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An ever-growing catalog of human variants is hosted in the ClinVar database. In this database, submissions on a variant are combined into a multisubmitter record; and in the case of discordance in variant classification between submitters, the record is labeled as conflicting. The current study used ClinVar data to identify characteristics that would make variants more likely to be associated with the conflict class of variants. Furthermore, the Extreme Gradient Boosting algorithm was used to train classifier models to provide prediction of classification discordance for single submission variants in ClinVar database. Population allele frequency, the gene harboring the variant, variant type, consequence on protein, variant deleteriousness score, first submitter identity, and submission count were associated with conflict in variant classification. Using such features, the optimized classifier showed accuracy on the test set of 88% with the weighted average of precision, recall, and f1-score of 0.84, 0.88, and 0.85, respectively. There were pronounced associations between variant classification discordance and allele frequency, gene type, and the identity of the first submitter. The study provides the predicted discordance status for single-submitter variants deposited in ClinVar. This approach can be used to assess whether single-submitter variants are likely to be supported, or in conflict with, future entries; this knowledge may help laboratories with clinical variant assessment., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Uncontrolled growth of tumor cells is a key contributor to cancer-associated mortalities. Tumor growth is a biomechanical process whereby the cancer cells displace the surrounding matrix that provides mechanical resistance to the growing cells. The process of tumor growth and remodeling is regulated by material properties of both the cancer cells and their surrounding matrix, yet the mechanical interdependency between the two entities is not well understood. Herein, this work develops a microfluidic platform that precisely positions tumor spheroids within a hydrogel and mechanically probes the growing spheroids and surrounding matrix simultaneously. By using hydrostatic pressure to deform the spheroid-laden hydrogel along with confocal imaging and finite element (FE) analysis, this work deduces the material properties of the spheroid and the matrix in situ. For spheroids embedded within soft hydrogels, decreases in the Young's modulus of the matrix are detected at discrete locations accompanied by localized tumor growth. Contrastingly, spheroids within stiff hydrogels do not significantly decrease the Young's modulus of the surrounding matrix, despite exhibiting growth. Spheroids in stiff matrices leverage their high bulk modulus to grow and display a uniform volumetric expansion. Collectively, a quantitative platform is established and new insights into tumor growth within a stiff 3D environment are provided., (© 2022 Wiley-VCH GmbH.)

Background: We previously developed a biological assay to accurately predict BRCA1 (BRCA1 DNA repair associated) mutation status, based on gene expression profiles of Epstein-Barr virus-transformed lymphoblastoid cell lines. The original work was done using whole genome expression microarrays, and nearest shrunken centroids analysis. While these approaches are appropriate for model building, they are difficult to implement clinically, where more targeted testing and analysis are required for time and cost savings., Methods: Here, we describe adaptation of the original predictor to use the NanoString nCounter platform for testing, with analysis based on the k-top scoring pairs (k-TSP) method., Results: Assessing gene expression using the nCounter platform on a set of lymphoblastoid cell lines yielded 93.8% agreement with the microarray-derived data, and 87.5% overall correct classification of BRCA1 carriers and controls. Using the original gene expression microarray data used to develop our predictor with nearest shrunken centroids, we rebuilt a classifier based on the k-TSP method. This classifier relies on the relative expression of 10 pairs of genes, compared to the original 43 identified by nearest shrunken centroids (NSC), and was 96.2% concordant with the original training set prediction, with a 94.3% overall correct classification of BRCA1 carriers and controls., Conclusions: The k-TSP classifier was shown to accurately predict BRCA1 status using data generated on the nCounter platform and is feasible for initiating a clinical validation., (© American Association for Clinical Chemistry 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Muscle Invasive bladder cancer is known to have an abundance of mutations, particularly in DNA damage response and chromatin modification genes. The role of these mutations in the development and progression of the disease is not well understood. However, a mutually exclusive mutation pattern between gene pairs could suggest gene mutations of significance. For example, a mutually exclusive mutation pattern could suggest an epistatic relationship where the outcome of a mutation in one gene would have the same outcome as a mutation in a different gene. The significance of a mutually exclusive relationship was determined by establishing a normal distribution of the conditional probabilities for having a mutation in one gene and not the other as well as the reverse relationship for each gene pairing. Then these distributions were used to determine the sigma-magnitude of standard deviation by which the observed value differed from the expected, a value that can also be interpreted as the 'p-value'. This approach led to the identification of mutually exclusive mutation patterns in KDM6A and KMT2D as well as KDM6A and RB1 that suggested the observed mutation pattern did not happen by chance. Upon further investigation of these genes and their interactions, a potential similar outcome was identified that supports the concept of epistasis. Knowledge of these mutational interactions provides a better understanding of the mechanisms underlying muscle invasive bladder cancer development, and may direct therapeutic development exploiting genotoxic chemotherapy and synthetic lethality in these pathways., Competing Interests: The authors have declared that no competing interests exist.

