Optimizing Spectronaut Search Parameters to Improve Data Quality with Minimal Proteome Coverage Reductions in DIA Analyses of Heterogeneous Samples.

Data-independent acquisition has seen breakthroughs that enable comprehensive proteome profiling using short gradients. As the proteome coverage continues to increase, the quality of the data generated becomes much more relevant. Using Spectronaut, we show that the default search parameters can be easily optimized to minimize the occurrence of false positives across different samples. Using an immunological infection model system to demonstrate the impact of adjusting search settings, we analyzed Mus musculus macrophages and compared their proteome to macrophages spiked withCandida albicans. This experimental system enabled the identification of "false positives" as Candida albicans peptides and proteins should not be present in the Mus musculus-only samples. We show that adjusting the search parameters reduced "false positive" identifications by 89% at the peptide and protein level, thereby considerably increasing the quality of the data. We also show that these optimized parameters incurred a moderate cost, only reducing the overall number of "true positive" identifications across each biological replicate by <6.7% at both the peptide and protein level. We believe the value of our updated search parameters extends beyond a two-organism analysis and would be of great value to any DIA experiment analyzing heterogeneous populations of cell types or tissues.

Bronchiectasis with Chronic Rhinosinusitis Is Associated with Eosinophilic Airway Inflammation and Is Distinct from Asthma.

Rationale: Bronchiectasis is an airway inflammatory disease that is frequently associated with chronic rhinosinusitis (CRS). An eosinophilic endotype of bronchiectasis has recently been described, but detailed testing to differentiate eosinophilic bronchiectasis from asthma has not been performed. Objectives: This prospective observational study aimed to test the hypotheses that bronchiectasis with CRS is enriched for the eosinophilic phenotype in comparison with bronchiectasis alone and that the eosinophilic bronchiectasis phenotype exists as a separate entity from bronchiectasis associated with asthma. Methods: People with idiopathic or postinfectious bronchiectasis were assessed for concomitant CRS. We excluded people with asthma or primary ciliary dyskinesia and smokers. We assessed sputum and blood cell counts, nasal NO and fractional excreted NO, methacholine reactivity, skin allergy testing and total and specific immunoglobulin (Ig) E, cytokines in the sputum and serum, and the microbiome in the sputum and nasopharynx. Results: A total of 22 people with CRS (BE + CRS) and 17 without CRS (BE - CRS) were included. Sex, age, Reiff score, and bronchiectasis severity were similar. Median sputum eosinophil percentages were 0% (IQR, 0-1.5%) in BE - CRS and 3% (1-12%) in BE + CRS (P = 0.012). Blood eosinophil counts were predictive of sputum eosinophilia (counts ⩾3%; area under the receiver operating characteristic curve, 0.68; 95% confidence interval, 0.50-0.85). Inclusion of CRS improved the prediction of sputum eosinophilia by blood eosinophil counts (area under the receiver operating characteristic curve, 0.79; 95% confidence interval, 0.65-0.94). Methacholine tests were negative in 85.7% of patients in the BE - CRS group and 85.2% of patients in the BE + CRS group (P > 0.99). Specific IgE and skin testing were similar between the groups, but total IgE levels were increased in people with increased sputum eosinophils. Microbiome analysis demonstrated distinct microbiota in nasopharyngeal and airway samples in the BE + CRS and BE - CRS groups, without significant differences between groups. However, interactome analysis revealed altered interactomes in individuals with high sputum eosinophil counts and CRS. Conclusions: Bronchiectasis with CRS is associated with an eosinophilic airway inflammation that is distinct from asthma.

Successful radical surgical resection of a giant neurofibroma.

Large neurofibromas often cause significant patient morbidity and present a unique challenge to dermatologists and surgeons. Radical resection offers the lowest rate of recurrence but is not often pursued due to the high risk of intraoperative hemorrhage and difficulty in repairing large defects. Subtotal resection and debulking are more frequently performed, leading to higher rates of recurrence. This case highlights a particularly large neurofibroma and demonstrates how surgical techniques like preoperative embolization and advancement flaps can improve outcomes in the radical resection of large neurofibromas.

The putative role of the epipeptide EpeX in Bacillus subtilis intra-species competition.

