Epigenetic Regulation of DNA Repair Pathway Choice by MacroH2A1 Splice Variants Ensures Genome Stability.

The inactive X chromosome (Xi) is inherently susceptible to genomic aberrations. Replication stress (RS) has been proposed as an underlying cause, but the mechanisms that protect from Xi instability remain unknown. Here, we show that macroH2A1.2, an RS-protective histone variant enriched on the Xi, is required for Xi integrity and female survival. Mechanistically, macroH2A1.2 counteracts its structurally distinct and equally Xi-enriched alternative splice variant, macroH2A1.1. Comparative proteomics identified a role for macroH2A1.1 in alternative end joining (alt-EJ), which accounts for Xi anaphase defects in the absence of macroH2A1.2. Genomic instability was rescued by simultaneous depletion of macroH2A1.1 or alt-EJ factors, and mice deficient for both macroH2A1 variants harbor no overt female defects. Notably, macroH2A1 splice variant imbalance affected alt-EJ capacity also in tumor cells. Together, these findings identify macroH2A1 splicing as a modulator of genome maintenance that ensures Xi integrity and may, more broadly, predict DNA repair outcome in malignant cells.

Replication Stress Shapes a Protective Chromatin Environment across Fragile Genomic Regions.

Recent integrative epigenome analyses highlight the importance of functionally distinct chromatin states for accurate cell function. How these states are established and maintained is a matter of intense investigation. Here, we present evidence for DNA damage as an unexpected means to shape a protective chromatin environment at regions of recurrent replication stress (RS). Upon aberrant fork stalling, DNA damage signaling and concomitant H2AX phosphorylation coordinate the FACT-dependent deposition of macroH2A1.2, a histone variant that promotes DNA repair by homologous recombination (HR). MacroH2A1.2, in turn, facilitates the accumulation of the tumor suppressor and HR effector BRCA1 at replication forks to protect from RS-induced DNA damage. Consequently, replicating primary cells steadily accrue macroH2A1.2 at fragile regions, whereas macroH2A1.2 loss in these cells triggers DNA damage signaling-dependent senescence, a hallmark of RS. Altogether, our findings demonstrate that recurrent DNA damage contributes to the chromatin landscape to ensure the epigenomic integrity of dividing cells.

Saturation genome editing of 11 codons and exon 13 of BRCA2 coupled with chemotherapeutic drug response accurately determines pathogenicity of variants.

The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, we have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.

Periodontitis and brain magnetic resonance imaging markers of Alzheimer's disease and cognitive aging.

INTRODUCTION: We examined the association of clinical, microbiological, and host response features of periodontitis with MRI markers of atrophy/cerebrovascular disease in the Washington Heights Inwood Columbia Aging Project (WHICAP) Ancillary Study of Oral Health. METHODS: We analyzed 468 participants with clinical periodontal data, microbial plaque and serum samples, and brain MRIs. We tested the association of periodontitis features with MRI features, after adjusting for multiple risk factors for Alzheimer's disease/Alzheimer's disease-related dementia (AD/ADRD). RESULTS: In fully adjusted models, having more teeth was associated with lower odds for infarcts, lower white matter hyperintensity (WMH) volume, higher entorhinal cortex volume, and higher cortical thickness. Higher extent of periodontitis was associated with lower entorhinal cortex volume and lower cortical thickness. Differential associations emerged between colonization by specific bacteria/serum antibacterial IgG responses and MRI outcomes. DISCUSSION: In an elderly cohort, clinical, microbiological, and serological features of periodontitis were associated with MRI findings related to ADRD risk. Further investigation of causal associations is warranted.

Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells.

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

Loss of Brca1 and Trp53 in adult mouse mammary ductal epithelium results in development of hormone receptor-positive or hormone receptor-negative tumors, depending on inactivation of Rb family proteins.

