Genomic characterization of cervical lymph node metastases in papillary thyroid carcinoma following the Chornobyl accident.

Childhood radioactive iodine exposure from the Chornobyl accident increased papillary thyroid carcinoma (PTC) risk. While cervical lymph node metastases (cLNM) are well-recognized in pediatric PTC, the PTC metastatic process and potential radiation association are poorly understood. Here, we analyze cLNM occurrence among 428 PTC with genomic landscape analyses and known drivers (131I-exposed = 349, unexposed = 79; mean age = 27.9 years). We show that cLNM are more frequent in PTC with fusion (55%) versus mutation (30%) drivers, although the proportion varies by specific driver gene (RET-fusion = 71%, BRAF-mutation = 38%, RAS-mutation = 5%). cLNM frequency is not associated with other characteristics, including radiation dose. cLNM molecular profiling (N = 47) demonstrates 100% driver concordance with matched primary PTCs and highly concordant mutational spectra. Transcriptome analysis reveals 17 differentially expressed genes, particularly in the HOXC cluster and BRINP3; the strongest differentially expressed microRNA also is near HOXC10. Our findings underscore the critical role of driver alterations and provide promising candidates for elucidating the biological underpinnings of PTC cLNM.

Extrachromosomal Amplification of Human Papillomavirus Episomes Is a Mechanism of Cervical Carcinogenesis.

SIGNIFICANCE: Multimers of the HPV genome are generated in cervical tumors replicating as extrachromosomal episomes, which is associated with deletion and rearrangement of the HPV genome and provides a mechanism for oncogenesis without integration.

Development of Aging-Related Emphysematous and Lymphoma-Like Lesions is Enhanced by the Lack of Secretoglobin 3A2 in Mouse Lungs.

Background: Secretoglobin (SCGB) 3A2 is a novel bioactive molecule with anti-inflammatory and anti-fibrotic activities. SCGB3A2 also promotes the maturation of bronchial divergence and the lungs during embryonic development. However, much remains unknown concerning the roles of SCGB3A2 in diseases associated with aging. Methods: The lungs of Scgb3a2-knockout (KO) mice and their wild-type (WT) littermates were subjected to histological analysis, Victoria blue staining to evaluate of elastic fibers, and lung morphometric analysis during the postnatal period (birth to 8 weeks) and during aging (8 weeks to 2 years). Their spleens were also histologically evaluated. The expression of lung surfactant protein (SP) mRNAs was examined by quantitative reverse transcriptase-polymerase chain reaction. RNA sequencing (RNAseq) analysis was performed on 3-month-old KO and WT mouse lungs. Results: The alveolar spaces of KO mice continuously expanded between 0.5 and 2 years of age, accompanied by increases of the mean linear intercept and destructive index. KO mouse lungs displayed inflammation associated with lymphocyte aggregate starting at 1 year of age, and the inflammation was worse than that of WT mouse lungs. A high number of lymphoma-like cells were presented in 2-year-old KO mouse lungs. White pulp fusion was detected in the spleens of both WT and KO mice older than 0.5 years; however, the fusion was more severe in KO mice than in WT mice. The expression of surfactant protein (SP)-A, SP-B, SP-C, and SP-D mRNAs in KO mouse lungs decreased with age, and after 1 year of age, the expression of most SPs was significantly lower in KO mice than in WT mice. RNAseq demonstrated that the expression of immune system-related genes was highly altered in KO mouse lungs. Conclusion: SCGB3A2 may be required for maintaining homeostasis and immune activity in the lungs during aging. SCGB3A2 deficiency might increase the risk of emphysema of the lung.

Pathogenic germline IKZF1 variant alters hematopoietic gene expression profiles.

IKZF1 encodes Ikaros, a zinc finger-containing transcription factor crucial to the development of the hematopoietic system. Germline pathogenic variants in IKZF1 have been reported in patients with acute lymphocytic leukemia and immunodeficiency syndromes. Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by erythroid hypoplasia, associated with a spectrum of congenital anomalies and an elevated risk of certain cancers. DBA is usually caused by heterozygous pathogenic variants in genes that function in ribosomal biogenesis; however, in many cases the genetic etiology is unknown. We identified a germline IKZF1 variant, rs757907717 C > T, in identical twins with DBA-like features and autoimmune gastrointestinal disease. rs757907717 C > T results in a p.R381C amino acid change in the IKZF1 Ik-x isoform (p.R423C on isoform Ik-1), which we show is associated with altered global gene expression and perturbation of transcriptional networks involved in hematopoietic system development. These data suggest that this missense substitution caused a DBA-like syndrome in this family because of alterations in hematopoiesis, including dysregulation of networks essential for normal erythropoiesis and the immune system.