Recruitment of immune cells to a tumor is determined by the complex interplay between cellular and noncellular components of the tumor microenvironment. Ex vivo platforms that enable identification of key components that promote immune cell recruitment to the tumor could advance the field significantly. Herein, we describe the development of a perfusable multicellular tumor-on-a-chip platform involving different cell populations. Cancer cells, monocytes, and endothelial cells were spatially confined within a gelatin hydrogel in a controlled manner by using 3D photopatterning. The migration of the encapsulated endothelial cells against a chemokine gradient created an endothelial layer around the constructs. Using this platform, we examined the effect of cancer cell-monocyte interaction on T-cell recruitment, where T cells were dispersed within the perfused media and allowed to infiltrate. The hypoxic environment in the spheroid cultures recruited more T cells compared with dispersed cancer cells. Moreover, the addition of monocytes to the cancer cells improved T-cell recruitment. The differences in T-cell recruitment were associated with differences in chemokine secretion including chemokines influencing the permeability of the endothelial barrier. This proof-of-concept study shows how integration of microfabrication, microfluidics, and 3D cell culture systems could be used for the development of tumor-on-a-chip platforms involving heterotypic cells and their application in studying recruitment of cells by the tumor-associated microenvironment. SIGNIFICANCE: This study describes how tumor-on-chip platforms could be designed to create a heterogeneous mix of cells and noncellular components to study the effect of the tumor microenvironment on immune cell recruitment., (©2019 American Association for Cancer Research.)

The human RAD9A protein is required for successful execution of the G2/M DNA damage checkpoint. Along with RAD1 and HUS1, RAD9A exists in a heterotrimeric ring-shaped complex which is necessary for activation of the CHK1 checkpoint kinase. RAD9A is also required for proper localization of both TopBP1 and the Claspin adaptor protein during the DNA damage response. We have shown large, RAD9A-dense nuclear foci containing several members of the homologous recombination pathway as well as BRCA1 and the DNA damage marker γH2AX. This RAD9A-dense body is closely associated with the inactive X in HeLa cells but not in other cell types analyzed including a Klinefelter's syndrome-derived line containing multiple Xi. We have also shown these foci disappear after cell synchronization but are enriched after treatment with the homologous recombination inhibitor pentoxifylline. We conclude these foci are the result of an active process, suspended in perturbed cells, that involves interaction between the cell cycle checkpoint and homologous recombination machinery., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We present the development of three-dimensional (3D) cardiac microtissues within a microfluidic device with the ability to quantify real-time contractile stress measurements in situ. Using a 3D patterning technology that allows for the precise spatial distribution of cells within the device, we created an array of 3D cardiac microtissues from neonatal mouse cardiomyocytes. We integrated the 3D micropatterning technology with microfluidics to achieve perfused cell-laden structures. The cells were encapsulated within a degradable gelatin methacrylate hydrogel, which was sandwiched between two polyacrylamide hydrogels. The polyacrylamide hydrogels were used as "stress sensors" to acquire the contractile stresses generated by the beating cardiac cells. The cardiac-specific response of the engineered 3D system was examined by exposing it to epinephrine, an adrenergic neurotransmitter known to increase the magnitude and frequency of cardiac contractions. In response to exogenous epinephrine the engineered cardiac tissues exhibited an increased beating frequency and stress magnitude. Such cost-effective and easy-to-adapt 3D cardiac systems with real-time functional readout could be an attractive technological platform for drug discovery and development.

