. Therefore, we propose that UBL3 may play a role in the sorting of proteins towards the photoreceptor outer segment, further explaining the development of PDE6D-associated retinal degeneration.

Evidence from patients with Parkinson's disease (PD) and our previously reported α-synuclein (SNCA) transgenic rat model support the idea that increased SNCA protein is a substantial risk factor of PD pathogenesis. However, little is known about the transcription control of the human SNCA gene in the brain in vivo. Here, we identified that the DYT6 gene product THAP1 (THAP domain-containing apoptosis-associated protein 1) and its interaction partner CTCF (CCCTC-binding factor) act as transcription regulators of SNCA . THAP1 controls SNCA intronic enhancers' activities, while CTCF regulates its enhancer-promoter loop formation. The SNCA intronic enhancers present neurodevelopment-dependent activities and form enhancer clusters similar to "super-enhancers" in the brain, in which the PD-associated single-nucleotide polymorphisms are enriched. Deletion of the SNCA intronic enhancer clusters prevents the release of paused RNA polymerase II from its promoter and subsequently reduces its expression drastically in the brain, which may provide new therapeutic approaches to prevent its accumulation and thus related neurodegenerative diseases defined as synucleinopathies.

Leukodystrophies are a heterogenous group of genetic disorders, characterised by abnormal development of cerebral white matter. Pelizaeus-Merzbacher disease is caused by mutations in PLP1, encoding major myelin-resident protein required for myelin sheath assembly. We report a missense variant p.(Ala109Asp) in MAL as causative for a rare, hypomyelinating leukodystrophy similar to Pelizaeus-Merzbacher disease. MAL encodes a membrane proteolipid that directly interacts with PLP1, ensuring correct distribution during myelin assembly. In contrast to wild-type MAL, mutant MAL was retained in the endoplasmic reticulum but was released following treatment with 4-phenylbutyrate. Proximity-dependent identification of wild-type MAL interactants implicated post-Golgi vesicle-mediated protein transport and protein localisation to membranes, whereas mutant MAL interactants suggested unfolded protein responses. Our results suggest that mislocalisation of MAL affects PLP1 distribution, consistent with known pathomechanisms for hypomyelinating leukodystrophies., (© 2022. The Author(s).)

Intraflagellar transport (IFT) is a microtubule-based system that supports the assembly and maintenance of cilia. The dysfunction of IFT leads to ciliopathies of variable severity. Two of the IFT-B components are the paralogue proteins TTC30A and TTC30B. To investigate whether these proteins constitute redundant functions, CRISPR/Cas9 was used to generate single TTC30A or B and double-knockout hTERT-RPE1 cells. Ciliogenesis assays showed the redundancy of both proteins while the polyglutamylation of cilia was affected in single knockouts. The localization of other IFT components was not affected by the depletion of a single paralogue. A loss of both proteins led to a severe ciliogenesis defect, resulting in no cilia formation, which was rescued by TTC30A or B. The redundancy can be explained by the highly similar interaction patterns of the paralogues; both equally interact with the IFT-B machinery. Our study demonstrates that a loss of one TTC30 paralogue can mostly be compensated by the other, thus preventing severe ciliary defects. However, cells assemble shorter cilia, which are potentially limited in their function, especially because of impaired polyglutamylation. A complete loss of both proteins leads to a deficit in IFT complex B integrity followed by disrupted IFT and subsequently no cilia formation.