BACKGROUND: Breast cancer is a heterogenous disease with several histological and molecular subtypes. Models that represent these subtypes are essential for translational research aimed at improving clinical strategy for targeted therapeutics. METHODS: Different combinations of genetic aberrations (Brca1 and Trp53 loss, and inhibition of proteins of the Rb family) were induced in the mammary gland by injection of adenovirus expressing Cre recombinase into the mammary ducts of adult genetically engineered mice. Mammary tumors with different genetic aberrations were classified into molecular subtypes based on expression of molecular markers and RNAseq analysis. In vitro potency assays and Western blots were used to examine their drug sensitivities. RESULTS: Induction of Brca1 and Trp53 loss in mammary ductal epithelium resulted in development of basal-like hormone receptor (HR)-negative mammary tumors. Inhibition of Rb and Trp53 loss or the combination of Rb, Trp53 and Brca1 aberrations resulted in development of luminal ductal carcinoma positive for ER, PR, and Her2 expression. HR positivity in tumors with Rb, Trp53 and Brca1 aberrations indicated that functionality of the Rb pathway rather than Brca1 status affected HR status in these models. Mammary tumor gene expression profiles recapitulated human basal-like or luminal B breast cancer signatures, but HR-positive luminal cancer models were endocrine resistant and exhibited upregulation of PI3K signaling and sensitivity to this pathway inhibition. Furthermore, both tumor subtypes were resistant to CDK4/6 inhibition. CONCLUSIONS: Examination of molecular expression profiles and drug sensitivities of tumors indicate that these breast cancer models can be utilized as a translational platform for evaluation of targeted combinations to improve chemotherapeutic response in patients that no longer respond to hormone therapy or that are resistant to CDK4/6 inhibition.

EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells.

BACKGROUND: Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells. METHODS: Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes. RESULTS: The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated ß-galactosidase (SA-ß-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice. CONCLUSIONS: Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.

5-Azacytidine prevents relapse and produces long-term complete remissions in leukemia xenografts treated with Moxetumomab pasudotox.

Moxetumomab pasudotox (Moxe) is a chimeric protein composed of an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A and kills CD22-expressing leukemia cells. It is very active in hairy-cell leukemia, but many children with relapsed/refractory acute lymphoblastic leukemia (ALL) either respond transiently or are initially resistant. Resistance to Moxe in cultured cells is due to low expression of diphthamide genes (DPH), but only two of six ALL blast samples from resistant patients had low DPH expression. To develop a more clinically relevant model of resistance, we treated NSG mice bearing KOPN-8 or Reh cells with Moxe. More than 99.9% of the cancer cells were killed by Moxe, but relapse occurred from discrete bone marrow sites. The resistant cells would no longer grow in cell culture and showed major chromosomal changes and changes in phenotype with greatly decreased CD22. RNA deep sequencing of resistant KOPN-8 blasts revealed global changes in gene expression, indicating dedifferentiation toward less-mature B cell precursors, and showed an up-regulation of myeloid genes. When Moxe was combined with 5-azacytidine, resistance was prevented and survival increased to over 5 months in the KOPN-8 model and greatly improved in the Reh model. We conclude that Moxe resistance in mice is due to a new mechanism that could not be observed using cultured cells and is prevented by treatment with 5-azacytidine.

Chromoanasynthesis as a cause of Jacobsen syndrome.

Jacobsen syndrome (MIM #147791) is a rare multisystem genomic disorder involving craniofacial abnormalities, intellectual disability, other neurodevelopmental defects, and terminal truncation of chromosome 11q, typically deleting ~170 to >340 genes. We describe the first case of Jacobsen syndrome caused by congenital chromoanasynthesis, an extreme form of complex chromosomal rearrangement. Six duplications and five deletions occurred on one copy of chromosome 11q with microhomology signatures in the breakpoint junctions, indicating an all-at-once replication-based rearrangement mechanism in a gametocyte or early post-zygotic cell. Eighteen genes were deleted from the Jacobsen region, including KIRREL3, which is associated with intellectual disability.

Interaction with PALB2 Is Essential for Maintenance of Genomic Integrity by BRCA2.