Techniques that can create three-dimensional (3D) structures to provide architectural support for cells have a significant impact in generating complex and hierarchically organized tissues/organs. In recent times, a number of technologies, including photopatterning, have been developed to create such intricate 3D structures. In this study, we describe an easy-to-implement photopatterning approach, involving a conventional fluorescent microscope and a simple photomask, to encapsulate cells within spatially defined 3D structures. We have demonstrated the ease and the versatility of this approach by creating simple to complex as well as multilayered structures. We have extended this photopatterning approach to incorporate and spatially organize multiple cell types, thereby establishing coculture systems. Such cost-effective and easy-to-use approaches can greatly advance tissue engineering strategies.

Genomic integrity is preserved by checkpoints, which act to delay cell cycle progression in the presence of DNA damage or replication stress. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex is a PCNA-like clamp that is loaded onto DNA at structures resulting from damage and is important for initiating and maintaining the checkpoint response. Rad9 possesses a C-terminal tail that is phosphorylated constitutively and in response to cell cycle position and DNA damage. Previous studies have identified tousled-like kinase 1 (TLK1) as a kinase that may modify Rad9. Here we show that Rad9 is phosphorylated in a TLK-dependent manner in vitro and in vivo, and that T355 within the C-terminal tail is the primary targeted residue. Phosphorylation of Rad9 at T355 is quickly reduced upon exposure to ionizing radiation before returning to baseline later in the damage response. We also show that TLK1 and Rad9 interact constitutively, and that this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, we demonstrate via siRNA-mediated depletion that TLK1 is required for progression through S-phase in normally cycling cells, and that cells lacking TLK1 display a prolonged G2/M arrest upon exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 has previously been shown to be transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, we propose that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn serves to regulate the DNA damage response.

The interaction between the 911 complex, via Rad9A, and Claspin is required for activation of the Chk1-mediated checkpoint response, along with ATR, TopBp1, and the 911 clamp loader complex Rad17/RFC. Despite the importance of the Rad9A-Claspin interaction in the cell cycle, this interaction has yet to be characterized. In this work we show this interaction persists in a variety of different conditions. During the course of this study we also determined the nuclear localization of Rad9A affected the localization of the Claspin protein, leading us to the conclusion that Rad9A is able to affect Claspin cellular localization. This was verified experimentally using a Rad9A-null cell line and reconstitution of Wt Rad9A. We also show that in meS cells the Rad9A paralog, Rad9B, is also capable of affecting Claspin localization. Together, these data suggest that Rad9 plays a role in locating Claspin to sites of DNA damage, facilitating its role during the Chk1-mediated checkpoint response. Since disruption of both Rad9A and Claspin has been shown to abolish Chk1 activation, we postulate that Rad9A-mediated Claspin localization is a vital step during checkpoint activation.

The least understood components of the DNA damage checkpoint are the DNA damage sensors. Genetic studies of Schizosaccharomyces pombe identified six yeast genes, Rad3, Rad17, Rad9, Rad1, Hus1, and Rad26, which encode proteins thought to sense DNA damage and activate the checkpoint-signaling cascade. It has been suggested that Rad9, Rad1 and Hus1 make a heterotrimeric complex forming a PCNA-like structure. In order to carry out structural and biophysical studies of the complex and its associated proteins, the cDNAs encoding full length human Rad9, Rad1 and Hus1 were cloned together into the pET28a vector using a one-step ligation procedure. Here we report successful tri-cistronic cloning, overexpression and purification of this three-protein complex using a single hexa-histidine tag. The trimeric protein complex of Rad9, Rad1 and Hus1 was purified to near homogeneity, yielding approximately 10mg of protein from one liter of Escherichia coli culture.

