Post-Translational Modifications of the DUX4 Protein Impact Toxic Function in FSHD Cell Models.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is caused by abnormal de-repression of the myotoxic transcription factor DUX4. Although the transcriptional targets of DUX4 are known, the regulation of DUX4 protein and the molecular consequences of this regulation are unclear. Here, we used in vitro models of FSHD to identify and characterize DUX4 post-translational modifications (PTMs) and their impact on the toxic function of DUX4. METHODS: We immunoprecipitated DUX4 protein and performed mass spectrometry to identify PTMs. We then characterized DUX4 PTMs and potential enzyme modifiers using mutagenesis, proteomics, and biochemical assays in HEK293 and human myoblast cell lines. RESULTS: We identified 17 DUX4 amino acids with PTMs, and generated 55 DUX4 mutants designed to prevent or mimic PTMs. Five mutants protected cells against DUX4-mediated toxicity and reduced the ability of DUX4 to transactivate FSHD biomarkers. These mutagenesis results suggested that DUX4 toxicity could be counteracted by serine/threonine phosphorylation and/or inhibition of arginine methylation. We therefore sought to identify modifying enzymes that could play a role in regulating DUX4 PTMs. We found several enzymes capable of modifying DUX4 protein in vitro, and confirmed that protein kinase A (PKA) and protein arginine methyltransferase (PRMT1) interact with DUX4. INTERPRETATION: These results support that DUX4 is regulated by PTMs and set a foundation for developing FSHD drug screens based mechanistically on DUX4 PTMs and modifying enzymes. ANN NEUROL 2023;94:398-413.

Using Fractal Iconography to Emulate Nature's Aesthetics.

This year's cover artists are members of a team of physicists and psy-chologists who create human-centered designs based on psychology experiments that investigate the positive impacts of viewing fractal patterns. These positive impacts include reduced physiological stress levels and enhanced cognitive skills. Here, the team explores the concept of 'fractal iconography' as an approach to employing computers to generate naturalistic art. Adopting this approach, three forms of fractal patterning ('fractal icons') are combined in a variety of ways to generate the rich complexity of nature's scenery. These composite fractals are remarkably effective at conveying nature's aesthetic power.

What is in? What is out? Updating the British Society for Cutaneous Allergy facial series.

BACKGROUND: Allergic contact dermatitis (ACD) to cosmetics is widely reported. To ensure we are accurately diagnosing ACD, patch test series should be continually reviewed to identify relevant and emerging allergens and highlight those that are outdated. The current British Society for Cutaneous Allergy (BSCA) facial series recommends 26 allergens and was last modified in 2012. OBJECTIVES: To review and update the BSCA facial series. METHODS: We retrospectively reviewed the results from 12 UK and Ireland patch test centres' facial series from January 2016 to December 2017. We recorded the number of allergens tested in each centre and the detection rate for each allergen. Using a 0·3% positive rate as the inclusion threshold, we established which allergens in the BSCA facial series had positive patch test rates < 0·3% and > 0·3%. Allergens not in the BSCA facial series that had a positive patch test rate > 0·3% were identified. RESULTS: Overall, 4224 patients were patch tested to the facial series. The number of allergens included in individual centres' facial series ranged from 24 to 66, with a total of 103 allergens tested across all centres. Twelve of the 26 allergens in the BSCA facial series had a positive patch test rate < 0·3% and 14 had a rate > 0·3%. Twenty-five allergens not recommended in the BSCA facial series had a positive patch test rate > 0·3%. CONCLUSIONS: This audit has highlighted the significant variation in practice that exists among patch test centres, despite a recommended facial series. The BSCA facial series has been updated and now contains 24 allergens. Fifteen allergens remain, 11 allergens have been dropped and nine new allergens have been added.

Atrial Fibrillation Genetic Risk and Ischemic Stroke Mechanisms.

