Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes.

A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental approaches using animal model systems. Zebrafish has become a powerful vertebrate genetic model system with availability due to the ease of genome editing and large-scale phenotype screening. Here, we generated zebrafish mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. Large-scale phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.

Development and operation of a digital platform for sharing pathology image data.

BACKGROUND: Artificial intelligence (AI) research is highly dependent on the nature of the data available. With the steady increase of AI applications in the medical field, the demand for quality medical data is increasing significantly. We here describe the development of a platform for providing and sharing digital pathology data to AI researchers, and highlight challenges to overcome in operating a sustainable platform in conjunction with pathologists. METHODS: Over 3000 pathological slides from five organs (liver, colon, prostate, pancreas and biliary tract, and kidney) in histologically confirmed tumor cases by pathology departments at three hospitals were selected for the dataset. After digitalizing the slides, tumor areas were annotated and overlaid onto the images by pathologists as the ground truth for AI training. To reduce the pathologists' workload, AI-assisted annotation was established in collaboration with university AI teams. RESULTS: A web-based data sharing platform was developed to share massive pathological image data in 2019. This platform includes 3100 images, and 5 pre-processing algorithms for AI researchers to easily load images into their learning models. DISCUSSION: Due to different regulations among countries for privacy protection, when releasing internationally shared learning platforms, it is considered to be most prudent to obtain consent from patients during data acquisition. CONCLUSIONS: Despite limitations encountered during platform development and model training, the present medical image sharing platform can steadily fulfill the high demand of AI developers for quality data. This study is expected to help other researchers intending to generate similar platforms that are more effective and accessible in the future.

A literature review of quality, costs, process-associated with digital pathology.

Digital pathology incorporates the acquisition, management, sharing, and interpretation of pathological information, including slides and data, in a digital environment. Digital slides are created using a scanning device to capture a high-resolution image on glass slides for analysis on a computer or a mobile device. Though digital pathology has drastically grown over the last 10 years and has created opportunities to support specialists, few have attempted to address its full-scale implementation in routine clinical practice. To incorporate new technologies in diagnostic processes, it is necessary to study their application, the value they provide to specialists, and their effects on improvements across the entire workflow, rather than studying a particular element. In this study, we aimed to identify what have the current digital pathology systems contributed to the pathological and diagnostic process. We retrieved articles published between 2010 and 2020 from the databases PubMed and Google Scholar. We explored how digital pathology systems can better utilize existing medical data and new technologies within the current diagnostic workflow. While the evidence concerning the efficacy and effectiveness of digital pathology is mounting, high-quality evidence regarding its impact on resource allocation and value for diagnosis is still needed to support clinical diagnosis and policy decision-making.

PAIP 2019: Liver cancer segmentation challenge.

Pathology Artificial Intelligence Platform (PAIP) is a free research platform in support of pathological artificial intelligence (AI). The main goal of the platform is to construct a high-quality pathology learning data set that will allow greater accessibility. The PAIP Liver Cancer Segmentation Challenge, organized in conjunction with the Medical Image Computing and Computer Assisted Intervention Society (MICCAI 2019), is the first image analysis challenge to apply PAIP datasets. The goal of the challenge was to evaluate new and existing algorithms for automated detection of liver cancer in whole-slide images (WSIs). Additionally, the PAIP of this year attempted to address potential future problems of AI applicability in clinical settings. In the challenge, participants were asked to use analytical data and statistical metrics to evaluate the performance of automated algorithms in two different tasks. The participants were given the two different tasks: Task 1 involved investigating Liver Cancer Segmentation and Task 2 involved investigating Viable Tumor Burden Estimation. There was a strong correlation between high performance of teams on both tasks, in which teams that performed well on Task 1 also performed well on Task 2. After evaluation, we summarized the top 11 team's algorithms. We then gave pathological implications on the easily predicted images for cancer segmentation and the challenging images for viable tumor burden estimation. Out of the 231 participants of the PAIP challenge datasets, a total of 64 were submitted from 28 team participants. The submitted algorithms predicted the automatic segmentation on the liver cancer with WSIs to an accuracy of a score estimation of 0.78. The PAIP challenge was created in an effort to combat the lack of research that has been done to address Liver cancer using digital pathology. It remains unclear of how the applicability of AI algorithms created during the challenge can affect clinical diagnoses. However, the results of this dataset and evaluation metric provided has the potential to aid the development and benchmarking of cancer diagnosis and segmentation.

