Detection and editing of the updated Arabidopsis plastid- and mitochondrial-encoded proteomes through PeptideAtlas.

Arabidopsis (Arabidopsis thaliana) ecotype Col-0 has plastid and mitochondrial genomes encoding over 100 proteins. Public databases (e.g. Araport11) have redundancy and discrepancies in gene identifiers for these organelle-encoded proteins. RNA editing results in changes to specific amino acid residues or creation of start and stop codons for many of these proteins, but the impact of RNA editing at the protein level is largely unexplored due to the complexities of detection. Here, we assembled the nonredundant set of identifiers, their correct protein sequences, and 452 predicted nonsynonymous editing sites of which 56 are edited at lower frequency. We then determined accumulation of edited and/or unedited proteoforms by searching ∼259 million raw tandem MS spectra from ProteomeXchange, which is part of PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/). We identified all mitochondrial proteins and all except 3 plastid-encoded proteins (NdhG/Ndh6, PsbM, and Rps16), but no proteins predicted from the 4 ORFs were identified. We suggest that Rps16 and 3 of the ORFs are pseudogenes. Detection frequencies for each edit site and type of edit (e.g. S to L/F) were determined at the protein level, cross-referenced against the metadata (e.g. tissue), and evaluated for technical detection challenges. We detected 167 predicted edit sites at the proteome level. Minor frequency sites were edited at low frequency at the protein level except for cytochrome C biogenesis 382 at residue 124 (Ccb382-124). Major frequency sites (>50% editing of RNA) only accumulated in edited form (>98% to 100% edited) at the protein level, with the exception of Rpl5-22. We conclude that RNA editing for major editing sites is required for stable protein accumulation.

Detection of the Arabidopsis Proteome and Its Post-translational Modifications and the Nature of the Unobserved (Dark) Proteome in PeptideAtlas.

This study describes a new release of the Arabidopsis thaliana PeptideAtlas proteomics resource (build 2023-10) providing protein sequence coverage, matched mass spectrometry (MS) spectra, selected post-translational modifications (PTMs), and metadata. 70 million MS/MS spectra were matched to the Araport11 annotation, identifying ∼0.6 million unique peptides and 18,267 proteins at the highest confidence level and 3396 lower confidence proteins, together representing 78.6% of the predicted proteome. Additional identified proteins not predicted in Araport11 should be considered for the next Arabidopsis genome annotation. This release identified 5198 phosphorylated proteins, 668 ubiquitinated proteins, 3050 N-terminally acetylated proteins, and 864 lysine-acetylated proteins and mapped their PTM sites. MS support was lacking for 21.4% (5896 proteins) of the predicted Araport11 proteome: the "dark" proteome. This dark proteome is highly enriched for E3 ligases, transcription factors, and for certain (e.g., CLE, IDA, PSY) but not other (e.g., THIONIN, CAP) signaling peptides families. A machine learning model trained on RNA expression data and protein properties predicts the probability that proteins will be detected. The model aids in discovery of proteins with short half-life (e.g., SIG1,3 and ERF-VII TFs) and for developing strategies to identify the missing proteins. PeptideAtlas is linked to TAIR, tracks in JBrowse, and several other community proteomics resources.

Filaggrin-Associated Atopic Skin, Eye, Airways, and Gut Disease, Modifying the Presentation of X-Linked Reticular Pigmentary Disorder (XLPDR).

