Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures.

Double-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.

Homozygous mutation in the Neurofascin gene affecting the glial isoform of Neurofascin causes severe neurodevelopment disorder with hypotonia, amimia and areflexia.

The Neurofascins (NFASCs) are a family of proteins encoded by alternative transcripts of NFASC that cooperate in the assembly of the node of Ranvier in myelinated nerves. Differential expression of NFASC in neurons and glia presents a remarkable example of cell-type specific expression of protein isoforms with a common overall function. In mice there are three NFASC isoforms: Nfasc186 and Nfasc140, located in the axonal membrane at the node of Ranvier, and Nfasc155, a glial component of the paranodal axoglial junction. Nfasc186 and Nfasc155 are the major isoforms at mature nodes and paranodes, respectively. Conditional deletion of the glial isoform Nfasc155 in mice causes severe motor coordination defects and death at 16-17 days after birth. We describe a proband with severe congenital hypotonia, contractures of fingers and toes, and no reaction to touch or pain. Whole exome sequencing revealed a homozygous NFASC variant chr1:204953187-C>T (rs755160624). The variant creates a premature stop codon in 3 out of four NFASC human transcripts and is predicted to specifically eliminate Nfasc155 leaving neuronal Neurofascin intact. The selective absence of Nfasc155 and disruption of the paranodal junction was confirmed by an immunofluorescent study of skin biopsies from the patient versus control. We propose that the disease in our proband is the first reported example of genetic deficiency of glial Neurofascin isoforms in humans and that the severity of the condition reflects the importance of the Nfasc155 in forming paranodal axoglial junctions and in determining the structure and function of the node of Ranvier.

Feasibility Study of a Novel Protease-Activated Fluorescent Imaging System for Real-Time, Intraoperative Detection of Residual Breast Cancer in Breast Conserving Surgery.

BACKGROUND: Obtaining tumor-free margins is critical to prevent recurrence after lumpectomy for breast cancer. Unfortunately, current approaches leave positive margins that require second surgeries in 20-40% of patients. We assessed the LUM Imaging System for real-time, intraoperative detection of residual tumor. METHODS: Breast lumpectomy cavity walls and excised specimens were assessed with the LUM Imaging System after 1 mg/kg intravenous LUM015, a protease-activatable fluorescent agent. Fluorescence at potential sites of residual tumor in lumpectomy cavity walls was evaluated intraoperatively with a sterile hand-held probe, with real-time predictive results displayed on a monitor intraoperatively, and later correlated with histopathology. RESULTS: In vivo lumpectomy cavities and excised specimens were imaged after LUM015 injection in 45 women undergoing breast cancer surgery. Invasive ductal and lobular cancers and intraductal cancer (DCIS) were included. A total of 570 cavity margin surfaces in 40 patients were used for algorithm development. Image analysis and display took approximately 1 s per 2.6-cm-diameter circular margin surface. All breast cancer subtypes could be distinguished from adjacent normal tissue. For all imaged cavity surfaces, sensitivity for tumor detection was 84%. Among 8 patients with positive margins after standard surgery, sensitivity for residual tumor detection was 100%; 2 of 8 were spared second surgeries because additional tissue was excised at sites of LUM015 signal. Specificity was 73%, with some benign tissues showing elevated fluorescent signal. CONCLUSIONS: The LUM015 agent and LUM Imaging System allow rapid identification of residual tumor in the lumpectomy cavity of breast cancer patients and may reduce rates of positive margins.

Correction to: Feasibility Study of a Novel Protease-Activated Fluorescent Imaging System for Real-Time, Intraoperative Detection of Residual Breast Cancer in Breast Conserving Surgery.

The article Feasibility Study of a Novel Protease-Activated Fluorescent Imaging System for Real-Time, Intraoperative Detection of Residual Breast Cancer in Breast Conserving Surgery, written by Barbara L. Smith et al., was originally published electronically on the publisher's internet portal on January 2, 2020, without open access.

Mapping of breakpoints in balanced chromosomal translocations by shallow whole-genome sequencing points to EFNA5, BAHD1 and PPP2R5E as novel candidates for genes causing human Mendelian disorders.

