The role of RBC antigen transgene integration sites on RBC biology in mice.

BACKGROUND: Transgenic mice expressing RBC specific antigens are widely used in mechanistic studies of RBC alloimmunization. Existing RBC donor strains have random transgene integration, potentially disrupting host elements that can confound biological interpretation. STUDY DESIGN AND METHODS: Integration site and genomic alterations were characterized by both targeted locus amplification and congenic backcrossing in the five most commonly used RBC alloantigen donor strains (KEL-K2hi , KEL-K2med , and KEL-K2lo , and KEL-K1). A targeted transgenic approach was developed to allow RBC specific transgene expression from a safe harbor locus (ROSA26). Alloimmune responses were assessed by transfusing alloantigen expressing RBCs into wild-type recipients and measuring alloantibodies by flow cytometry. RESULTS/FINDINGS: Four of the five analyzed strains had at least one gene disrupted by the transgene integration but none of the disrupted genes are known to be involved in RBC biology. The integration of KEL-K2med potentially altered the immunological properties of RBCs, although the biological significance of the observed changes is unclear. The ROSA26 targeted approach resulted in a single copy of the transgene that maintains RBC specific expression without random disruption of genomic elements. CONCLUSION: These findings provide a detailed characterization of genomic disruption by transgene integration found in commonly used RBC donor strains that is relevant to numerous previous publications as well as future studies. With the possible exception of KEL-K2med , transgene integration is not predicted to affect RBC biology in existing models, and new models can avoid this concern using the described targeted transgenic approach.

Protein kinase CK2 phosphorylation of SAPS3 subunit increases PP6 phosphatase activity with Aurora A kinase.

Protein Ser/Thr phosphatase-6 (PP6) regulates pathways for activation of NF-kB, YAP1 and Aurora A kinase (AURKA). PP6 is a heterotrimer comprised of a catalytic subunit, one of three different SAPS subunits and one of three different ankyrin-repeat ANKRD subunits. Here, we show FLAG-PP6C expressed in cells preferentially binds endogenous SAPS3, and the complex is active with the chemical substrate DiFMUP. SAPS3 has multiple acidic sequence motifs recognized by protein kinase CK2 (CK2) and SAPS3 is phosphorylated by purified CK2, without affecting its associated PP6 phosphatase activity. However, HA3-SAPS3-PP6 phosphatase activity using pT288 AURKA as substrate is significantly increased by phosphorylation with CK2. The substitution of Ala in nine putative phosphorylation sites in SAPS3 was required to prevent CK2 activation of the phosphatase. Different CK2 chemical inhibitors equally increased phosphorylation of endogenous AURKA in living cells, consistent with reduction in PP6 activity. CRISPR/Cas9 deletion or siRNA knockdown of SAPS3 resulted in highly activated endogenous AURKA, and a high proportion of cells with abnormal nuclei. Activation of PP6 by CK2 can form a feedback loop with bistable changes in substrates.

Selective toxicity of caffeic acid in hepatocellular carcinoma cells.

Caffeic acid is a natural phytochemical structurally similar to other cinnamic acids. In this study we found caffeic acid (CA) but not ferulic, sinapic or cinnamic acids inhibited proliferation of hepatocellular carcinoma cells (HCC) and reduced cell numbers by inducing apoptosis. Only transient exposure to CA was required for these lethal effects that are associated with disruption of mitochondrial membrane potential and induction of reactive oxygen species. By comparison, primary hepatocytes resisted CA toxicity for nearly 48 h, consistent with selective sensitivity of HCC to CA. These results support use of CA as an anti-tumor agent to inhibit HCC, especially if delivered by locoregional catheterization in an embolization procedure.

Deregulation of F-box proteins and its consequence on cancer development, progression and metastasis.

F-box proteins are substrate receptors of the SCF (SKP1-Cullin 1-F-box protein) E3 ubiquitin ligase that play important roles in a number of physiological processes and activities. Through their ability to assemble distinct E3 ubiquitin ligases and target key regulators of cellular activities for ubiquitylation and degradation, this versatile group of proteins is able to regulate the abundance of cellular proteins whose deregulated expression or activity contributes to disease. In this review, we describe the important roles of select F-box proteins in regulating cellular activities, the perturbation of which contributes to the initiation and progression of a number of human malignancies.