BACKGROUND: X-linked reticular pigmentary disorder (XLPDR) is a rare condition characterized by skin hyperpigmentation, ectodermal features, multiorgan inflammation, and recurrent infections. All probands identified to date share the same intronic hemizygous POLA1 hypomorphic variant (NM_001330360.2(POLA1):c.1393-354A > G) on the X chromosome. Previous studies have supported excessive type 1 interferon (IFN) inflammation and natural killer (NK) cell dysfunction in disease pathogenesis. Common null polymorphisms in filaggrin (FLG) gene underlie ichthyosis vulgaris and atopic predisposition. CASE: A 9-year-old boy born to non-consanguineous parents developed eczema with reticular skin hyperpigmentation in early infancy. He suffered recurrent chest infections with chronic cough, clubbing, and asthma, moderate allergic rhinoconjunctivitis with keratitis, multiple food allergies, and vomiting with growth failure. Imaging demonstrated bronchiectasis, while gastroscopy identified chronic eosinophilic gastroduodenitis. Interestingly, growth failure and bronchiectasis improved over time without specific treatment. METHODS: Whole-genome sequencing (WGS) using Illumina short-read sequencing was followed by both manual and orthogonal automated bioinformatic analyses for single-nucleotide variants, small insertions/deletions (indels), and larger copy number variations. NK cell cytotoxic function was assessed using 51Cr release and degranulation assays. The presence of an interferon signature was investigated using a panel of six interferon-stimulated genes (ISGs) by QPCR. RESULTS: WGS identified a de novo hemizygous intronic variant in POLA1 (NM_001330360.2(POLA1):c.1393-354A > G) giving a diagnosis of XLPDR, as well as a heterozygous nonsense FLG variant (NM_002016.2(FLG):c.441del, NP_0020.1:p.(Arg151Glyfs*43)). Compared to healthy controls, the IFN signature was elevated although the degree moderated over time with the improvement in his chest disease. NK cell functional studies showed normal cytotoxicity and degranulation. CONCLUSION: This patient had multiple atopic manifestations affecting eye, skin, chest, and gut, complicating the presentation of XLPDR. This highlights that common FLG polymorphisms should always be considered when assessing genotype-phenotype correlations of other genetic variation in patients with atopic symptoms. Additionally, while the patient exhibited an enhanced IFN signature, he does not have an NK cell defect, suggesting this may not be a constant feature of XLPDR.

[ISOLATED FOURTH EXTENSOR COMPARTMENT TENOSYNOVITIS DUE TO EPIDEMIC RELATED CHANGES IN THE WORKPLACE].

INTRODUCTION: We report on cases of isolated fourth extensor compartment tenosynovitis without evidence of systemic inflammation that occurred in the context of alteration in the work environment due to the COVID-19 epidemic. Early identification of the deleterious effects of virtual/technologically-dependent work from home can aid in treatment and prevention of these conditions. We describe the phenomenon and suggest a treatment approach.

Enhanced BMP signaling in Cathepsin K-positive tendon progenitors induces heterotopic ossification.

Heterotopic ossification (HO) is abnormal bone growth in soft tissues that results from injury, trauma, and rare genetic disorders. Bone morphogenetic proteins (BMPs) are critical osteogenic regulators which are involved in HO. However, it remains unclear how BMP signaling interacts with other extracellular stimuli to form HO. To address this question, using the Cre-loxP recombination system in mice, we conditionally expressed the constitutively activated BMP type I receptor ALK2 with a Q207D mutation (Ca-ALK2) in Cathepsin K-Cre labeled tendon progenitors (hereafter "Ca-Alk2:Ctsk-Cre"). Ca-Alk2:Ctsk-Cre mice were viable but they formed spontaneous HO in the Achilles tendon. Histological and molecular marker analysis revealed that HO is formed via endochondral ossification. Ectopic chondrogenesis coincided with enhanced GLI1 production, suggesting that elevated Hedgehog (Hh) signaling is involved in the pathogenesis of HO. Interestingly, focal adhesion kinase, a critical mediator for the mechanotransduction pathway, was also activated in Ca-Alk2:Ctsk-Cre mice. Our findings suggest that enhanced BMP signaling may elevate Hh and mechanotransduction pathways, thereby causing HO in the regions of the Achilles tendon.

Enhanced BMP signaling through ALK2 attenuates keratinocyte differentiation.