BACKGROUND: Mapping the breakpoints in de novo balanced chromosomal translocations (BCT) in symptomatic individuals provides a unique opportunity to identify in an unbiased way the likely causative genetic defect and thus find novel human disease candidate genes. Our aim was to fine-map breakpoints of de novo BCTs in a case series of nine patients. METHODS: Shallow whole-genome mate pair sequencing (SGMPS) together with long-range PCR and Sanger sequencing. In one case (BCT disrupting BAHD1 and RET) cDNA analysis was used to verify expression of a fusion transcript in cultured fibroblasts. RESULTS: In all nine probands 11 disrupted genes were found, that is, EFNA5, EBF3, LARGE, PPP2R5E, TXNDC5, ZNF423, NIPBL, BAHD1, RET, TRPS1 and SLC4A10. Five subjects had translocations that disrupted genes with so far unknown (EFNA5, BAHD1, PPP2R5E, TXNDC5) or poorly delineated impact on the phenotype (SLC4A10, two previous reports of BCT disrupting the gene). The four genes with no previous disease associations (EFNA5, BAHD1, PPP2R5E, TXNDC5), when compared with all human genes by a bootstrap test, had significantly higher pLI (p<0.017) and DOMINO (p<0.02) scores indicating enrichment in genes likely to be intolerant to single copy damage. Inspection of individual pLI and DOMINO scores, and local topologically associating domain structure suggested that EFNA5, BAHD1 and PPP2R5E were particularly good candidates for novel disease loci. The pathomechanism for BAHD1 may involve deregulation of expression due to fusion with RET promoter. CONCLUSION: SGMPS in symptomatic carriers of BCTs is a powerful approach to delineate novel human gene-disease associations.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.

Novel de novo mutation affecting two adjacent aminoacids in the EED gene in a patient with Weaver syndrome.

Overgrowth, macrocephaly, accelerated osseous maturation, variable intellectual disability, and characteristic facial features are the main symptoms of Weaver syndrome, a rare condition caused by mutations in EZH2 gene. Recently, in four patients with Weaver-like symptoms without mutations in EZH2 gene, pathogenic variants in EED were described. We present another patient clinically diagnosed with Weaver syndrome in whom WES revealed an EED de novo mutation affecting two neighboring aminoacids, NM_003797.3:c.917_919delinsCGG/p.(Arg306_Asn307delinsThrAsp) located in one allele (in cis). Our observation, together with previous reports suggests that EED gene testing is warranted in patients with the overgrowth syndrome features and suspicion of Weaver syndrome with normal results of EZH2 gene sequencing.

Evidence for HNRNPH1 being another gene for Bain type syndromic mental retardation.

The HNRNPH2-associated disease (mental retardation, X-linked, syndromic, Bain type [MRXSB, MIM #300986]) is caused by de novo mutations in the X-linked HNRNPH2 gene. MRXSB has been described in six female patients with dysmorphy, developmental delay, intellectual disability, autism, hypotonia and seizures. The reported HNRNPH2 mutations were clustered in the small domain encoding nuclear localization signal; in particular, the p.Arg206Trp was found in four independent de novo events. HNRNPH1 is a conserved autosomal paralogue of HNRNPH2 with a similar function in regulation of pre-mRNAs splicing but so far it has not been associated with human disease. We describe a boy with a disease similar to MRXSB in whom a novel de novo mutation c.616C>T (p.Arg206Trp) in HNRNPH1 was found (ie, the exact paralogue of the recurrent HNRNPH2 mutation). We propose that defective function of HNRNPH2 and HNRNPH1 nuclear localization signal has similar clinical consequences. An important difference between the two diseases is that the HNRNPH1-associated syndrome may occur in boys (as in the case of our proband) which is well explained by the autosomal (chr5q35.3) rather than X-linked localization of the HNRNPH2 gene.

Neurodevelopmental phenotype caused by a de novo PTPN4 single nucleotide variant disrupting protein localization in neuronal dendritic spines.