BACKGROUND AND PURPOSE: Atrial fibrillation (AF) is a leading cause of cardioembolic stroke, but the relationship between AF and noncardioembolic stroke subtypes are unclear. Because AF may be unrecognized, and because AF has a substantial genetic basis, we assessed for predisposition to AF across ischemic stroke subtypes. METHODS: We examined associations between AF genetic risk and Trial of Org 10172 in Acute Stroke Treatment stroke subtypes in 2374 ambulatory individuals with ischemic stroke and 5175 without from the Wellcome Trust Case-Control Consortium 2 using logistic regression. We calculated AF genetic risk scores using single-nucleotide polymorphisms associated with AF in a previous independent analysis across a range of preselected significance thresholds. RESULTS: There were 460 (19.4%) individuals with cardioembolic stroke, 498 (21.0%) with large vessel, 474 (20.0%) with small vessel, and 814 (32.3%) individuals with strokes of undetermined cause. Most AF genetic risk scores were associated with stroke, with the strongest association (P=6×10-4) attributed to scores of 944 single-nucleotide polymorphisms (each associated with AF at P<1×10-3 in a previous analysis). Associations between AF genetic risk and stroke were enriched in the cardioembolic stroke subset (strongest P=1.2×10-9, 944 single-nucleotide polymorphism score). In contrast, AF genetic risk was not significantly associated with noncardioembolic stroke subtypes. CONCLUSIONS: Comprehensive AF genetic risk scores were specific for cardioembolic stroke. Incomplete workups and subtype misclassification may have limited the power to detect associations with strokes of undetermined pathogenesis. Future studies are warranted to determine whether AF genetic risk is a useful biomarker to enhance clinical discrimination of stroke pathogeneses.

Tag-Count Analysis of Large-Scale Proteomic Data.

Label-free quantitative methods are advantageous in bottom-up (shotgun) proteomics because they are robust and can easily be applied to different workflows without additional cost. Both label-based and label-free approaches are routinely applied to discovery-based proteomics experiments and are widely accepted as semiquantitative. Label-free quantitation approaches are segregated into two distinct approaches: peak-abundance-based approaches and spectral counting (SpC). Peak abundance approaches like MaxLFQ, which is integrated into the MaxQuant environment, require precursor peak alignment that is computationally intensive and cannot be routinely applied to low-resolution data. Not limited by these constraints, SpC approaches simply use the number of peptide identifications corresponding to a given protein as a measurement of protein abundance. We show here that spectral counts from multidimensional proteomic data sets have a mean-dispersion relationship that can be modeled in edgeR. Furthermore, by simulating spectral counts, we show that this approach can routinely be applied to large-scale discovery proteomics data sets to determine differential protein expression.

Pathoscope: species identification and strain attribution with unassembled sequencing data.

Emerging next-generation sequencing technologies have revolutionized the collection of genomic data for applications in bioforensics, biosurveillance, and for use in clinical settings. However, to make the most of these new data, new methodology needs to be developed that can accommodate large volumes of genetic data in a computationally efficient manner. We present a statistical framework to analyze raw next-generation sequence reads from purified or mixed environmental or targeted infected tissue samples for rapid species identification and strain attribution against a robust database of known biological agents. Our method, Pathoscope, capitalizes on a Bayesian statistical framework that accommodates information on sequence quality, mapping quality, and provides posterior probabilities of matches to a known database of target genomes. Importantly, our approach also incorporates the possibility that multiple species can be present in the sample and considers cases when the sample species/strain is not in the reference database. Furthermore, our approach can accurately discriminate between very closely related strains of the same species with very little coverage of the genome and without the need for multiple alignment steps, extensive homology searches, or genome assembly--which are time-consuming and labor-intensive steps. We demonstrate the utility of our approach on genomic data from purified and in silico "environmental" samples from known bacterial agents impacting human health for accuracy assessment and comparison with other approaches.

Identification of replication-dependent and replication-independent linker histone complexes: Tpr specifically promotes replication-dependent linker histone stability.