Accumulated studies have suggested that bone morphogenetic proteins (BMPs) are critical for skin development. However, it remains elusive how BMP signaling via ALK2 (aka ACVR1), one of the important BMP type I receptors, regulates keratinocyte differentiation. To address this question, we utilized a genetic system that enhances BMP signaling via ALK2 in an epidermis-specific manner in mice (hereafter ca-Alk2:K14-Cre). Ca-Alk2:K14-Cre mice displayed a sticky and hairless skin phenotype with a thinner epidermis incapable of differentiating. Although cellular proliferation and survival were comparable between wild-type and ca-Alk2:K14-Cre mice, skin differentiation was severely hampered in ca-Alk2:K14-Cre mice. To uncover the mechanism of altered keratinocyte differentiation, we performed a transcriptome analysis. As a result, we found that the expression levels of cell cycle inhibitor p21 were increased in ca-Alk2:K14-Cre mice. Our findings suggest that aberrant BMP signaling via ALK2 positively regulates p21 expression that attenuates keratinocyte differentiation, and further highlights the critical role of BMP signaling in skin development.

Dominant negative OTULIN-related autoinflammatory syndrome.

OTU deubiquitinase with linear linkage specificity (OTULIN) regulates inflammation and cell death by deubiquitinating linear ubiquitin chains generated by the linear ubiquitin chain assembly complex (LUBAC). Biallelic loss-of-function mutations causes OTULIN-related autoinflammatory syndrome (ORAS), while OTULIN haploinsuffiency has not been associated with spontaneous inflammation. However, herein, we identify two patients with the heterozygous mutation p.Cys129Ser in OTULIN. Consistent with ORAS, we observed accumulation of linear ubiquitin chains, increased sensitivity to TNF-induced death, and dysregulation of inflammatory signaling in patient cells. While the C129S mutation did not affect OTULIN protein stability or binding capacity to LUBAC and linear ubiquitin chains, it did ablate OTULIN deubiquitinase activity. Loss of activity facilitated the accumulation of autoubiquitin chains on LUBAC. Altered ubiquitination of LUBAC inhibits its recruitment to the TNF receptor signaling complex, promoting TNF-induced cell death and disease pathology. By reporting the first dominant negative mutation driving ORAS, this study expands our clinical understanding of OTULIN-associated pathology.

Health benefits and costs of screening for colorectal cancer in people on dialysis or who have received a kidney transplant.

BACKGROUND: Despite the higher risk of colorectal cancer (CRC) in people with chronic kidney disease, it remains uncertain whether early detection through screening is cost-effective in this setting. We aimed to determine the costs and health benefits of CRC screening in people on dialysis or who have received a kidney transplant. METHODS: Using a government health perspective, three probabilistic Markov models were constructed to compare the cost-effectiveness and cost-utility of annual immunochemical faecal occult blood test (iFOBT) screening against no-screening in a cohort of 1000 patients (age 50-70 years) on dialysis and with kidney transplants. A series of one-way, multi-way and probabilistic sensitivity analyses were conducted to assess the robustness of the model structure and the extent in which the model's assumptions were sensitive to the uncertainties within the input variables. RESULTS: The incremental cost-effectiveness ratios (ICERs) of CRC screening compared with no-screening were $138 828 per quality-adjusted life year [QALY; $122 977 per life year saved (LYS)], $121 973 per QALY ($ 85 095 per LYS) and $44 790 per QALY ($25 036 per LYS) for dialysis patients not listed on the transplant waiting list, patients on the transplant waiting list and patients with kidney transplants, respectively. The test specificity of iFOBT, the starting age of screening and cancer prevalence were influential factors that determined the overall cost-effectiveness of screening in this setting. CONCLUSION: Screening for CRC using iFOBT may reduce cancer-specific mortality in patients on dialysis and with kidney transplants. However, the benefits and costs of screening CRCs in patients on dialysis, especially for those deemed not suitable for transplantation, greatly exceeded the typical thresholds for acceptable cost-effectiveness.

Mapping the Arabidopsis thaliana proteome in PeptideAtlas and the nature of the unobserved (dark) proteome; strategies towards a complete proteome.