Protein tyrosine phosphatase non-receptor type 4 (PTPN4) encodes non-receptor protein tyrosine phosphatase implicated in synaptic plasticity and innate immune response. The only report of PTPN4-associated disease described a neurodevelopmental disorder associated with a whole gene deletion. We describe a child with developmental delay, autistic features, hypotonia, increased immunoglobulin E and dental problems with a novel mosaic de novo variant in PTPN4 (hg19 chr2:g.120620188 T > C, NM_002830.3:p.[Leu72Ser]/c.215T>C) located in domain that controls protein subcellular distribution. Studies in mouse hippocampal neurons transfected with non-mutated or mutated human PTPN4 showed that despite their similar expression in neurons the mutated protein was absent from dendritic spines. Next, we studied patient's primary blood mononuclear cells' response to lipopolysaccharide stimulation and found no difference from control in phosphorylation of TBK1 and IRF3 (involved in Toll-like receptor 4 signaling) and induction of cytokines' messenger RNA. We conclude that the PTPN4 p.(Leu72Ser) variant is a likely cause of neurodevelopmental symptoms of our proband whereas its role in immune dysfunction requires further studies.

Cytoplasmic Cyclin E and Phospho-Cyclin-Dependent Kinase 2 Are Biomarkers of Aggressive Breast Cancer.

Cyclin E and its co-activator, phospho-cyclin-dependent kinase 2 (p-CDK2), regulate G1 to S phase transition and their deregulation induces oncogenesis. Immunohistochemical assessments of these proteins in cancer have been reported but were based only on their nuclear expression. However, the oncogenic forms of cyclin E (low molecular weight cyclin E or LMW-E) in complex with CDK2 are preferentially mislocalized to the cytoplasm. Here, we used separate nuclear and cytoplasmic scoring systems for both cyclin E and p-CDK2 expression to demonstrate altered cellular accumulation of these proteins using immunohistochemical analysis. We examined the specificity of different cyclin E antibodies and evaluated their concordance between immunohistochemical and Western blot analyses in a panel of 14 breast cell lines. Nuclear versus cytoplasmic staining of cyclin E readily differentiated full-length from LMW-E, respectively. We also evaluated the expression of cyclin E and p-CDK2 in 1676 breast carcinoma patients by immunohistochemistry. Cytoplasmic cyclin E correlated strongly with cytoplasmic p-CDK2 (P < 0.0001), high tumor grade, negative estrogen/progesterone receptor status, and human epidermal growth factor receptor 2 positivity (all P < 0.0001). In multivariable analysis, cytoplasmic cyclin E plus phosphorylated CDK2 (as one variable) predicted breast cancer recurrence-free and overall survival. These results suggest that cytoplasmic cyclin E and p-CDK2 can be readily detected with immunohistochemistry and used as clinical biomarkers for aggressive breast cancer.

Thrombospondin-1 induction in the diabetic myocardium stabilizes the cardiac matrix in addition to promoting vascular rarefaction through angiopoietin-2 upregulation.

RATIONALE: Diabetes mellitus is associated with cardiac fibrosis. Matricellular proteins are induced in fibrotic conditions and modulate fibrogenic and angiogenic responses by regulating growth factor signaling. OBJECTIVE: Our aim was to test the hypothesis that the prototypical matricellular protein thrombospondin (TSP)-1, a potent angiostatic molecule and crucial activator of transforming growth factor-ß, may play a key role in remodeling of the diabetic heart. METHODS AND RESULTS: Obese diabetic db/db mice exhibited marked myocardial TSP-1 upregulation in the interstitial and perivascular space. To study the role of TSP-1 in remodeling of the diabetic heart, we generated and characterized db/db TSP-1(-/-) (dbTSP) mice. TSP-1 disruption did not significantly affect weight gain and metabolic function in db/db animals. When compared with db/db animals, dbTSP mice had increased left ventricular dilation associated with mild nonprogressive systolic dysfunction. Chamber dilation in dbTSP mice was associated with decreased myocardial collagen content and accentuated matrix metalloproteinase-2 and -9 activity. TSP-1 disruption did not affect inflammatory gene expression and activation of transforming growth factor-ß/small mothers against decapendaplegic signaling in the db/db myocardium. In cardiac fibroblasts populating collagen pads, TSP-1 incorporation into the matrix did not activate transforming growth factor-ß responses, but inhibited leptin-induced matrix metalloproteinase-2 activation. TSP-1 disruption abrogated age-associated capillary rarefaction in db/db mice, attenuating myocardial upregulation of angiopoietin-2, a mediator that induces vascular regression. In vitro, TSP-1 stimulation increased macrophage, but not endothelial cell, angiopoietin-2 synthesis. CONCLUSIONS: TSP-1 upregulation in the diabetic heart prevents chamber dilation by exerting matrix-preserving actions on cardiac fibroblasts and mediates capillary rarefaction through effects that may involve angiopoietin-2 upregulation.