Human breast cancer susceptibility gene, BRCA2, encodes a 3418-amino acid protein that is essential for maintaining genomic integrity. Among the proteins that physically interact with BRCA2, Partner and Localizer of BRCA2 (PALB2), which binds to the N-terminal region of BRCA2, is vital for its function by facilitating its subnuclear localization. A functional redundancy has been reported between this N-terminal PALB2-binding domain and the C-terminal DNA-binding domain of BRCA2, which undermines the relevance of the interaction between these two proteins. Here, we describe a genetic approach to examine the functional significance of the interaction between BRCA2 and PALB2 by generating a knock-in mouse model of Brca2 carrying a single amino acid change (Gly25Arg, Brca2G25R) that disrupts this interaction. In addition, we have combined Brca2G25R homozygosity as well as hemizygosity with Palb2 and Trp53 heterozygosity to generate an array of genotypically and phenotypically distinct mouse models. Our findings reveal defects in body size, fertility, meiotic progression, and genome stability, as well as increased tumor susceptibility in these mice. The severity of the phenotype increased with a decrease in the interaction between BRCA2 and PALB2, highlighting the significance of this interaction. In addition, our findings also demonstrate that hypomorphic mutations such as Brca2G25R have the potential to be more detrimental than the functionally null alleles by increasing genomic instability to a level that induces tumorigenesis, rather than apoptosis.

BRCA2 minor transcript lacking exons 4-7 supports viability in mice and may account for survival of humans with a pathogenic biallelic mutation.

The breast cancer gene, BRCA2, is essential for viability, yet patients with Fanconi anemia-D1 subtype are born alive with biallelic mutations in this gene. The hypomorphic nature of the mutations is believed to support viability, but this is not always apparent. One such mutation is IVS7+2T>G, which causes premature protein truncation due to skipping of exon 7. We previously identified a transcript lacking exons 4-7, which restores the open-reading frame, encodes a DNA repair proficient protein and is expressed in IVS7+2T>G carriers. However, because the exons 4-7 encoded region contains several residues required for normal cell-cycle regulation and cytokinesis, this transcript's ability to support viability can be argued. To address this, we generated a Brca2 knock-in mouse model lacking exons 4-7 and demonstrated that these exons are dispensable for viability as well as tumor-free survival. This study provides the first in vivo evidence of the functional significance of a minor transcript of BRCA2 that can play a major role in the survival of humans who are homozygous for a clearly pathogenic mutation. Our results highlight the importance of assessing protein function restoration by premature truncating codon bypass by alternative splicing when evaluating the functional significance of variants such as nonsense and frame-shift mutations that are assumed to be clearly pathogenic. Our findings will impact not only the assessment of variants that map to this region, but also influence counseling paradigms and treatment options for such mutation carriers.

Periodontal status among elderly inhabitants of northern Manhattan: The WHICAP ancillary study of oral health.

AIM: We conducted a cross-sectional study of the prevalence, extent and severity of periodontitis in a tri-ethnic cohort of ≥65 year-old participants of the Washington-Heights Inwood Community Aging Project (WHICAP). METHODS: 1,130 individuals (57% of eligible invitees) participated in a full-mouth periodontal examination that included assessments of bleeding on probing, pocket depth and clinical attachment loss (CAL) at six sites/tooth. RESULTS: Participants had a mean age of 75.4 years (SD 6.7), were predominantly female (66.6%) and Hispanic (44.7%), and of middle/low educational attainment (~82%). The prevalence of edentulism was 14.7%, and an average of 17.1 teeth (SD 8.0) was present among the dentate. The prevalence of moderate/severe periodontitis according to the CDC/AAP definition was 77.5%. Pockets ≥6 mm were found in 50.2% of the sample, affecting an average of 5.7% of teeth/person. Corresponding figures for CAL≥5 mm were 71.4% and 23.6%, respectively. In multivariable models, male gender, being Black or Hispanic, and no dental visit within the prior year were associated with higher proportion of teeth with CAL ≥5 mm. CONCLUSIONS: The prevalence, extent and severity of periodontitis were higher than the US national average in this urban elderly sample, suggesting substantial unmet periodontal treatment needs.

Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction.

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene, resulting in absent or defective gp91phox protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91phox expression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed, involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1-13 minigene into CYBB exon 1 resulted in no detectable gp91phox expression or ROS activity in iPSC-derived granulocytes. In contrast, targeted insertion of an exon 2-13 minigene into exon 2 restored both gp91phox and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2-13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore, it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression.

Thymic expression of a T-cell receptor targeting a tumor-associated antigen coexpressed in the thymus induces T-ALL.