Nephrotic syndrome is characterized by urinary excretion of plasma proteases or proteasuria. There is a lack of data on the quantity, activity status and identity of these aberrantly filtered proteases. We established a fluorescence-based substrate assay to quantify protease activity in urine samples from healthy and nephrotic humans and mice. Protease class activity was determined after addition of specific inhibitors. Individual proteases were identified by tandem mass spectrometry (MS/MS). In spot urine samples from 10 patients with acute nephrotic syndrome of various etiology, urinary protease activity was significantly increased compared to that of healthy persons (753 ± 178 vs. 244 ± 65 relative units, p < 0.05). The corresponding proteases were highly sensitive to inhibition by the serine protease inhibitors AEBSF (reduction by 85 ± 6% and 72 ± 8%, respectively) and aprotinin (83 ± 9% vs. 25 ± 6%, p < 0.05). MS/MS of all urinary proteins or after AEBSF purification showed that most of them were active serine proteases from the coagulation and complement cascade. These findings were recapitulated in mice, pointing to a similar pathophysiology. In conclusion, nephrotic syndrome leads to increased urinary excretion of active plasma proteases which can be termed proteasuria. Serine proteases account for the vast majority of urinary protease activity in health and nephrotic syndrome. SIGNIFICANCE STATEMENT: In this study, we found that nephrotic urine samples of humans and mice have a significantly increased protease activity compared to healthy urine samples, using a universal pentapeptide substrate library. This was driven by increased excretion of aprotinin-sensitive serine proteases. With tandem mass spectrometry, we provide a comprehensive and systematic overview of all urinary proteases or the "urine proteasome". We identified renally expressed proteases in health and addition of proteases from the coagulation and complement cascade in the nephrotic state. These results set the basis to study the role of urinary proteases at both health and nephrotic syndrome to find diagnostic markers of renal disease as well as possible therapeutic targets., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45 -/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

The current study compared differences in health-related quality of life (HRQOL) between youth with new-onset epilepsy with and without elevated psychological symptoms at time of epilepsy diagnosis within an integrated behavioral health and epilepsy service. Patients received both behavioral health and epilepsy care during clinic visits. A retrospective chart review was conducted between July 2011 and December 2015. Caregivers completed the Behavior Assessment System for Children-2: Parent Rating Scale (BASC-2: PRS) to assess psychological symptoms at the diagnostic visit, along with completing the Pediatric Quality of Life Inventory (PedsQL™ 4.0) at the diagnostic visit and each subsequent epilepsy clinic visit during the first year of treatment. Latent growth curve modeling was used to identify HRQOL changes over the first year of treatment. Health-related quality of life was significantly lower for youth with elevated psychological symptoms at diagnosis and over the first year of treatment compared with those without psychological symptoms. For those with elevated internalizing, inattention, withdrawal, and atypical symptoms at diagnosis, greater HRQOL improvements were detected over the first year of treatment compared with those without elevated psychological symptoms at the diagnostic visit. Within integrated behavioral health and epilepsy routine care, targeted psychological interventions can improve HRQOL over the first year of treatment, particularly for those with premorbid psychological symptoms., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: The objectives of this study were to identify executive functioning (EF) phenotypes in youth with epilepsy and to examine whether phenotypes differ on psychosocial and medical outcomes (i.e., absence/presence of seizures in the past three months), health-related quality of life (HRQOL), and emotional and behavioral functioning., Methods: Youth 5-18 years with diagnosed epilepsy and caregivers completed a battery of questionnaires as part of a larger national validation of the Pediatric Quality of Life (PedsQL) Epilepsy Module. The primary measure of interest was the Behavior Rating Inventory of Executive Function-Parent Form. Medical chart reviews and demographic data were also collected. Latent class analysis was used to identify EF phenotypes. Chi-square and analyses of covariance (ANCOVA) were conducted to examine EF phenotype group differences on seizure outcomes, HRQOL, and behavioral and emotional functioning., Results: Two-hundred and thirty-seven children with epilepsy (M age  = 11.2 years; 56% female; 60% White: Non-Hispanic; 55% experienced seizures in the past three months) and their caregivers participated. Four EF phenotypes were identified: Group 1 - No EF deficits (45% of sample), Group 2 - Global EF deficits (29% of sample), Group 3 - Behavioral Regulation + Working Memory deficits (8% of sample), and Group 4 - Metacognitive deficits (17% of sample). No significant EF phenotype group differences were found for seizure characteristics. The ANCOVAs indicated significant EF phenotype group differences on HRQOL (parent-reported Impact, Cognitive, Sleep, EF, and Mood/Behavior and child-reported Cognitive, Sleep, EF, and Mood/Behavior subscales; ps < .001) and emotional and behavioral functioning (Externalizing, Internalizing, and Behavioral Symptom Index; ps < .001), with the Global EF deficits (Group 2) and Behavioral Regulation + Working Memory deficits groups (Group 3) demonstrating the greatest level of impairment., Conclusion: Phenotypic variability in EF is significantly related to patient-reported outcomes. Interventions addressing EF deficits need to be individualized to a child's particular EF phenotype to achieve optimal outcomes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.