BACKGROUND: There are 11 variants of linker histone H1 in mammalian cells. Beyond their shared abilities to stabilize and condense chromatin, the H1 variants have been found to have non-redundant functions, the mechanisms of which are not fully understood. Like core histones, there are both replication-dependent and replication-independent linker histone variants. The histone chaperones and other factors that regulate linker histone dynamics in the cell are largely unknown. In particular, it is not known whether replication-dependent and replication-independent linker histones interact with distinct or common sets of proteins. To better understand linker histone dynamics and assembly, we used chromatography and mass spectrometry approaches to identify proteins that are associated with replication-dependent and replication-independent H1 variants. We then used a variety of in vivo analyses to validate the functional relevance of identified interactions. RESULTS: We identified proteins that bind to all linker histone variants and proteins that are specific for only one class of variant. The factors identified include histone chaperones, transcriptional regulators, RNA binding proteins and ribosomal proteins. The nuclear pore complex protein Tpr, which was found to associate with only replication-dependent linker histones, specifically promoted their stability. CONCLUSION: Replication-dependent and replication-independent linker histone variants can interact with both common and distinct sets of proteins. Some of these factors are likely to function as histone chaperones while others may suggest novel links between linker histones and RNA metabolism. The nuclear pore complex protein Tpr specifically interacts with histone H1.1 and H1.2 but not H1x and can regulate the stability of these replication-dependent linker histones.

The non-canonical hydroxylase structure of YfcM reveals a metal ion-coordination motif required for EF-P hydroxylation.

EF-P is a bacterial tRNA-mimic protein, which accelerates the ribosome-catalyzed polymerization of poly-prolines. In Escherichia coli, EF-P is post-translationally modified on a conserved lysine residue. The post-translational modification is performed in a two-step reaction involving the addition of a ß-lysine moiety and the subsequent hydroxylation, catalyzed by PoxA and YfcM, respectively. The ß-lysine moiety was previously shown to enhance the rate of poly-proline synthesis, but the role of the hydroxylation is poorly understood. We solved the crystal structure of YfcM and performed functional analyses to determine the hydroxylation mechanism. In addition, YfcM appears to be structurally distinct from any other hydroxylase structures reported so far. The structure of YfcM is similar to that of the ribonuclease YbeY, even though they do not share sequence homology. Furthermore, YfcM has a metal ion-coordinating motif, similar to YbeY. The metal ion-coordinating motif of YfcM resembles a 2-His-1-carboxylate motif, which coordinates an Fe(II) ion and forms the catalytic site of non-heme iron enzymes. Our findings showed that the metal ion-coordinating motif of YfcM plays an essential role in the hydroxylation of the ß-lysylated lysine residue of EF-P. Taken together, our results suggested the potential catalytic mechanism of hydroxylation by YfcM.

Multiplatform single-sample estimates of transcriptional activation.

Over the past two decades, many biotechnology platforms have been developed for high-throughput gene expression profiling. However, because each platform is subject to technology-specific biases and produces distinct raw-data distributions, researchers have experienced difficulty in integrating data across platforms. Data integration is crucial to data-generating consortiums, researchers transitioning to newer profiling technologies, and individuals seeking to aggregate data across experiments. We address this need with our Universal exPression Code (UPC) approach, which corrects for platform-specific background noise using models that account for the genomic base composition and length of target regions; this approach also uses a mixture model to estimate whether a gene is active in a particular profiling sample. The latter produces standardized UPC values on a zero-to-one scale, so that they can be interpreted consistently, irrespective of profiling technology, thus enabling downstream analysis pipelines to be developed in a platform-agnostic manner. The UPC method can be applied to one- and two-channel expression microarrays and to next-generation sequencing data (RNA sequencing). Furthermore, UPCs are derived using information from within a given sample only--no ancillary samples are required at processing time. Thus, UPCs are suitable for personalized-medicine workflows where samples must be processed individually rather than in batches. In a variety of analyses and comparisons, UPCs perform comparably to other methods designed specifically for microarrays or RNA sequencing in most settings. Software for calculating UPCs is freely available at www.bioconductor.org/packages/release/bioc/html/SCAN.UPC.html.

Common mutation causes cystinosis in the majority of black South African patients.