We have isolated a murine cDNA, Mrad9, that is orthologous to the fission yeast rad9+ and human HRAD9 genes. Mrad9 encodes a 389 amino acid long, 42,032 Dalton protein that is 27% identical and 56% similar to Rad9p, and 82% identical and 88% similar to HRAD9, at the amino acid level. Expression of the Mrad9 cDNA in Schizosaccharomyces pombe rad9::ura4+ cells restores nearly wild-type levels of hydroxyurea resistance and early S phase checkpoint control to mutant fission yeast cell populations. However, UV resistance is only minimally restored, and mutant cells remain sensitive to gamma radiation. Mrad9 genomic DNA was isolated from a mouse 129/SvEv library. The Mrad9 gene was local ized to a 15-kbp genomic DNA fragment, and contains 10 exons separated by 9 introns. Northern blot analysis indicates that the gene is expressed in many different tissues of the adult mouse, but the mRNA is most abundant in the heart and present at very low levels in the liver. These studies demonstrate the existence of a murine orthologue of the fission yeast rad9+ gene and underscore at least the partial evolutionary conservation of rad9+-dependent checkpoint control mechanisms.

DNA polymerase alpha was studied in a direct gap-filling assay. Using a defined template, DNA synthesis was primed from the M13 17-mer universal primer and blocked by an oligonucleotide hybridized 56 nucleotides downstream of the primer. DNA polymerase alpha filled this gap to completion. A time course of the reaction showed that in 50% of the substrate molecules, gaps were filled to completion within 10 min. In another 35% of the molecules the final nucleotide was lacking after 10 min. This nucleotide was added at a reduced rate, and was not incorporated into all of the molecules even after 6 h. The reduced rate of incorporation of the final nucleotide is reflected in an increased Km for de novo incorporation of one nucleotide at a single nucleotide gap (0.7 microM), as opposed to the Km for de novo incorporation of one nucleotide into singly primed M13 DNA (0.18 microM). DNA polymerase alpha purified from murine cells infected with the parvovirus minute virus of mice, and HeLa cell DNA polymerase alpha 2, exhibited the same kinetics of gap filling as did DNA polymerase alpha purified from uninfected Ehrlich ascites murine tumor cells. T4 DNA polymerase filled gaps to completion in this assay. Escherichia coli DNA polymerase I Klenow fragment quantitatively displaced the downstream oligonucleotide, and extended nascent DNA chains for an additional 100 nucleotides. Nicks and single-nucleotide gaps produced in gap-filling reactions by murine DNA polymerase alpha and T4 DNA polymerase were sealed by T4 DNA ligase.

We find that the purified murine DNA polymerase.alpha-primase complex exhibits the greatest affinity for DNA templates in which CCC occurs 10 nucleotides downstream of a DNA primase initiation site (Km = 6.6 +/- 0.3 pM). Templates with 3'-CCA-5' at this position are less efficiently utilized (Km = 16 +/- 4 pM). Point mutations that disrupt the 3'-CC(C/A)-5' motif further decrease the affinity for DNA approximately 7-fold (Km = 105 +/- 58 pM). Mutations at the primase start site reduce Vmax 2-fold. Template pyrimidines are required for priming, and initiation with ATP is preferred to initiation with GTP. We conclude that a component of the DNA polymerase.alpha-primase complex recognizes a 3'-CC(C/A)-5' motif in the DNA template, downstream of a primase start site, and that this interaction controls site selection and frequency of initiation by DNA primase.

The distribution of termination and initiation sites in a 5081-nucleotide minute virus of mice DNA template being copied by a highly purified mouse DNA polymerase alpha-DNA primase complex in the presence of GTP has been examined. The 3'-hydroxyl termini (17 in all) were clustered at six sites that were located 2-14 nucleotides upstream of C2A2C2, C2AC3, or C2A2T2 sequences. When either [alpha-32P]- or [gamma-32P]GTP was included in the DNA polymerase reaction mixtures, nascent DNA became radiolabeled. Analysis of the 32P-labeled material following treatment of the DNA with tobacco acid pyrophosphatase, bacterial alkaline phosphatase, or ribonuclease T1 revealed the presence of oligoribonucleotide chains averaging 5-7 nucleotides long and beginning with 5' GTP residues. Eight presumptive DNA primase initiation sites were located opposite C4 or C5 sequences 3-9 nucleotides upstream of one of the three closely related hexanucleotides C2A2C2, C2AC3, and C2A2T2. RNA-DNA junctions were found 3-10 nucleotides downstream of DNA primase initiation sites. The results indicate that hexanucleotides having the general formula C2A1-2(C2-3/T2), herein referred to as psi, are involved in promoting termination of DNA synthesis and/or de novo initiation of RNA-primed DNA chains by DNA polymerase alpha-primase.