CRISPR/Cas9-mediated gene editing allows manipulation of a gene of interest in its own chromosomal context. When applied to the analysis of protein interactions and in contrast to exogenous expression of a protein, this can be studied maintaining physiological stoichiometry, topology, and context. We have used CRISPR/Cas9-mediated genomic editing to investigate Cluap1/IFT38, a component of the intraflagellar transport complex B (IFT-B). Cluap1 has been implicated in human development as well as in cancer progression. Cluap1 loss of function results in early developmental defects with neural tube closure, sonic hedgehog signaling and left-right defects. Herein, we generated an endogenously tagged Cluap1 for protein complex analysis, which was then correlated to the corresponding interactome determined by ectopic expression. Besides IFT-B complex components, new interacting proteins like Ephrin-B1 and TRIP6, which are known to be involved in cytoskeletal arrangement and protein transport, were identified. With the identification of platelet-derived growth factor A (PDGFA) and coiled-coil domain-containing protein 6 (CCDC6) two new interactions were discovered, which link Cluap1 to ciliogenesis and cancer development. The CRISPR/Cas9-mediated knockout of Cluap1 revealed a new phenotype affecting the actin cytoskeleton. Together, these data provide first evidence for a role of Cluap1 not only for cilia assembly and maintenance but also for cytoskeletal rearrangement and intracellular transport processes., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: To examine baseline psychological functioning and antiepileptic drug (AED) behavioral side effects in new onset epilepsy and determine, by age, whether baseline psychological functioning predicts AED behavioral side effects 1 month following AED initiation., Methods: A retrospective chart review was conducted between July 2011 and December 2014 that included youths with new onset epilepsy. As part of routine interdisciplinary care, caregivers completed the Behavior Assessment System for Children, 2nd Edition: Parent Rating Scale to report on baseline psychological functioning at the diagnostic visit and the Pediatric Epilepsy Side Effects Questionnaire to identify AED behavioral side effects at the 1-month follow-up clinic visit following AED initiation. Children (age = 2-11 years) and adolescents (age = 12-18 years) were examined separately., Results: A total of 380 youths with new onset epilepsy (M age  = 8.9 ± 4.3 years; 83.4% Caucasian; 34.8% focal epilepsy, 41.1% generalized epilepsy, 23.7% unclassified epilepsy) were included. Seventy percent of youths had at-risk or clinically elevated baseline psychological symptoms. Children had significantly greater AED behavioral side effects (M = 25.08 ± 26.36) compared to adolescents (M = 12.36 ± 17.73), regardless of AED. Valproic acid demonstrated significantly greater behavioral side effects compared to all other AEDs, with the exception of levetiracetam. Higher hyperactivity/impulsivity at baseline significantly predicted higher AED behavioral side effects 1 month after AED initiation in both age groups., Significance: Younger children seem to be more prone to experience behavioral side effects, and these are likely to be higher if youths with epilepsy have baseline hyperactivity/impulsivity. Baseline psychological screening, specifically hyperactivity, can be used as a precision medicine tool for AED selection., (Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.)