Novel Human Cytomegalovirus Viral Chemokines, vCXCL-1s, Display Functional Selectivity for Neutrophil Signaling and Function.

Human CMV (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable genes in the HCMV genome. Why HCMV has evolved this hypervariability and how this affects the virus' dissemination and pathogenesis is unknown. Because the vCXCL-1 hypervariability maps to important binding and activation domains, we hypothesized that vCXCL-1s differentially activate neutrophils, which could contribute to HCMV dissemination, pathogenesis, or both. To test whether these viral chemokines affect neutrophil function, we generated vCXCL-1 proteins from 11 different clades from clinical isolates from infants infected congenitally with HCMV. All vCXCL-1s were able to induce calcium flux at a concentration of 100 nM and integrin expression on human peripheral blood neutrophils, despite differences in affinity for the CXCR1 and CXCR2 receptors. In fact, their affinity for CXCR1 or CXCR2 did not correlate directly with chemotaxis, G protein-dependent and independent (ß-arrestin-2) activation, or secondary chemokine (CCL22) expression. Our data suggest that vCXCL-1 polymorphisms affect the binding affinity, receptor usage, and differential peripheral blood neutrophil activation that could contribute to HCMV dissemination and pathogenesis.

A human short open reading frame (sORF)-encoded polypeptide that stimulates DNA end joining.

The recent discovery of numerous human short open reading frame (sORF)-encoded polypeptides (SEPs) has raised important questions about the functional roles of these molecules in cells. Here, we show that a 69-amino acid SEP, MRI-2, physically interacts with the Ku heterodimer to stimulate DNA double-strand break ligation via nonhomologous end joining. The characterization of MRI-2 suggests that this SEP may participate in DNA repair and underscores the potential of SEPs to serve important biological functions in mammalian cells.

Serial Circulating Tumor DNA Analysis with a Tumor-Naïve Next-Generation Sequencing Panel Detects Minimal Residual Disease and Predicts Outcome in Ovarian Cancer.

Circulating tumor DNA (ctDNA) may aid in personalizing ovarian cancer therapeutic options. Here, we aimed to assess the clinical utility of serial ctDNA testing using tumor-naïve, small-sized next-generation sequencing (NGS) panels. A total of 296 patients, including 201 with ovarian cancer and 95 with benign or borderline disease, were enrolled. Samples were collected at baseline (initial diagnosis or surgery) and every 3 months after that, resulting in a total of 811 blood samples. Patients received adjuvant therapy based on the current standard of care. Cell-free DNA was extracted and sequenced using an NGS panel of 9 genes: TP53, BRCA1, BRCA2, ARID1A, CCNE1, KRAS, MYC, PIK3CA, and PTEN. Pathogenic somatic mutations were identified in 69.2% (139/201) of patients with ovarian cancer at baseline but not in those with benign or borderline disease. Detection of ctDNA at baseline and/or at 6 months follow-up was predictive of progression-free survival (PFS). PFS was significantly poorer in patients with detectable pathogenic mutations at baseline that persisted at follow-up than in patients that converted from having detectable ctDNA at baseline to being undetectable at follow-up; survival did not differ between patients without pathogenic ctDNA mutations in baseline or follow-up samples and those that converted from ctDNA positive to negative. Disease recurrence was also detected earlier with ctDNA than with conventional radiologic assessment or CA125 monitoring. These findings demonstrate that serial ctDNA testing could effectively monitor patients and detect minimal residual disease, facilitating early detection of disease progression and tailoring of adjuvant therapies for ovarian cancer treatment. SIGNIFICANCE: In ovarian cancer, serial circulating tumor DNA testing is a highly predictive marker of patient survival, with a significantly improved recurrence detection lead time compared with conventional monitoring tools.

Transcriptome-wide association study and eQTL colocalization identify potentially causal genes responsible for human bone mineral density GWAS associations.