This study describes a new release of the Arabidopsis thaliana PeptideAtlas proteomics resource providing protein sequence coverage, matched mass spectrometry (MS) spectra, selected PTMs, and metadata. 70 million MS/MS spectra were matched to the Araport11 annotation, identifying ∼0.6 million unique peptides and 18267 proteins at the highest confidence level and 3396 lower confidence proteins, together representing 78.6% of the predicted proteome. Additional identified proteins not predicted in Araport11 should be considered for building the next Arabidopsis genome annotation. This release identified 5198 phosphorylated proteins, 668 ubiquitinated proteins, 3050 N-terminally acetylated proteins and 864 lysine-acetylated proteins and mapped their PTM sites. MS support was lacking for 21.4% (5896 proteins) of the predicted Araport11 proteome - the 'dark' proteome. This dark proteome is highly enriched for certain ( e.g. CLE, CEP, IDA, PSY) but not other ( e.g. THIONIN, CAP,) signaling peptides families, E3 ligases, TFs, and other proteins with unfavorable physicochemical properties. A machine learning model trained on RNA expression data and protein properties predicts the probability for proteins to be detected. The model aids in discovery of proteins with short-half life ( e.g. SIG1,3 and ERF-VII TFs) and completing the proteome. PeptideAtlas is linked to TAIR, JBrowse, PPDB, SUBA, UniProtKB and Plant PTM Viewer.

Selective dysregulation of the FcgammaIIB receptor on memory B cells in SLE.

The inappropriate expansion and activation of autoreactive memory B cells and plasmablasts contributes to loss of self-tolerance in systemic lupus erythematosus (SLE). Defects in the inhibitory Fc receptor, FcgammaRIIB, have been shown to contribute to B cell activation and autoimmunity in several mouse models of SLE. In this paper, we demonstrate that expression of FcgammaRIIB is routinely up-regulated on memory B cells in the peripheral blood of healthy controls, whereas up-regulation of FcgammaRIIB is considerably decreased in memory B cells of SLE patients. This directly correlates with decreased FcgammaRIIB-mediated suppression of B cell receptor-induced calcium (Ca2+) response in those B cells. We also found substantial overrepresentation of African-American patients among those who failed to up-regulate FcgammaRIIB. These results suggest that the inhibitory receptor, FcgammaRIIB, may be impaired at a critical checkpoint in SLE in the regulation of memory B cells; thus, FcgammaRIIB represents a novel target for therapeutic interventions in this disease.

Prolactinoma and systemic lupus erythematosus: do serum prolactin levels matter?

The lactogenic hormone prolactin is produced in part by cells of the immune system and serves as an upregulator of immune function. Hyperprolactinemia is common in patients with systemic lupus erythematosus (SLE), raising the possibility that the hormone contributes to the excessive immune response in the disease. The highest levels of circulating prolactin occur in association with prolactin-secreting tumors, but prolactinomas have only rarely been encountered in patients with SLE. We present here three patients with SLE and prolactinomas. As with the previously reported six patients, there was no consistency in the presence of findings related to prolactin excess or in the coincidence of hyperprolactinemia with flares of SLE disease activity. We speculate that this may be due to genetic differences in the response to prolactin and/or to the presence of variant prolactin isoforms detected in the clinical immunoassay that have reduced or absent biologic activity.

Effects of immediate feedback on learning auditory perceptual voice quality evaluation.

The study investigated the effect of immediate feedback in training listeners to perceive subtle differences in voice quality, a perceptual skill that is important for speech-language pathologists. Sixty naive listeners were randomly assigned to a feedback group (Group F), a no feedback group (Group NF), and a no training group acting as a control group (Group C). The task was to evaluate the severity of a perceptual voice quality (breathiness) by using a reference-matching paradigm. All participants took part in three rating sessions (pre-training, 2 days after training and 1 week after training). Group F and Group NF participated in a training session immediately after the first rating session, where Group F practiced with immediate feedback given and Group NF practice with no immediate feedback given. The results showed that Group F and Group NF had significant improvement after training, but Group F did not retain the improvement in the third rating session. The use of a reference-matching training paradigm without giving frequent immediate feedback is suggested for auditory-perceptual voice evaluation training. The most effective frequency of immediate feedback is yet to be determined.