Objective: To examine differences in health care charges following a pediatric epilepsy diagnosis based on changes in health-related quality of life (HRQOL)., Methods: Billing records were obtained for 171 youth [M (SD) age = 8.9 (4.1) years] newly diagnosed with epilepsy. Differences in health care charges among HRQOL groups (stable low, declining, improving, or stable high as determined by PedsQL(™) scores at diagnosis and 12 months after diagnosis) were examined., Results: Patients with persistently low or declining HRQOL incurred higher total health care charges in the year following diagnosis (g = .49, g = .81) than patients with stable high HRQOL after controlling for epilepsy etiology, seizure occurrence, and insurance type. These relationships remained consistent after excluding health care charges for behavioral medicine or neuropsychology services (g = .49, g = .80)., Conclusions: Monitoring HRQOL over time may identify youth with epilepsy at particular risk for higher health care charges., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Pediatric Psychology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To estimate first-year health care charges for youth with newly diagnosed epilepsy seen within an interdisciplinary pediatric epilepsy team and examine demographic, clinical, and psychosocial predictors of annual charges., Methods: Retrospective chart review was conducted to extract medical, hospital, and physician billing data from the year following an epilepsy diagnosis for 258 patients (aged 2-18 years) seen in a New Onset Seizure Clinic between July 2011 and December 2012. Descriptive statistics were used to estimate per-patient total first-year charges and health care utilization patterns (e.g., hospitalizations, emergency department visits, outpatient visits). Univariate analyses examined differences in health care charges between demographic, clinical, and psychosocial factors. Predictors of health care charges were examined using hierarchical multiple regression analysis., Results: The estimated per-patient total first-year health care charge was $20,084 (95% confidence interval [CI] $16,491-$23,677). Charges were higher for patients who reported having seizures since diagnosis ($25,509; 95% CI $20,162-$30,856) and were associated with more antiepileptic drug side effects (r = 0.18; 95% CI 0.03-0.32). Controlling for demographic and clinical factors, poorer baseline health-related quality of life was associated with higher per-patient health care charges (B = -445.40; 95% CI -865 to -25)., Conclusions: The economic impact of pediatric epilepsy in the year following diagnosis is substantial. Cost reduction efforts would be optimized by improving seizure control and targeting health-related quality of life, an outcome amenable to behavioral intervention., (© 2015 American Academy of Neurology.)

Objective: To examine the influence of parent and family general and epilepsy-related stress on longitudinal generic and epilepsy-specific health-related quality of life (HRQOL) for children with new-onset epilepsy, while controlling for demographic characteristics, disease factors, and antiepileptic drug (AED) adherence., Methods: This prospective, longitudinal study included 124 children with new-onset epilepsy (mean age 7.2 years, standard deviation [SD] 2.9 years). Parents completed questionnaires on parenting stress, perceived stigma, fears and concerns, and HRQOL at 1, 13, and 25 months after diagnosis. Adherence to AEDs was assessed using electronic monitors. A medical chart review was conducted at each visit to obtain seizure and side effect data., Results: Higher levels of general and epilepsy-specific parent and family stress, fears and concerns, and perceived stigma negatively affected child generic and epilepsy-specific HRQOL, above and beyond disease and demographic factors. General parenting and family stress affected child generic and epilepsy-specific HRQOL more in the first year of disease management than at 2 years after diagnosis. Higher fears and concerns predicted higher epilepsy-specific HRQOL at 13 months postdiagnosis, whereas 2 years postdiagnosis, higher fears and concerns predicted lower epilepsy-specific HRQOL. Several demographic (i.e., age) and disease-related variables (i.e., side effects and AED adherence) influenced child generic and epilepsy-specific HRQOL. Although some findings were consistent across generic and epilepsy-specific HRQOL measures, others were unique., Significance: Modifiable parent factors (i.e., general and disease-specific parent and family stress, perceived stigma) impact HRQOL for children with new-onset epilepsy differently over the first 2 years postdiagnosis. Psychosocial interventions to improve HRQOL within the first year postdiagnosis should address parenting and family stress, overall coping, and anticipatory guidance on managing epilepsy. Interventions targeting adherence, perceived stigma, and fears and concerns could improve HRQOL. Promoting parent management of stress, fears/concerns, and perceived stigma may lead to improved child HRQOL outcomes. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here., (Wiley Periodicals, Inc. © 2014 International League Against Epilepsy.)