Fate of nondiagnostic thyroid fine needle aspirations.

BACKGROUND: Thyroid nodules may be detected during the workup of thyroid hormone abnormalities and as incidental findings during unrelated imaging studies. The diagnosis of a thyroid nodule is mainly established by performing fine needle aspiration (FNA) under ultrasound guidance. Thyroid nodules are classified as nondiagnostic, defined in the Bethesda System for Reporting Thyroid Cytopathology as samples with excess blood, cyst fluid only, and lack of thyroid follicular cells. The current study evaluates a series of nondiagnostic FNAs to assess whether repeat sampling improves yield and what patient management, and outcomes are after a nondiagnostic FNA. METHODS: Thyroid FNAs from 2016 to 2023 were retrieved from our institution archives. All cases were performed under ultrasound guidance and with rapid on-site evaluation. Cases were assigned the Bethesda System Category. Nondiagnostic FNAs were further reviewed for repeat FNA procedures, potential molecular testing, or diagnostic resections. RESULTS: In total 3104 thyroid FNAs were reviewed, with 153 (4.9%) being nondiagnostic. Of the 154 FNAs, there were 129 patients with an average age of 60 and a male-to-female ratio of 1:3.2. Of the 130 patients, there were 50 patients who underwent 55 repeat FNAs. Thirty-seven (67%) of the repeats were benign, 13 (24%) were nondiagnostic again, and 5 (9%) were atypia of undetermined significance (AUS). Molecular testing was performed on repeat FNAs diagnosed AUS. Four cases showed no mutations and had a high likelihood of being benign. One case did have an NRAS Q61R mutation, and resection revealed a noninvasive follicular thyroid neoplasm with papillary-like nuclear features. Seventeen (13% of all cases) with nondiagnostic FNA were resected. Twelve (71%) thyroidectomies showed benign adenomatous nodules. The remainder showed incidental papillary thyroid microcarcinoma (0.1 cm), an infarcted follicular adenoma, a noninvasive follicular thyroid neoplasm with papillary-like nuclear features, and metastatic renal cell carcinoma (2×). CONCLUSION: Thyroid nodules with nondiagnostic cytology are reassuring of being highly likely a benign nodule. Only 5 of the 55 (9%) repeat FNAs yielded abnormalities, with only one of those being truly a follicular neoplasm (confirmed by molecular testing and resection). No primary thyroid malignancies have been identified in follow-up (repeat FNA or surgery). Clinical and ultrasound follow-up may be more appropriate management for nondiagnostic thyroid FNAs.

Cytomorphologic and molecular characterization of spindle cell carcinoid tumors of the lung.

BACKGROUND: Spindle cell carcinoid tumor (SCCT) is a rare variant of lung carcinoid tumor consisting predominantly or exclusively of spindle cells. To the authors' knowledge, this is the first study to date investigating the molecular characteristics of SCCTs. METHODS: Eighty-five carcinoid tumors initially diagnosed by fine-needle aspiration over a period of 10 years were reviewed. The final diagnostic classification was based on resection specimens. Six SCCTs were identified and characterized based on cytomorphology, and immunohistochemical and molecular features. RESULTS: Most patients with SCCT were Caucasian (100.0%), women (83.3%), asymptomatic (66.7%), and nonsmokers (83.3%). The median age at diagnosis was 78.0 years (range, 58.2-80.3 years). A higher proportion of patients who had SCCT were diagnosed with distant metastasis. The smears were cellular and demonstrated clean backgrounds without necrosis or mitotic activity. SCCTs comprised of bipolar-to-elongated cells with finely granular chromatin, inconspicuous nucleoli, scant cytoplasm, and minimal atypia or pleomorphism. The tumor cells sometimes appeared boomerang-shaped and might mimic granulomas or blood vessels. SCCTs showed strong expression for pan-cytokeratin, synaptophysin, chromogranin, and CD56, with weak TTF-1 and a very low Ki-67 proliferation index. All SCCTs had low tumor mutational burden and were microsatellite-stable. One case showed multiple whole-gene losses in chromosome 11, whereas another harbored duplication in ARID1A. Two cases demonstrated gains in chromosomes 17, one of which also showed gains in chromosome 18. None had a single nucleotide mutation. CONCLUSIONS: SCCT is a rare subset of lung carcinoid tumors. These tumors harbor unique cytologic, prognostic, and molecular features that may have significant diagnostic and clinical implications.