Genome-wide association studies (GWASs) for bone mineral density (BMD) in humans have identified over 1100 associations to date. However, identifying causal genes implicated by such studies has been challenging. Recent advances in the development of transcriptome reference datasets and computational approaches such as transcriptome-wide association studies (TWASs) and expression quantitative trait loci (eQTL) colocalization have proven to be informative in identifying putatively causal genes underlying GWAS associations. Here, we used TWAS/eQTL colocalization in conjunction with transcriptomic data from the Genotype-Tissue Expression (GTEx) project to identify potentially causal genes for the largest BMD GWAS performed to date. Using this approach, we identified 512 genes as significant using both TWAS and eQTL colocalization. This set of genes was enriched for regulators of BMD and members of bone relevant biological processes. To investigate the significance of our findings, we selected PPP6R3, the gene with the strongest support from our analysis which was not previously implicated in the regulation of BMD, for further investigation. We observed that Ppp6r3 deletion in mice decreased BMD. In this work, we provide an updated resource of putatively causal BMD genes and demonstrate that PPP6R3 is a putatively causal BMD GWAS gene. These data increase our understanding of the genetics of BMD and provide further evidence for the utility of combined TWAS/colocalization approaches in untangling the genetics of complex traits.

Genome-Wide Association Study for Ultraviolet-B Resistance in Soybean (Glycine max L.).

The depletion of the stratospheric ozone layer is a major environmental issue and has increased the dosage of ultraviolet-B (UV-B) radiation reaching the Earth's surface. Organisms are negatively affected by enhanced UV-B radiation, and especially in crop plants this may lead to severe yield losses. Soybean (Glycine max L.), a major legume crop, is sensitive to UV-B radiation, and therefore, it is required to breed the UV-B-resistant soybean cultivar. In this study, 688 soybean germplasms were phenotyped for two categories, Damage of Leaf Chlorosis (DLC) and Damage of Leaf Shape (DLS), after supplementary UV-B irradiation for 14 days. About 5% of the germplasms showed strong UV-B resistance, and GCS731 was the most resistant genotype. Their phenotypic distributions showed similar patterns to the normal, suggesting UV-B resistance as a quantitative trait governed by polygenes. A total of 688 soybean germplasms were genotyped using the Axiom® Soya 180K SNP array, and a genome-wide association study (GWAS) was conducted to identify SNPs significantly associated with the two traits, DLC and DLS. Five peaks on chromosomes 2, 6, 10, and 11 were significantly associated with either DLC or DLS, and the five adjacent genes were selected as candidate genes responsible for UV-B resistance. Among those candidate genes, Glyma.02g017500 and Glyma.06g103200 encode cryptochrome (CRY) and cryptochrome 1 (CRY1), respectively, and are known to play a role in DNA repair during photoreactivation. Real-time quantitative RT-PCR (qRT-PCR) results revealed that CRY1 was expressed significantly higher in the UV-B-resistant soybean compared to the susceptible soybean after 6 h of UV-B irradiation. This study is the first GWAS report on UV-B resistance in soybean, and the results will provide valuable information for breeding UV-B-resistant soybeans in preparation for climate change.

Identification of a novel trait associated with phytotoxicity of an insecticide etofenprox in soybean.

Synthetic insecticides are widely used to control pests in various crop fields. Especially in soybean [Glycine max (L.) Merr.] fields, the insecticide etofenprox, which is a pyrethroid derivative, has been used to manage hemiptera pests. To date, soybean phytotoxicity response has not been reported to etofenprox derivatives, two Korean cultivars, Danbaek and Kwangan, were first identified to show leaf shape shrinkage damage after etofenprox application. We confirmed that the causal substance for phytotoxicity is etofenprox and that it had dosage effects. Through genetic analysis using three F2 populations, sensitivity to etofenprox is confirmed to be managed by a single dominant gene, and that gene is the same in Danbaek and Kwangan. Although further genetic research is required to identify the gene responsible for sensitivity to etofenprox, the results of this study will help to elucidate the interaction between plants and chemicals when breeding new cultivars or developing pesticides.