Background: Inconsistent relationships between weight and psychosocial functioning may be due to discrepancies between objective measures of weight and children's perceptions of weight. The current study compared the predictive validity of actual versus perceived weight in children to determine which is the strongest predictor of psychosocial functioning., Methods: Ninety-eight youth (ages 8-17) completed measures of perceived weight and psychosocial functioning (i.e., depressive symptoms, peer victimization, and self-esteem) while attending a well-child visit. Height and weight were obtained from the medical record and used to classify children as healthy weight, overweight, or obese. Actual and perceived weight percentiles were entered simultaneously in regression analyses predicting psychosocial functioning., Results: A disproportionate number of overweight (70%) and obese (40.6%) youth reported a perceived weight in the healthy range (below 85(th) BMI percentile). Perceived weight was predictive of depressive symptoms whereas actual weight was not. No relationship between weight (perceived or actual) was found for peer victimization or self-esteem., Conclusions: Weight underestimation is common in children, particularly among youth who are overweight and obese. Perceived, but not actual, weight was predictive of depressive symptoms, highlighting the importance of weight perceptions among youth across the weight spectrum.

Background: Behavioral interventions targeting children with overweight have been successful in facilitating weight loss; however, there is concern that these programs produce disordered eating attitudes among youth., Objective: The purpose of this research was to determine whether youth with overweight receiving one of two behavioral interventions were more likely to report an increase in disordered eating attitudes over time compared to a waitlist control and to determine psychosocial predictors of eating-disordered attitudes at 6-month follow-up., Design: Participants were randomized to one of two behavioral lifestyle interventions or a waitlist control. Data were collected at baseline, post-treatment, and 6-month follow-up., Participants/setting: Participants were 68 youths with overweight, aged 8 to 13 years, and their parent(s) who lived in rural north central Florida. The project ran from January 2006 to January 2008., Intervention: Each treatment condition consisted of 12 group sessions over 16 weeks., Main Outcome Measures: Parents completed a demographic form and the Child Feeding Questionnaire. Children completed the Children's Eating Attitudes Test, Schwartz Peer Victimization Scale, and Children's Body Image Scale., Statistical Analyses Performed: Mixed 2×2 analyses of variance were used to examine the effect of treatment on eating attitudes. Hierarchical linear regression was used to assess whether baseline levels of psychosocial variables predicted disordered eating attitudes at follow-up, controlling for baseline eating attitudes and treatment condition., Results: Youth who participated in the behavioral interventions did not report significant increases in disordered eating attitudes over time compared to the waitlist control. Across all conditions, higher levels of body dissatisfaction, peer victimization, parent restrictive feeding practices, and concern for child weight at baseline predicted higher levels of disordered eating attitudes at follow-up., Conclusions: These findings do not provide evidence that behavioral interventions lead to an increase in unhealthy eating attitudes and behaviors. Future research should examine the effects of incorporating eating disorder prevention in pediatric weight management programs., (Copyright © 2010 American Dietetic Association. Published by Elsevier Inc. All rights reserved.)

Human immunodeficiency virus type 1 (HIV-1) protease was expressed in Escherichia coli as a fusion protein with the N-terminal sequence of IGF-2. The protein accumulated in inclusion bodies as a 40:60 mixture of unprocessed fusion protein and processed protein. A simple purification procedure was developed that yielded 30-40 mg of active protease per liter of fermentation broth with a recovery of 30-40%. The purification process involved the selective extraction of HIV-1 protease from E. coli inclusion bodies with 50% acetic acid and fractional diafiltration to remove impurities and low-molecular-weight protease-related fragments. No chromatographic steps were employed, yet the HIV-1 protease produced by this procedure was greater than 95% pure by SDS-PAGE, reverse-phase HPLC, and N-terminal sequence analysis.