Lung adenocarcinomas with isolated TP53 mutation: A comprehensive clinical, cytopathologic and molecular characterization.

BACKGROUND: TP53 mutation is present in about 50.8% of lung adenocarcinomas, frequently in combination with other genetic alterations. However, a rare subset harbors the TP53 mutation alone. METHODS: Next-generation sequencing was performed in 844 lung adenocarcinomas diagnosed by fine needle aspiration. Fourteen cases (1.7%) showed isolated TP53 alteration and were subjected to a comprehensive analysis. RESULTS: The average age at diagnosis was 65.7 years (range 48-79); 9 males and 5 females. All were smokers with an average pack-year of 40.7 (range 10-70). Ten had metastases, mostly in the brain (n = 4) and pleura (n = 4). After a follow-up period of up to 102 months, 9 died, 3 were alive free of disease, 1 was alive with disease, and 1 was lost to follow-up. The median survival was 12.2 months. Most tumors exhibited poor differentiation, composed of solid sheets with moderate to severe atypia, increased mitotic activity, and necrotic background. Half were positive for TTF-1 and showed p53 overexpression. PD-L1 was positive in 5 cases. Most alterations were missense mutations in exons 5-8, and this mutation type was associated with p53 overexpression. Tumors with combined missense mutation and truncated protein had higher PD-L1 expression along with a trend towards an increase in tumor mutational burden (TMB). CEBPA deletion of undetermined significance was the most common copy number alteration. CONCLUSION: Isolated TP53 mutation was seen in association with smoking, high-grade cytomorphologic features, adverse prognosis, and recurrent CEBPA deletions. These tumors tend to have strong PD-L1 expression and high TMB, suggesting potential benefit from immune checkpoint inhibitors. Hence, the recognition of this molecular group has prognostic and therapeutic implications.

Exposure of Keratinocytes to Candida Albicans in the Context of Atopic Milieu Induces Changes in the Surface Glycosylation Pattern of Small Extracellular Vesicles to Enhance Their Propensity to Interact With Inhibitory Siglec Receptors.

Candida albicans (C. albicans) infection is a potential complication in the individuals with atopic dermatitis (AD) and can affect clinical course of the disease. Here, using primary keratinocytes we determined that atopic milieu promotes changes in the interaction of small extracellular vesicles (sEVs) with dendritic cells and that this is further enhanced by the presence of C. albicans. sEV uptake is largely dependent on the expression of glycans on their surface; modelling of the protein interactions indicated that recognition of this pathogen through C. albicans-relevant pattern recognition receptors (PRRs) is linked to several glycosylation enzymes which may in turn affect the expression of sEV glycans. Here, significant changes in the surface glycosylation pattern, as determined by lectin array, could be observed in sEVs upon a combined exposure of keratinocytes to AD cytokines and C. albicans. This included enhanced expression of multiple types of glycans, for which several dendritic cell receptors could be proposed as binding partners. Blocking experiments showed predominant involvement of the inhibitory Siglec-7 and -9 receptors in the sEV-cell interaction and the engagement of sialic acid-containing carbohydrate moieties on the surface of sEVs. This pointed on ST6 ß-Galactoside α-2,6-Sialyltransferase 1 (ST6GAL1) and Core 1 ß,3-Galactosyltransferase 1 (C1GALT1) as potential enzymes involved in the process of remodelling of the sEV surface glycans upon C. albicans exposure. Our results suggest that, in combination with atopic dermatitis milieu, C. albicans promotes alterations in the glycosylation pattern of keratinocyte-derived sEVs to interact with inhibitory Siglecs on antigen presenting cells. Hence, a strategy aiming at this pathway to enhance antifungal responses and restrict pathogen spread could offer novel therapeutic options for skin candidiasis in AD.

TGF-ß signaling in fibrosis.