Anastrozole-Induced Dermatitis: Report of a Woman with an Anastrozole-Associated Dermatosis and a Review of Aromatase Inhibitor-Related Cutaneous Adverse Events.

Anastrozole is an aromatase inhibitor. Anastrozole competitively inhibits the aromatase enzyme, which synthesizes estrogen. It is used for estrogen receptor-positive breast cancers. A woman with breast cancer and anastrozole-induced dermatitis is described; the cutaneous side effects associated with aromatase inhibitors are also reviewed. Skin-related adverse events associated with aromatase inhibitor use are uncommon and may be delayed in appearance; the time of onset ranges from less than 5 days to 6 months (median 2 months). They present as either vasculitis, erythema nodosum, subacute cutaneous lupus erythematosus, or other dermatoses. Some patients demonstrate cutaneous lesions at either the original site of the breast malignancy or in areas that were previously treated with surgery or radiotherapy. The skin reactions are usually treated with discontinuation of the aromatase inhibitor; topical corticosteroids or oral corticosteroids or both are also used for some patients. Our patient's drug reaction occurred 2 months after starting the anastrozole and improved after a course of oral and topical corticosteroids. She was able to be switched to an alternate aromatase inhibitor without a recurrence of her adverse skin reaction.

Dilemma in management of hemorrhagic myositis in dermatomyositis.

Dermatomyositis (DM) is a rare inflammatory disorder affecting the muscle and skin. DM patients can present with spontaneous muscle hemorrhage, a potentially fatal complication. The best practice for management of hemorrhagic myositis in these patients remains unclear. Here we discuss the case of a patient who presented with progressive muscle weakness and intermittent rash that was diagnosed with dermatomyositis. During admission, she developed spontaneous hemorrhagic myositis of the right pectoralis major treated with surgical evacuation. She also developed a spontaneous left anterior thigh hematoma which was treated conservatively. She recovered and showed no evidence of recurrent bleeding at either location. We performed a literature review and identified ten cases of spontaneous hemorrhage in DM patients, with a 60% mortality rate among reported cases. Given the high mortality rate associated with spontaneous hemorrhage in DM patients, it is important for physicians to be aware of the diagnosis, workup, and management strategies.

Blue Nevus Developing at a Combined Tetanus, Diphtheria, and Pertussis (Tdap) Vaccination Site: Case Report and Summary of Vaccine-Associated Cutaneous Lesions.

Skin reaction may develop at the site of vaccine administration. A 54-year-old woman who developed a cellular blue nevus at the site of the combined tetanus, diphtheria, and acellular pertussis (Tdap) vaccine injection four years prior to presentation is described. In addition to blue nevus, other reactions at combined tetanus, diphtheria, and pertussis vaccine injection sites include abscess, deep reactive nodular infiltrates of mixed inflammation, and necrotizing granuloma. In conclusion, blue nevus can be added to the list of cutaneous events that can occur at Tdap vaccination sites.

Evidence-based, Skin-directed Treatments for Cutaneous Chronic Graft-versus-host Disease.

Chronic graft-versus host disease (cGVHD) occurs in 30% to 70% of patients undergoing allogeneic hematopoietic cell transplantation (HCT). Cutaneous cGVHD affects 75% of cGVHD patients, causing discomfort, limiting the range of movement, and increasing the risk of wound infections. Furthermore, systemic immunosuppression is often needed to treat cGVHD and long-term use can lead to adverse events. Optimal use of skin-directed therapies is integral to the management of cutaneous cGVHD and may decrease the amount of systemic immunosuppression required. This study reviewed English-language articles published from 1990 to 2017 that evaluated the effect of skin-directed treatments for cutaneous cGVHD. A total of 201 papers were identified, 164 articles were screened, 46 were read, and 18 publications were utilized in the review. Skin-directed treatments for cGVHD included topical steroids, topical calcineurin inhibitors, psoralen with ultraviolet A (PUVA) irradiation, ultraviolet A1 (UVA1) irradiation, and ultraviolet B (UVB) irradiation. We report the number of complete remissions, partial remissions, and systemic immunosuppression reduction in each study, as available. Twenty-two out of 30 (73.3%) patients experienced overall improvement with topical calcineurin inhibitors. At least 26 out of 76 patients (34.2%) receiving PUVA experienced complete remission, and 30 out of 76 patients (39.5%) experienced partial remission. In UVA1 studies, 44 out of 52 (84.6%) patients experienced overall improvement. In UVB studies, nine out of 14 patients (64.3%) experienced complete remission and four out of 14 patients (28.6%) experienced partial remission. As more HCTs are performed, more individuals will develop cGVHD. Awareness and optimal use of skin-directed therapies for cutaneous cGVHD may help improve patient outcomes and quality of life.