Tissue factor pathway inhibitor is an inhibitor of the extrinsic coagulation pathway. Evaluation of the pharmacological effects of tissue factor pathway inhibitor in animal models has been limited by the high cost and low availability of mammalian tissue culture produced protein. In order to circumvent this obstacle, a 277-amino-acid nonglycosylated tissue factor pathway inhibitor variant possessing an N-terminal alanine was expressed in recombinant E. coli using the tac promoter expression system. High-level expression in recombinant E. coli resulted in the accumulation of ala-tissue factor pathway inhibitor in inclusion bodies. Active protein was produced by solubilization of the inclusion bodies in 8 M urea, purification of the full-length molecule by cation exchange chromatography, and renaturation in 6 M urea. Fractionation of crude refold mixtures using cation exchange chromatography yielded a purified nonglycosylated tissue factor pathway inhibitor possessing in vitro prothrombin time activity comparable to inhibitor purified from mammalian cell lines.

Physiotherapy involves manifold measures that are partly not clearly separated from other methods of therapy. In most cases the skin or mucosa are not injured so that the danger of infectious complications is relatively low. On the other hand, patients with severe underlying diseases which are problematic from the aspect of hygiene in connection with infections, may have to be treated (for example, intensive-care patients, patients with decubital ulcers etc.) In such patients physiotherapy must comply with hygienic precautions that are employed with other diseases and treatments to prevent the transfer of pathogens. Special rules govern the composition of the water and the servicing of equipment used in hydrotherapy (German DIN standard specification No. 1964). Guidelines for the water used in municipal swimming pools and baths are in preparation. Other rules to be observed concern the recognition, treatment and prevention of nosocomial infections. In addition, every establishment must set up definite rules for regular measures to prevent infections and to monitor these measures, especially with regard to cleaning and disinfecting.

Active human tissue plasminogen activator variant kringle-2-serine protease (K2 + SP domains; referred to as MB1004) was synthesized as a secreted protein in Escherichia coli, isolated, and characterized. MB1004 is a relatively large and complex protein, approximately 38 kDa in size and containing nine disulfide bonds. MB1004 without a pro region was secreted into the periplasm of E. coli by fusing the protein to the PhoA leader peptide expressed from the tac promoter. Approximately 1% (20 micrograms/L broth) of the secreted MB1004 was purified from E. coli homogenates as a soluble, active enzyme by using a combination of lysine and Erythrina inhibitor affinity chromatography. Purified MB1004 was monomeric and single-chain, and the N-terminus was identical with the predicted amino acid sequence. The specific activity of purified MB1004 from E. coli was compared against the equivalent recombinant material purified from mammalian cells that was naturally glycosylated (MB1004G) or deglycosylated after treatment with N-glycanase (MB1004N). Results from four different in vitro assays showed that MB1004 and MB1004N had similar activities. Both exhibited 4-12-fold higher specific activity than MB1004G in plasminogen activation assays. These results suggest that an inaccurate picture of specific activity can be obtained if the effects of glycosylation are not considered. By utilization of secretion in E. coli, nonglycosylated MB1004 was purified without in vitro refolding and was shown to be suitable for structure-function studies.

6 metabolically healthy control persons, 6 patients with liver cirrhosis, and 6 patients with diabetes were infused for 48 hours with a 20 % (w/v) glucose solution. The infusion rate was 0,25 g glucose/kg body weight and hour. A constant surveillance of the metabolical status was performed by control of 39 different laboratory values. In all 3 groups, glucose infusion alone was sufficient for basal metabolic rate. This was shown by the inhibition of lipolysis as well as in the decreased catabolism. Only the diabetic group exhibited high blood-sugar values and an energy loss up to 10 % of the administered glucose. We could not observe any side effects.

A simple and rapid colorimetric assay for measuring the high affinity Ca2+-ATPase activity in subcellular fractions is presented. With this method a one-step addition of a malachite green/molybdate/polyvinyl alcohol reagent to the assay mixture at the end of the incubation period is all that is required for the spectrophotometric quantification of the phosphomolybdate-malachite green complex. The presence of polyvinyl alcohol allows the quantification of released phosphate without having to separate it from protein. We have validated this assay by characterizing the high affinity Ca2+-ATPase activity in isolated rat liver microsomes. Comparable Ca2+-ATPase activities in rat liver microsomes and adipocyte plasma membranes were found when measured with this colorimetric assay and an isotopic assay. This method is applicable to the measurement of other types of ATPase activities.