Bacteria engage in competitive interactions with neighbours that can either be of the same or different species. Multiple mechanisms are deployed to ensure the desired outcome and one tactic commonly implemented is the production of specialised metabolites. The Gram-positive bacterium Bacillus subtilis uses specialized metabolites as part of its intra-species competition determinants to differentiate between kin and non-kin isolates. It is, however, unknown if the collection of specialized metabolites defines competitive fitness when the two isolates start as a close, interwoven community that grows into a densely packed colony biofilm. Moreover, the identity of specialized metabolites that have an active role in defining the outcome of an intra-species interaction has not been revealed. Here, we determine the competition outcomes that manifest when 21 environmental isolates of B. subtilis are individually co-incubated with the model isolate NCIB 3610 in a colony biofilm. We correlated these data with the suite of specialized metabolite biosynthesis clusters encoded by each isolate. We found that the epeXEPAB gene cluster was primarily present in isolates with a strong competitive phenotype. This cluster is responsible for producing the epipeptide EpeX. We demonstrated that EpeX is a competition determinant of B. subtilis in an otherwise isogenic context for NCBI 3610. However, when we competed the NCIB 3610 EpeX-deficient strain against our suite of environmental isolates we found that the impact of EpeX in competition is isolate-specific, as only one of the 21 isolates showed increased survival when EpeX was lacking. Taken together, we have shown that EpeX is a competition determinant used by B. subtilis that impacts intra-species interactions but only in an isolate-specific manner.

Folliculocentric lymphocytic hypersensitivity reactions in CLL/SLL patients: A unique clinicopathologic entity amongst non-specific hypersensitivity reactions.

Background: Cutaneous hypersensitivity eruptions in chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) are a clinically and histologically heterogeneous group that can either precede, occur with, or follow the development of a hematologic malignancy. Therefore, establishing the diagnosis requires careful clinical and pathologic correlation and an understanding of the broad spectrum of presentations. Data is lacking on the correlation of skin disease with molecular/cytogenetic risk profiling of the tumor. Objectives: The aims of this study were to characterize the clinical, histological, and genetic aberrations in recurrent cutaneous hypersensitivity reactions in patients with CLL/SLL. Methods: A single site academic retrospective chart review of medical records, histopathology, molecular and cytogenetic data in CLL/SLL patients who developed biopsy-proven cutaneous hypersensitivity reactions. Results: Five hundred one new diagnoses of CLL/SLL with 73 patients requiring cutaneous biopsies for skin lesions or rashes were identified. With exclusion criteria, 20 biopsies were identified from 17 patients (mean age, 69.6 years, females = 9) with unexplained cutaneous eruptions. These were commonly pruritic, erythematous papules above the waist. Most biopsies had a prominent superficial, deep dermal eosinophilic infiltrate (85%), with a robust T-cell predominant dermal infiltrate in 40%. Five out of 17 patients (29%) had a predominately folliculocentric CD4+ T-cell infiltrate; all occurring on the head and neck. Overall, the prevalence of cutaneous hypersensitivity eruptions requiring biopsy was 3.4% (n = 17), and the prevalence of folliculocentric CD4+ T-cell infiltrate was 1% (n = 5). Conclusion: Cutaneous hypersensitivity reactions in CLL/SLL are heterogeneous; however, folliculotropic CD4+ T-cell infiltrates may be seen in a small but distinct clinical subset of patients. Commonly tested cytogenetic aberrations in CLL/SLL do not appear to be correlated with the presence of cutaneous hypersensitivity reactions.

The adenomatous polyposis coli protein 30 years on.

Mutations in the gene encoding the Adenomatous polyposis coli protein (APC) were discovered as driver mutations in colorectal cancers almost 30 years ago. Since then, the importance of APC in normal tissue homeostasis has been confirmed in a plethora of other (model) organisms spanning a large evolutionary space. APC is a multifunctional protein, with roles as a key scaffold protein in complexes involved in diverse signalling pathways, most prominently the Wnt signalling pathway. APC is also a cytoskeletal regulator with direct and indirect links to and impacts on all three major cytoskeletal networks. Correspondingly, a wide range of APC binding partners have been identified. Mutations in APC are extremely strongly associated with colorectal cancers, particularly those that result in the production of truncated proteins and the loss of significant regions from the remaining protein. Understanding the complement of its role in health and disease requires knowing the relationship between and regulation of its diverse functions and interactions. This in turn requires understanding its structural and biochemical features. Here we set out to provide a brief overview of the roles and function of APC and then explore its conservation and structure using the extensive sequence data, which is now available, and spans a broad range of taxonomy. This revealed conservation of APC across taxonomy and new relationships between different APC protein families.