BACKGROUND: The mutations responsible for cystinosis in South African patients are currently unknown. A pertinent question is whether they are similar to those described elsewhere in the world. METHODS: Children who were being managed for cystinosis in the Western Cape Province of South Africa between 2002 and 2013 were studied. All underwent molecular analysis to detect sequence variations in the cystinosis gene. RESULTS: This cohort study included 20 patients, 13 of whom were Xhosa-speaking black South Africans and seven were Cape Coloureds (mixed race); none were Caucasian. All had nephropathic infantile-type cystinosis with evidence of proximal tubulopathy, with glycosuria and renal phosphate wasting. Diagnosis was confirmed in 19 cases by demonstrating an elevated cystine concentration in leukocytes. Molecular analysis of the cystinosin gene revealed that 19 patients had a G > A mutation in intron 11 (CTNS-c.971-12G > A p.D324AfsX44) which caused an out-of-frame 10-bp insertion. Of these 19 patients, 16 were homozygous for this mutation, which was the most frequent mutation identified in the alleles of the black South African and Cape Coloured patients (96 and 71 %, respectively). CONCLUSION: We recommend that black South African and Cape Coloured patients presenting with cystinosis be tested for CTNS-c.971-12G > A in the first instance, with the possibility of prenatal testing being offered to at-risk families.

Histone H1 phosphorylation in breast cancer.

Breast cancer is the second leading cause of cancer-related deaths in women. The need for new clinical biomarkers in breast cancer is necessary to further predict prognosis and therapeutic response. In this article, the LC-MS histone H1 phosphorylation profiles were established for three distinct breast cancer cell lines. The results show that the extent of H1 phosphorylation can distinguish between the different cell lines. The histone H1 from the metastatic cell line, MDA-MB-231, was subjected to chemical derivitization and LC-MS/MS analysis. The results suggest that the phosphorylation at threonine 146 is found on both histone H1.2 and histone H1.4. Cell lines were then treated with an extracellular stimulus, estradiol or kinase inhibitor LY294002, to monitor changes in histone H1 phosphorylation. The data show that histone H1 phosphorylation can increase and decrease in response to extracellular stimuli. Finally, primary breast tissues were stained for the histone H1 phosphorylation at threonine 146. Variable staining patterns across tumor grades and subtypes were observed with pT146 labeling correlating with tumor grade. These results establish the potential for histone H1 phosphorylation at threonine 146 as a clinical biomarker in breast cancer.

Insight into the molecular recognition of spermine by DNA quadruplexes from an NMR study of the association of spermine with the thrombin-binding aptamer.

The preferred residence sites and the conformation of DNA-bound polyamines are central to understanding the regulatory roles of polyamines. To this end, we have used a series of selective (13)C-edited and selective total correlation spectroscopy-edited one-dimensional (1D) nuclear Overhauser effect spectroscopy NMR experiments to determine a number of intramolecular (1)H nuclear Overhauser effect (NOE) connectivities in (13)C-labelled spermine bound to the thrombin-binding aptamer. The results provide evidence that the aptamer-bound spermine adopts a conformation that optimizes electrostatic and hydrogen bond contacts with the aptamer backbone. The distance between the nitrogen atoms of the central aminobutyl is reduced by an increase in the population of gauche conformers at the C6-C7 bonds, which results in either a curved or S-shaped spermine conformation. Molecular modelling contributes insight toward the mode of spermine binding of these spermine structures within the narrow grooves of DNA quadruplexes. In each case, the N5 ammonium group makes hydrogen bonds with two nearby phosphates across the narrow groove. Our results have implications for the understanding of chromatin structure and the rational design of quadruplex-binding drugs.

Patients' experience of necrotising otitis externa: a qualitative study.

BACKGROUND: Necrotising otitis externa is a serious infective condition. Patients are typically frail, diagnostic delay is common and severe pain is a key feature. This study aimed to qualitatively analyse patient-centred data to identify key themes in the patient's experience. METHODS: Open-ended questionnaires were sent to 28 patients. Responses were qualitatively analysed using a grounded theory approach. Iterative cycles were used to develop codes using a constant comparison technique. Emerging categories were refined to identify core themes. RESULTS: Four main themes emerged: severe pain, mental health, quality of life and diagnostic delays. CONCLUSION: This is the first study to explore patients' perspectives in necrotising otitis externa. It indicates a need to raise awareness of necrotising otitis externa, and to improve symptom management, pain control and quality of life. This valuable information can be used to identify research priorities, guide service improvements, improve clinical care and feed into the development of a Core Outcome Set for necrotising otitis externa.