Transforming growth factor ß (TGF-ß) is a central mediator of fibrogenesis. TGF-ß is upregulated and activated in fibrotic diseases and modulates fibroblast phenotype and function, inducing myofibroblast transdifferentiation while promoting matrix preservation. Studies in a wide range of experimental models have demonstrated the involvement of the canonical activin receptor-like kinase 5/Smad3 pathway in fibrosis. Smad-independent pathways may regulate Smad activation and, under certain conditions, may directly transduce fibrogenic signals. The profibrotic actions of TGF-ß are mediated, at least in part, through induction of its downstream effector, connective tissue growth factor. In light of its essential role in the pathogenesis of fibrosis, TGF-ß has emerged as an attractive therapeutic target. However, the pleiotropic and multifunctional effects of TGF-ß and its role in tissue homeostasis, immunity and cell proliferation raise concerns regarding potential side effects that may be caused by TGF-ß blockade. This minireview summarizes the role of TGF-ß signaling pathways in the fibrotic response.

Applications of nanodosimetry in particle therapy planning and beyond.

This topical review summarizes underlying concepts of nanodosimetry. It describes the development and current status of nanodosimetric detector technology. It also gives an overview of Monte Carlo track structure simulations that can provide nanodosimetric parameters for treatment planning of proton and ion therapy. Classical and modern radiobiological assays that can be used to demonstrate the relationship between the frequency and complexity of DNA lesion clusters and nanodosimetric parameters are reviewed. At the end of the review, existing approaches of treatment planning based on relative biological effectiveness (RBE) models or dose-averaged linear energy transfer are contrasted with an RBE-independent approach based on nandosimetric parameters. Beyond treatment planning, nanodosimetry is also expected to have applications and give new insights into radiation protection dosimetry.

High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS.

DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such as cancer. Therefore, there is a great need for a precise, sensitive, and universal method for DSB detection, to enable both the study of their mechanisms of formation and repair as well as to explore their therapeutic potential. We provide a detailed protocol for our recently developed ultrasensitive and genome-wide DSB detection method: immobilized direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (i-BLESS), which relies on the encapsulation of cells in agarose beads and labeling breaks directly and specifically with biotinylated linkers. i-BLESS labels DSBs with single-nucleotide resolution, allows detection of ultrarare breaks, takes 5 d to complete, and can be applied to samples from any organism, as long as a sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach to enable the measurement of absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Such normalization using qDSB-Seq is especially useful for the evaluation of spontaneous DSB levels and the estimation of DNA damage induced rather uniformly in the genome (e.g., by irradiation or radiomimetic chemotherapeutics).

Histopathologic and clinical outcomes of Milan System categories "non-diagnostic" and "non-neoplastic" of salivary gland fine needle aspirations.

INTRODUCTION: The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) specifies six categories with estimated risks of malignancy (ROM) and suggested management. The estimated ROM is 25% for Non-Diagnostic (ND) category, and 10% for Non-Neoplastic (NN). This study aimed to investigate histopathologic and clinical outcomes of MSRSGC categories ND and NN at the authors' institution. MATERIALS AND METHODS: Cytopathology fine needle aspiration reports from 2008-2020 were searched for the word "salivary", "parotid", and "submandibular". Cases fitting Non-Diagnostic (ND) and Non-Neoplastic (NN) categories were identified. Follow-up cyto-/histopathologic and clinical data were extracted. RESULTS: There were 43 ND and 46 NN cases. The average age was 58.3 years. Neoplastic lesions were found in 13 of 43 (30%) ND and 3 of 46 (6.5%) NN. The rate of malignancy in ND category was 14.0% (6/43) and 0% (0/46) in NN category. Four cases in ND (9.3%) and 6 (13.0%) in NN had no neoplasm and instead had an underlying reactive condition (e.g., chronic sialadenitis) or inflammatory lesion (e.g., lymphoepithelial cyst) on histologic follow-up. There was no follow-up pathology in 46.5% NDs (20/43) and 82.6% NNs (38/46); however, no lesions were apparent clinically with a mean follow-up of 3 years and 1.5 years, respectively. CONCLUSIONS: MSRSGC categories ND and NN are helpful for reporting salivary gland FNA results. With proper clinical and radiologic correlation, ROM of NN is low; however, ROM of ND remains significant. Repeat FNA after correlation for ND cases seems prudent as neoplasms and malignancies may have gone undetected.