Effectiveness of Interventions for Breast Cancer Screening in African American Women: A Meta-Analysis.

OBJECTIVE: The purpose of this study was to report the results of a meta-analysis conducted on the effects of clinical trials in breast cancer screening for African American women between 1997 and 2017. DATA SOURCES: Articles published in English and in the United States, between January 1997 and March 2017, were eligible for inclusion if they (1) conducted psychosocial, behavioral, or educational interventions designed to increase screening mammography rates in predominantly African American women of all ages; (2) utilized a randomized, controlled trial (RCT) design; and (3) reported quantitative screening rates following the intervention. STUDY DESIGN: Randomized clinical trials on breast cancer screening in African American women, published between January 1997 and March 2017, were selected from database searches. DATA COLLECTION METHODS: Data collected included effect size of screening versus comparison interventions, intervention characteristics, and a number of study characteristics to explore potential moderators. Search results yielded 327 articles, of which 14 met inclusion criteria and were included in analyses. PRINCIPAL FINDINGS: Findings indicated that screening interventions for African American women were significantly more likely to result in mammography than control (OR = 1.56 [95 percent CI = 1.27-1.93], p < .0001). Although no patient or study characteristics significantly moderated screening efficacy, the most effective interventions were those specifically tailored to meet the perceived risk of African American women. CONCLUSIONS: Screening interventions are at least minimally effective for promoting mammography among African American women, but research in this area is limited to a small number of studies. More research is needed to enhance the efficacy of existing interventions and reduce the high morbidity and mortality rate of this underserved population.

Reconstituting development of pancreatic intraepithelial neoplasia from primary human pancreas duct cells.

Development of systems that reconstitute hallmark features of human pancreatic intraepithelial neoplasia (PanINs), the precursor to pancreatic ductal adenocarcinoma, could generate new strategies for early diagnosis and intervention. However, human cell-based PanIN models with defined mutations are unavailable. Here, we report that genetic modification of primary human pancreatic cells leads to development of lesions resembling native human PanINs. Primary human pancreas duct cells harbouring oncogenic KRAS and induced mutations in CDKN2A, SMAD4 and TP53 expand in vitro as epithelial spheres. After pancreatic transplantation, mutant clones form lesions histologically similar to native PanINs, including prominent stromal responses. Gene expression profiling reveals molecular similarities of mutant clones with native PanINs, and identifies potential PanIN biomarker candidates including Neuromedin U, a circulating peptide hormone. Prospective reconstitution of human PanIN development from primary cells provides experimental opportunities to investigate pancreas cancer development, progression and early-stage detection.

Does Sense of Control Predict Depression Among Individuals After Psychiatric Hospital Discharge?

Sense of control is known to be related to depression. Yet, few studies have examined the role of sense of control as related to depression for discharged psychiatric patients. In this study the longitudinal relationship between sense of control and depressive mood was examined using the MacArthur Violence Risk Assessment Study, a 6-wave, 1-year study of 948 ethnically diverse postdischarge psychiatric patients. Sense of control was decomposed into 2 components (i.e., a time-invariant as well as a time-varying component) and so as to examine which component of sense of control would more accurately explain this relationship. Results demonstrated that time-varying sense of control significantly predicted changes in depressive mood during the transition to community environment. Time-invariant sense of control, however, was not significantly related to changes in depressive mood. Findings of this study hold important implications for intervention practice with people before or after psychiatric discharge, including the need for incorporation of therapeutic and psychoeducational efforts that bolster sense of control.

Hyper-Acetylation of Histone H3K56 Limits Break-Induced Replication by Inhibiting Extensive Repair Synthesis.

Break-induced replication (BIR) has been implicated in restoring eroded telomeres and collapsed replication forks via single-ended invasion and extensive DNA synthesis on the recipient chromosome. Unlike other recombination subtypes, DNA synthesis in BIR likely relies heavily on mechanisms enabling efficient fork progression such as chromatin modification. Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR). The basis for deficiency in BIR and gene conversion with long gap synthesis in hst3Δ hst4Δ cells can be traced to a defect in extensive DNA synthesis. Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants. Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR.

Repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome.