Deletion of Sod1 in Motor Neurons Exacerbates Age-Related Changes in Axons and Neuromuscular Junctions in Mice.

Whole-body knock-out of Cu,Zn superoxide dismutase (Sod1KO) results in accelerated, age-related loss of muscle mass and function associated with neuromuscular junction (NMJ) breakdown similar to sarcopenia. In order to determine whether altered redox in motor neurons underlies this phenotype, an inducible neuron-specific deletion of Sod1 (i-mnSod1KO) was compared with wild-type (WT) mice of different ages (adult, mid-age, and old) and whole-body Sod1KO mice. Nerve oxidative damage, motor neuron numbers and structural changes to neurons and NMJ were examined. Tamoxifen-induced deletion of neuronal Sod1 from two months of age. No specific effect of a lack of neuronal Sod1 was seen on markers of nerve oxidation (electron paramagnetic resonance of an in vivo spin probe, protein carbonyl, or protein 3-nitrotyrosine contents). i-mnSod1KO mice showed increased denervated NMJ, reduced numbers of large axons and increased number of small axons compared with old WT mice. A large proportion of the innervated NMJs in old i-mnSod1KO mice displayed a simpler structure than that seen in adult or old WT mice. Thus, previous work showed that neuronal deletion of Sod1 induced exaggerated loss of muscle in old mice, and we report that this deletion leads to a specific nerve phenotype including reduced axonal area, increased proportion of denervated NMJ, and reduced acetyl choline receptor complexity. Other changes in nerve and NMJ structure seen in the old i-mnSod1KO mice reflect aging of the mice.

Complete deficiency of the sixth complement component (C6Q0), susceptibility to Neisseria meningitidis infections and analysis of the frequencies of C6Q0 gene defects in South Africans.

Complete complement component 6 deficiency (C6Q0) is a co-dominant genetic disease presenting as increased susceptibility to invasive Neisseria meningitidis infections. Affected individuals have two affected alleles which can be homozygous or compound heterozygous for the particular gene defects they carry. This disorder has been diagnosed relatively frequently in Western Cape South Africans. Affected patients are prescribed penicillin prophylaxis. In 2004 we commenced a clinical follow-up study of 46 patients. Of these, 43 had family age-matched C6 sufficient controls. Participants were classified as either (i) well, or (ii) having a serious illness (SI) or died (D). An SI was a long-term illness that did not allow the performance of normal daily activities. Among 43 patients, 21 were well and 22 were SI/D, while among 43 matched controls, 35 were well and eight were SI/D. This difference is highly significant. Among all 46 C6Q0 patients, those who had had recurrent infection had significantly more SI/D than those who had suffered none or one infection. Thus, this work demonstrates the long-term serious outcome of repeated meningococcal disease (MD) episodes. We investigated the frequencies of four C6Q0 pathogenic mutations known to affect Cape patients (828delG, 1138delC, 821delA and 1879delG) in 2250 newborns. A total of 103 defective alleles (2·28%) and three affected C6Q0 individuals were detected. For all defects combined, 5·24 affected subjects (C6Q0) are expected among 10,000 individuals. What is still unknown is the number of C6Q0 individuals who suffer MD or other infectious diseases.

Skeletal muscle transcriptomics identifies common pathways in nerve crush injury and ageing.

Motor unit remodelling involving repeated denervation and re-innervation occurs throughout life. The efficiency of this process declines with age contributing to neuromuscular deficits. This study investigated differentially expressed genes (DEG) in muscle following peroneal nerve crush to model motor unit remodelling in C57BL/6 J mice. Muscle RNA was isolated at 3 days post-crush, RNA libraries were generated using poly-A selection, sequenced and analysed using gene ontology and pathway tools. Three hundred thirty-four DEG were found in quiescent muscle from (26mnth) old compared with (4-6mnth) adult mice and these same DEG were present in muscle from adult mice following nerve crush. Peroneal crush induced 7133 DEG in muscles of adult and 699 DEG in muscles from old mice, although only one DEG (ZCCHC17) was found when directly comparing nerve-crushed muscles from old and adult mice. This analysis revealed key differences in muscle responses which may underlie the diminished ability of old mice to repair following nerve injury.