T-cell receptors (TCRs) and chimeric antigen receptors recognizing tumor-associated antigens (TAAs) can now be engineered to be expressed on a wide array of immune effectors. Engineered receptors targeting TAAs have most commonly been expressed on mature T cells, however, some have postulated that receptor expression on immune progenitors could yield T cells with enhanced potency. We generated mice (survivin-TCR-transgenic [Sur-TCR-Tg]) expressing a TCR recognizing the immunodominant epitope (Sur20-28) of murine survivin during early stages of thymopoiesis. Spontaneous T-cell acute lymphoblastic leukemia (T-ALL) occurred in 100% of Sur-TCR-Tg mice derived from 3 separate founders. The leukemias expressed the Sur-TCR and signaled in response to the Sur20-28 peptide. In preleukemic mice, we observed increased cycling of double-negative thymocytes expressing the Sur-TCR and increased nuclear translocation of nuclear factor of activated T cells, consistent with TCR signaling induced by survivin expression in the murine thymus. ß2M(-/-) Sur-TCR-Tg mice, which cannot effectively present survivin peptides on class I major histocompatibility complex, had significantly diminished rates of leukemia. We conclude that TCR signaling during the early stages of thymopoiesis mediates an oncogenic signal, and therefore expression of signaling receptors on developing thymocytes with specificity for TAAs expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis.

Mesothelioma patient derived tumor xenografts with defined BAP1 mutations that mimic the molecular characteristics of human malignant mesothelioma.

BACKGROUND: The development and evaluation of new therapeutic approaches for malignant mesothelioma has been sparse due, in part, to lack of suitable tumor models. METHODS: We established primary mesothelioma cultures from pleural and ascitic fluids of five patients with advanced mesothelioma. Electron microscopy and immunohistochemistry (IHC) confirmed their mesothelial origin. Patient derived xenografts were generated by injecting the cells in nude or SCID mice, and malignant potential of the cells was analyzed by soft agar colony assay. Molecular profiles of the primary patient tumors, early passage cell cultures, and patient derived xenografts were assessed using mutational analysis, fluorescence in situ hybridization (FISH) analysis and IHC. RESULTS: Primary cultures from all five tumors exhibited morphologic and IHC features consistent to those of mesothelioma cells. Mutations of BAP1 and CDKN2A were each detected in four tumors. BAP1 mutation was associated with the lack of expression of BAP1 protein. Three cell cultures, all of which were derived from BAP1 mutant primary tumors, exhibited anchorage independent growth and also formed tumors in mice, suggesting that BAP1 loss may enhance tumor growth in vivo. Both early passage cell cultures and mouse xenograft tumors harbored BAP1 mutations and CDKN2A deletions identical to those found in the corresponding primary patient tumors. CONCLUSIONS: The mesothelioma patient derived tumor xenografts with mutational alterations that mimic those observed in patient tumors which we established can be used for preclinical development of novel drug regimens and for studying the functional aspects of BAP1 biology in mesothelioma.

Mice null for the deubiquitinase USP18 spontaneously develop leiomyosarcomas.

BACKGROUND: USP18 (ubiquitin-specific protease 18) removes ubiquitin-like modifier interferon stimulated gene 15 (ISG15) from conjugated proteins. USP18 null mice in a FVB/N background develop tumors as early as 2 months of age. These tumors are leiomyosarcomas and thus represent a new murine model for this disease. METHODS: Heterozygous USP18 +/- FVB/N mice were bred to generate wild-type, heterozygous and homozygous cohorts. Tumors were characterized immunohistochemically and two cell lines were derived from independent tumors. Cell lines were karyotyped and their responses to restoration of USP18 activity assessed. Drug testing and tumorigenic assays were also performed. USP18 immunohistochemical staining in a large series of human leiomyosacomas was examined. RESULTS: USP18 -/- FVB/N mice spontaneously develop tumors predominantly on the back of the neck with most tumors evident between 6-12 months (80 % penetrance). Immunohistochemical characterization of the tumors confirmed they were leiomyosarcomas, which originate from smooth muscle. Restoration of USP18 activity in sarcoma-derived cell lines did not reduce anchorage dependent or independent growth or xenograft tumor formation demonstrating that these cells no longer require USP18 suppression for tumorigenesis. Karyotyping revealed that both tumor-derived cell lines were aneuploid with extra copies of chromosomes 3 and 15. Chromosome 15 contains the Myc locus and MYC is also amplified in human leiomyosarcomas. MYC protein levels were elevated in both murine leiomyosarcoma cell lines. Stabilized P53 protein was detected in a subset of these murine tumors, another feature of human leiomyosarcomas. Immunohistochemical analyses of USP18 in human leiomyosarcomas revealed a range of staining intensities with the highest USP18 expression in normal vascular smooth muscle. USP18 tissue array analysis of primary leiomyosarcomas from 89 patients with a clinical database revealed cases with reduced USP18 levels had a significantly decreased time to metastasis (P = 0.0441). CONCLUSIONS: USP18 null mice develop leiomyosarcoma recapitulating key features of clinical leiomyosarcomas and patients with reduced-USP18 tumor levels have an unfavorable outcome. USP18 null mice and the derived cell lines represent clinically-relevant models of leiomyosarcoma and can provide insights into both leiomyosarcoma biology and therapy.