Clinicohistopathologic challenges and traps in the diagnosis of nail unit melanoma.

Melanoma of the nail apparatus is challenging to diagnose for both dermatologists and dermatopathologists. Misdiagnosis or delayed diagnosis of nail unit melanoma can have fatal consequences and legal ramifications. This review educates dermatopathologists on challenges and traps they should be aware of to avoid misdiagnosis of nail unit melanoma. We present illustrative difficult cases that introduce several themes regarding challenges in the diagnosis of nail unit melanoma: specimens with subtle histopathologic findings, challenges in immunoperoxidase interpretation, and how clinical knowledge and surgical procedural knowledge are mandatory to make the diagnosis. Dermatopathologists will be aware of when and how to suspect nail unit melanoma in unusual circumstances.

A call for nail clipping histopathology to become an essential component of the routine evaluation of melanonychia: Benefitting patients as a triage and surgical planning maneuver.

We call on dermatologists and dermatopathologists to include nail clipping histopathology as an essential component of the routine evaluation of melanonychia. This manuscript demonstrates a case where an adult woman with broad melanonychia of the right thumbnail declined a nail matrix biopsy, but was amenable to a nail clipping.The nail clipping showed pigmentation, melanocyte remnants, and small cavities in the nail plate. These features have been published previously by our group as a clue to nail unit melanoma within nail clippings.This patient was rapidly triaged for nail matrix biopsy, which demonstrated nail unit melanoma in situ. Every patient with melanonychia can benefit from a nail clipping by examination of the location of the pigmentation within the nail plate for surgical planning, and if melanocyte remnants are detected, the nail clipping also serves as a rapid triage mechanism for nail matrix biopsy to evaluate for nail unit melanoma. Fontana-stained sections will highlight the pigmentation in the nail plate, and its location in the nail plate can easily be described by the dermatopathologist. Nail clippings performed in the setting of clinically apparent melanonychia may show helpful histopathologic findings of pigmented fungi, hemorrhage, external pigmentation, features of other pigmented nail unit tumors, as well as other entities. Nail clipping histopathology can provide extensive information in the evaluation of melanonychia with minimal discomfort for a patient, and little disruption to a physician's clinic flow. With this additional case of a nail unit melanoma diagnosed after initial concern found in a nail clipping, as well as other information in the literature, it is clear that melanocyte remnants found in nail clippings are reliable concerning features related to nail unit melanoma in adults. With knowledge of these histopathologic features in nail clippings and the significance of melanocyte remnants, the dermatopathologist can play a crucial role in the use of a nail clipping as a life-saving diagnostic maneuver. Accordingly, given the potential benefit to patients in this setting, as well as other uses of a nail clipping in the evaluation of melanonychia, we call on dermatologists and dermatopathologists to innovate the routine evaluation of melanonychia through the routine employment of nail clippings for histopathologic evaluation.

Pleomorphic acquired digital fibrokeratoma: A novel clinicopathologic entity.

A 26-year-old male presented with a 2-year history of a hyperkeratotic growth from the left index finger. Histopathology was consistent with an acquired digital fibrokeratoma with changes of a pleomorphic fibroma. Lesional cells were negative for CD34, Rb, and p53, and were positive for FXIIIa. We introduce the pleomorphic acquired digital fibrokeratoma as a novel clinicopathologic entity.

Mycetomalike Skin Infection Due to Gordonia bronchialis in an Immunocompetent Patient.

Gordonia bronchialis is a partially acid-fast, gram-positive rod that has been found in a variety of nosocomial infections, most frequently sternal wound infection following coronary artery bypass surgery. We report a case of a mycetomalike infection due to G bronchialis in an immunocompetent patient with complete resolution after 3 months of oral antibiotics.

Defining Composition and Function of the Rhizosphere Microbiota of Barley Genotypes Exposed to Growth-Limiting Nitrogen Supplies.