Deletion of the SAPS1 subunit of protein phosphatase 6 in mice increases radiosensitivity and impairs the cellular DNA damage response.

Cellular responses to DNA damage include activation of DNA-dependent protein kinase (DNA-PK) through, among others, the serine/threonine protein phosphatase 6 (PP6). We previously showed that recognition of DNA-PKcs is mediated by the SAPS1 PP6 regulatory subunit. Here, we report and characterize a SAPS1 null mouse and investigate the effects of deletion on DNA damage signaling and repair. Strikingly, neither SAPS1-null animals nor cells derived from them show gross defects, unless subjected to DNA damage by radiation or chemical agents. The overall survival of SAPS1-null animals following whole body irradiation is significantly shortened as compared to wild-type mice, and the clonogenic survival of null cells subjected to ionizing radiation is reduced. The dephosphorylation of DNA damage/repair markers, such as γH2AX, p53 and Kap1, is diminished in SAPS1-null cells as compared to wild-type controls. Our results demonstrate that loss of SAPS1 confers sensitivity to DNA damage and confirms previously reported cellular phenotypes of SAPS1 knock-down in human glioma cells. The results support a role for PP6 regulatory subunit SAPS1 in DNA damage responses, and offer a novel target for sensitization to enhance current tumor therapies, with a potential for limited deleterious side effects.

LMO7 deficiency reveals the significance of the cuticular plate for hearing function.

Sensory hair cells, the mechanoreceptors of the auditory and vestibular systems, harbor two specialized elaborations of the apical surface, the hair bundle and the cuticular plate. In contrast to the extensively studied mechanosensory hair bundle, the cuticular plate is not as well understood. It is believed to provide a rigid foundation for stereocilia motion, but specifics about its function, especially the significance of its integrity for long-term maintenance of hair cell mechanotransduction, are not known. We discovered that a hair cell protein called LIM only protein 7 (LMO7) is specifically localized in the cuticular plate and the cell junction. Lmo7 KO mice suffer multiple cuticular plate deficiencies, including reduced filamentous actin density and abnormal stereociliar rootlets. In addition to the cuticular plate defects, older Lmo7 KO mice develop abnormalities in inner hair cell stereocilia. Together, these defects affect cochlear tuning and sensitivity and give rise to late-onset progressive hearing loss.

Combined Treatment with Low-Level Laser and rhBMP-2 Promotes Differentiation and Mineralization of Osteoblastic Cells under Hypoxic Stress.

BACKGROUND: The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia. METHODS: MC3T3-E1 cells were cultured under 1% oxygen tension for 72 h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48 h. Cells were treated with rhBMP-2 at 50 ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14 days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14 days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved. RESULTS: The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha (HIF-1α), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation. CONCLUSION: Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxic-cultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.

Impact of periodontitis as representative of chronic inflammation on long-term clinical outcomes in patients with atrial fibrillation.

Objectives: Relationship between atrial fibrillation (AF) and inflammation was shown in previous studies. However, there was limited data about the association between the periodontitis and AF in the long-term follow-up. The aim of this study was to evaluate the impact of periodontitis on long-term clinical outcomes in patients with AF. Methods: The Kosin University echocardiography, ECG and periodontitis database were reviewed from 2013 to 2015 to identify patients with AF. Those patients were divided into two groups according to the presence of periodontitis and clinical events including any arrhythmic attack, thromboembolic and bleeding and death were collected during a median of 18 months. Results: Among 227 patients with AF, 47 (20.7%) patients had periodontitis. Major adverse cardiac events (MACE) were significantly higher in patients with periodontitis compared with those without periodontitis (p<0.001). Arrhythmias including AF, atrial tachycardia, atrial premature beat, ventricular tachycardia and ventricular premature beat also occurred in 44 (93.6%) patients, which was higher significantly higher incidence in patients with periodontitis than in those without periodontitis (p<0.001). In univariate analysis, age, CHA2DS2-VASc, left atrial volume index (LAVi) and periodontitis were significantly associated with arrhythmic events and MACE including bleeding events, thromboembolic events, arrhythmic events and mortality. In multivariate analysis, LAVi (p=0.005) and periodontitis (p<0.001) were independent risk factors for arrhythmic events and periodontitis (p<0.001) for MACE at the long-term follow-up. Conclusions: The periodontitis as representative of chronic inflammation was an independent predictor of arrhythmic events and MACE in patients with AF.