We used the I-SceI endonuclease to produce DNA double-strand breaks (DSBs) and observed that a fraction of these DSBs were repaired by insertion of sequences, which we termed "templated sequence insertions" (TSIs), derived from distant regions of the genome. These TSIs were derived from genic, retrotransposon, or telomere sequences and were not deleted from the donor site in the genome, leading to the hypothesis that they were derived from reverse-transcribed RNA. Cotransfection of RNA and an I-SceI expression vector demonstrated insertion of RNA-derived sequences at the DNA-DSB site, and TSIs were suppressed by reverse-transcriptase inhibitors. Both observations support the hypothesis that TSIs were derived from RNA templates. In addition, similar insertions were detected at sites of DNA DSBs induced by transcription activator-like effector nuclease proteins. Whole-genome sequencing of myeloma cell lines revealed additional TSIs, demonstrating that repair of DNA DSBs via insertion was not restricted to experimentally produced DNA DSBs. Analysis of publicly available databases revealed that many of these TSIs are polymorphic in the human genome. Taken together, these results indicate that insertional events should be considered as alternatives to gross chromosomal rearrangements in the interpretation of whole-genome sequence data and that this mutagenic form of DNA repair may play a role in genetic disease, exon shuffling, and mammalian evolution.

A three-dimensional model of the yeast genome.

Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes. Interphase chromosomes are not positioned randomly within the nucleus, but instead adopt preferred conformations. Disparate DNA elements co-localize into functionally defined aggregates or 'factories' for transcription and DNA replication. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among transfer RNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

Hs.137007 is a novel epigenetic marker hypermethylated and up-regulated in breast cancer.

This study was conducted to mine novel breast-specific unigenes and analyze their epigenetic regulation in breast cancer. Differential digital display and methylation analysis identified the Hs.137007 gene containing a Kelch domain as a candidate novel epigenetic marker. In 50 pairs of breast cancer tissues and nearby normal tissues the methylation level of the 14 CpG sites at the promoter region (-778 to -485) of the gene was higher in cancer tissues (72-93%) than in normal tissues (31-83%), with a high correlation rate (p<0.05). End-point RT-PCR and real-time RT-PCR revealed that Hs.137007 was up-regulated in cancer tissues. A clear relationship between high methylation levels and up-regulated expression was also observed in the cultured breast cell lines. The MCF7 (90-100%) and MDAMB468 (100%) cancer cell lines that showed higher methylation than the BT549 (20-90%) and 184B5 (10-100%) at the 14 CpGs also showed elevated gene expression. Taken together, these results indicate that the Hs.137007 gene is a novel gene specifically expressed in the breast that can be utilized as an epigenetic marker of breast cancer.

Development of ELISAs for the class-specific determination of organophosphorus pesticides.

Enzyme-linked immunosorbent assays (ELISAs) for the class-specific determination of organophosphorus (OP) pesticides were developed from monoclonal antibodies raised against haptens with the functional group common to OP pesticides. To develop antigen-coated, indirect, competitive ELISAs, four haptens with different spacer arm structures were used to prepare antibodies, while eight haptens were tested for use as coating antigens. A total of 32 ELISAs were developed with one selected as the most suitable one based on average IC(50) and % CV values. The chosen ELISA showed class-selective response to O,O-diethyl phosphorothioate and phosphorodithioate OP pesticides with negligible cross-reactivity to other types of pesticides. Average IC(50) and % CV values of this ELISA for the 12 OP pesticides were 89 ng/mL and 96%, respectively. Compared to ELISAs previously developed with the same objective, the current ELISA demonstrates better sensitivity based on much lower mean IC(50) values in addition to improved class-selective determination based on considerably lower % CV values as well as precise discrimination against other types of pesticides.

Characterization of mouse synaptic vesicle-2-associated protein (Msvop) specifically expressed in the mouse central nervous system.

In this study, we identified and characterized a mouse brain-enriched unigene (msvop) after performing digital differential display. Msvop was the mouse ortholog of Xenopus synaptic vesicle-2-associated protein (svop), the molecular characteristics of which were unknown. The 3125-bp full-length cDNA encoded a 548-aa protein of approximately 60 kDa. A strong promoter element was found in the -200 to -100 bp region in both NG108-15 and HEK293 cells. RT-PCR and in situ hybridization analysis confirmed that msvop was strictly expressed in the mouse central nervous system. In adult brains, msvop was highly expressed in the hippocampus and cerebellum. When the gene was transfected into HEK293 cells in a GFP fusion vector, the protein was specifically localized in the cytosol. These results indicate that msvop is a central nervous system-specific gene and could be utilized for elucidating gene regulation in neuronal cells.