The microbiota populating the rhizosphere, the interface between roots and soil, can modulate plant growth, development, and health. These microbial communities are not stochastically assembled from the surrounding soil, but their composition and putative function are controlled, at least partially, by the host plant. Here, we use the staple cereal barley as a model to gain novel insights into the impact of differential applications of nitrogen, a rate-limiting step for global crop production, on the host genetic control of the rhizosphere microbiota. Using a high-throughput amplicon sequencing survey, we determined that nitrogen availability for plant uptake is a factor promoting the selective enrichment of individual taxa in the rhizosphere of wild and domesticated barley genotypes. Shotgun sequencing and metagenome-assembled genomes revealed that this taxonomic diversification is mirrored by a functional specialization, manifested by the differential enrichment of multiple Gene Ontology terms, of the microbiota of plants exposed to nitrogen conditions limiting barley growth. Finally, a plant soil feedback experiment revealed that host control of the barley microbiota underpins the assembly of a phylogenetically diverse group of bacteria putatively required to sustain plant performance under nitrogen-limiting supplies. Taken together, our observations indicate that under nitrogen conditions limiting plant growth, host-microbe and microbe-microbe interactions fine-tune the host genetic selection of the barley microbiota at both taxonomic and functional levels. The disruption of these recruitment cues negatively impacts plant growth. IMPORTANCE The microbiota inhabiting the rhizosphere, the thin layer of soil surrounding plant roots, can promote the growth, development, and health of their host plants. Previous research indicated that differences in the genetic composition of the host plant coincide with variations in the composition of the rhizosphere microbiota. This is particularly evident when looking at the microbiota associated with input-demanding modern cultivated varieties and their wild relatives, which have evolved under marginal conditions. However, the functional significance of these differences remains to be fully elucidated. We investigated the rhizosphere microbiota of wild and cultivated genotypes of the global crop barley and determined that nutrient conditions limiting plant growth amplify the host control on microbes at the root-soil interface. This is reflected in a plant- and genotype-dependent functional specialization of the rhizosphere microbiota, which appears to be required for optimal plant growth. These findings provide novel insights into the significance of the rhizosphere microbiota for plant growth and sustainable agriculture.

Identifying plant genes shaping microbiota composition in the barley rhizosphere.

A prerequisite to exploiting soil microbes for sustainable crop production is the identification of the plant genes shaping microbiota composition in the rhizosphere, the interface between roots and soil. Here, we use metagenomics information as an external quantitative phenotype to map the host genetic determinants of the rhizosphere microbiota in wild and domesticated genotypes of barley, the fourth most cultivated cereal globally. We identify a small number of loci with a major effect on the composition of rhizosphere communities. One of those, designated the QRMC-3HS, emerges as a major determinant of microbiota composition. We subject soil-grown sibling lines harbouring contrasting alleles at QRMC-3HS and hosting contrasting microbiotas to comparative root RNA-seq profiling. This allows us to identify three primary candidate genes, including a Nucleotide-Binding-Leucine-Rich-Repeat (NLR) gene in a region of structural variation of the barley genome. Our results provide insights into the footprint of crop improvement on the plant's capacity of shaping rhizosphere microbes.

Histological Transformation and Clonal Relationship of Subcutaneous Marginal Zone B-Cell Lymphoma and Diffuse Large B-Cell Lymphoma.

ABSTRACT: Histological transformation (HT) is an exceptionally uncommon and poorly understood event where a low-grade or indolent B-cell lymphoma transforms into a more aggressive entity, typically diffuse large B-cell lymphoma (DLBCL). The pathogenesis is unclear; however, HT is associated with a worse prognosis. This article reports a unique case of marginal zone lymphoma (MZL) limited to skin/subcutis (confirmed with PET-CT) that subsequently developed DLBCL, followed by nodal MZL. We explored phenotypic, molecular genetic, and cytogenetic findings in subcutaneous MZL with HT to DLBCL and subsequent progression to systemic MZL. Shared clonal peaks between the tumors were demonstrated through immunoglobulin heavy chain PCR, and genomic microarray analysis revealed both unique genomic abnormalities and shared regions of copy-neutral loss of heterozygosity in all specimens. BCL-2 expression was present in the original subcutaneous MZL, lost on conversion to Primary cutaneous diffuse large B cell lymphoma (PCDLBCL)-NOS, and regained during subsequent transformation to systemic MZL. The PCDLBCL-NOS did not demonstrate FISH rearrangements for MYC, BCL2, and BCL6. Here, we describe the histologic, immunophenotypic, and cytogenetic abnormalities of the clonally related transformation of subcutaneous MZL, PCDLBCL-NOS, and eventual systemic MZL. The predominantly subcutaneous presentation of MZL may be associated with a more aggressive outcome and raises consideration for careful evaluation of patients who present with this pattern.