Six-month outcomes in dental patients identified with hyperglycaemia: a randomized clinical trial.

AIM: To assess an approach to improving behavioural and glycaemic outcomes in dental patients who present with diabetes risk factors and previously unrecognized hyperglycaemia. METHODS: We randomized 101 individuals identified with potential diabetes or pre-diabetes into two interventions. In the basic/control intervention, participants were informed about their diabetes risk factors and blood test result, and advised to see a physician. In the enhanced/test intervention, patients received a detailed explanation of findings and their implications, a written report for the physician, and were contacted at 2 and 4 months to inquire whether medical follow-up had occurred. At a 6-month re-evaluation, outcome measures included visit to physician, positive lifestyle changes and reduction in HbA1c. RESULTS: Seventy-three subjects returned for the 6-month visit. The two intervention groups did not significantly differ in any of the outcome variables. Eighty-four percent of subjects reported having visited a physician post-randomization, and 49% reported at least one positive lifestyle change as a result of our intervention. In subjects identified with potential diabetes (baseline HbA1c ≥ 6.5%), HbA1c was reduced 1.46 ± 0.28% compared to baseline (p < 0.01). CONCLUSION: Diabetes risk assessment and education by dental professionals of affected individuals unaware of their status may contribute to improved patient outcomes.

Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay.

Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.

A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells.

Sister chromatids contain identical DNA sequence but are chiral with respect to both their helical handedness and their replication history. Emerging evidence from various model organisms suggests that certain stem cells segregate sister chromatids nonrandomly to either maintain genome integrity or to bias cellular differentiation in asymmetric cell divisions. Conventional methods for tracing of old vs. newly synthesized DNA strands generally lack resolution for individual chromosomes and employ halogenated thymidine analogs with profound cytotoxic effects on rapidly dividing cells. Here, we present a modified chromosome orientation fluorescence in situ hybridization (CO-FISH) assay, where identification of individual chromosomes and their replication history is achieved in subsequent hybridization steps with chromosome-specific DNA probes and PNA telomere probes. Importantly, we tackle the issue of BrdU cytotoxicity and show that our method is compatible with normal mouse ES cell biology, unlike a recently published related protocol. Results from our CO-FISH assay show that mitotic segregation of mouse chromosome 7 is random in ES cells, which contrasts previously published results from our laboratory and settles a controversy. Our straightforward protocol represents a useful resource for future studies on chromatid segregation patterns of in vitro-cultured cells from distinct model organisms.

Periodontal findings in individuals with newly identified pre-diabetes or diabetes mellitus.

AIM: To assess the periodontal status and number of missing teeth in patients with newly identified pre-diabetes or diabetes mellitus. METHODS: A total of 1097 subjects with previously undiagnosed diabetes were available for study, and were categorized into normoglycaemic, potentially pre-diabetes or potentially diabetes groups based on a point-of-care (POC) HbA1c test. RESULTS: In fully adjusted models, significant differences were observed between all groups for the per cent of teeth with at least one site with a probing depth of ≥5 mm. For bleeding on probing, there were significant differences between diabetes and pre-diabetes (p = 0.001), and between diabetes and normoglycaemic groups (p = 0.002). For missing teeth, there were significant differences between the pre-diabetes and normoglycaemic groups (p = 0.034), and the diabetes and normoglycaemic groups (p = 0.004). CONCLUSIONS: Individuals with previously unidentified pre-diabetes demonstrate a level of periodontal destruction between that observed for normoglycaemic individuals and persons with diabetes. These data emphasize the association of oral findings to dysglycaemia, and suggest that periodontal disease and tooth loss can be early complications of diabetes mellitus.