Osseointegration of magnesium-incorporated sand-blasted acid-etched implant in the dog mandible: Resonance frequency measurements and histomorphometric analysis.

The aim of this pilot study was to investigate the bone responses of novel magnesium (Mg)-incorporated sand-blasted and acid-etched (SLA) titanium (Ti) implant in an experimental animal model. Novel Mg-incorporated SLA Ti implant was obtained via vacuum arc source ion implantation method and Mg-ions were implanted into the SLA implant surface. Control group consisted of two commercial implants; resorbable blasting media (RBM) and SLA. Twelve implants from each group were placed into the mandibles of 6 mongrel dogs. Experimental animals were divided into 2 groups of 3 animals, with 4 weeks and 8 weeks healing time points. Resonance frequency analysis was performed at the time of fixture installation, 1, 2, 4, and 8 weeks after installation. Bone to implant contact (BIC) measurements were assessed at the 4 and 8 weeks healing time points. The overall implant survival rate was 97.2%. The Mg-incorporated SLA Ti implants showed more rapid osseointegration than control group implants at follow-up periods of 4 weeks. Histomorphometric analysis showed a tendency for BIC% values of Mg-incorporated SLA Ti implant to be higher than that of other the implant groups. The results of this study suggest that Mg-incorporated SLA Ti implant may be effective in enhancing the bone responses by rapid osseointegration in early healing periods.

TDP1 promotes assembly of non-homologous end joining protein complexes on DNA.

The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage and 3'-blocking DNA lesions, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 stimulated DNA binding by XLF and physically interacted with XLF to form TDP1:XLF:DNA complexes. TDP1:XLF interactions preferentially stimulated TDP1 activity on dsDNA as compared to ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Because Ku70/80 and XLF are the first factors recruited to the DSB at the onset of NHEJ, our data suggest a role for TDP1 during the early stages of mammalian NHEJ.

Polymorphisms within human cytomegalovirus chemokine (UL146/UL147) and cytokine receptor genes (UL144) are not predictive of sequelae in congenitally infected children.

Human cytomegalovirus (HCMV) viral chemokine, UL146, and TNF alpha-like receptor UL144 genes show a high degree of hypervariability in clinical isolates. These proteins are predicted to be immune modulators and may contribute to the pathogenesis of HCMV infections. We analyzed the UL146 and UL144 genetic variation of 51 HCMV isolates from congenitally infected children and 13 isolates from children in childcare. There was no statistically significant correlation between UL146 and UL144 genotypes and HCMV disease and/or sequelae. However, there were some groups that had a relatively large proportion of asymptomatic outcomes. These included UL146 group 8 (7/8 asymptomatic) and UL146 group 10 (3/3 asymptomatic). UL144 group B had 11/15 (73%) asymptomatic. UL146 and UL144 genes remained stable in serial isolates from children in daycare for intervals up to three years. These results indicate that most UL146 and UL144 genotypes do not predict clinical sequelae following congenital HCMV infections.

Growth of mycobacteria on carbon monoxide and methanol.

Several mycobacterial strains, such as Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy. These bacteria, except for M. tuberculosis, also utilized methanol as the sole carbon and energy source. A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH of Mycobacterium sp. strain JC1 (formerly Acinetobacter sp. strain JC1 [J. W. Cho, H. S. Yim, and Y. M. Kim, Kor. J. Microbiol. 23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO. Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, except M. tuberculosis, grown on CO. The mycobacteria grown on methanol, except for M. gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibited N,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities. Cells grown on methanol were also found to have dihydroxyacetone synthase. Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of the Mycobacterium sp. strain JC1 enzymes.