Radiation-related genomic profile of papillary thyroid carcinoma after the Chernobyl accident.

The 1986 Chernobyl nuclear power plant accident increased papillary thyroid carcinoma (PTC) incidence in surrounding regions, particularly for radioactive iodine (131I)-exposed children. We analyzed genomic, transcriptomic, and epigenomic characteristics of 440 PTCs from Ukraine (from 359 individuals with estimated childhood 131I exposure and 81 unexposed children born after 1986). PTCs displayed radiation dose-dependent enrichment of fusion drivers, nearly all in the mitogen-activated protein kinase pathway, and increases in small deletions and simple/balanced structural variants that were clonal and bore hallmarks of nonhomologous end-joining repair. Radiation-related genomic alterations were more pronounced for individuals who were younger at exposure. Transcriptomic and epigenomic features were strongly associated with driver events but not radiation dose. Our results point to DNA double-strand breaks as early carcinogenic events that subsequently enable PTC growth after environmental radiation exposure.

1q21.1 deletion and a rare functional polymorphism in siblings with thrombocytopenia-absent radius-like phenotypes.

Thrombocytopenia-absent radii (TAR) syndrome, characterized by neonatal thrombocytopenia and bilateral radial aplasia with thumbs present, is typically caused by the inheritance of a 1q21.1 deletion and a single-nucelotide polymorphism in RBM8A on the nondeleted allele. We evaluated two siblings with TAR-like dysmorphology but lacking thrombocytopenia in infancy. Family NCI-107 participated in an IRB-approved cohort study and underwent comprehensive clinical and genomic evaluations, including aCGH, whole-exome, whole-genome, and targeted sequencing. Gene expression assays and electromobility shift assays (EMSAs) were performed to evaluate the variant of interest. The previously identified TAR-associated 1q21.1 deletion was present in the affected siblings and one healthy parent. Multiple sequencing approaches did not identify previously described TAR-associated SNPs or mutations in relevant genes. We discovered rs61746197 A > G heterozygosity in the parent without the deletion and apparent hemizygosity in both siblings. rs61746197 A > G overlaps a RelA-p65 binding motif, and EMSAs indicate the A allele has higher transcription factor binding efficiency than the G allele. Stimulation of K562 cells to induce megakaryocyte differentiation abrogated the shift of both reference and alternative probes. The 1q21.1 TAR-associated deletion in combination with the G variant of rs61746197 on the nondeleted allele is associated with a TAR-like phenotype. rs61746197 G could be a functional enhancer/repressor element, but more studies are required to identify the specific factor(s) responsible. Overall, our findings suggest a role of rs61746197 A > G and human disease in the setting of a 1q21.1 deletion on the other chromosome.

Meiotic Knockdown and Complementation Reveals Essential Role of RAD51 in Mouse Spermatogenesis.

Meiotic homologous recombination (HR) is important for proper chromosomal segregation during gametogenesis and facilitates evolutionary adaptation via genomic reshuffling. In most eukaryotes, HR is mediated by two recombinases, the ubiquitous RAD51 and the meiosis-specific DMC1. The role of RAD51 in mammalian meiosis is unclear and study of its function is limited due to embryonic lethality of RAD51 knockouts. Here, we developed an in vivo meiotic knockdown and protein complementation system to study RAD51 during mouse spermatogenesis. We show that RAD51 is crucial during meiotic prophase and its loss leads to depletion of late prophase I spermatocytes through a p53-dependent apoptotic pathway. This phenotype is distinct from that observed in the DMC1 knockdown. Our meiotic knockdown and complementation system establishes an experimental platform for mechanistic studies of meiotic proteins with unknown functions or essential genes for which a testis-specific knockout is not possible.

Activation of an AMP-activated protein kinase is involved in post-diapause development of Artemia franciscana encysted embryos.

BACKGROUND: Cysts of Artemia can remain in a dormant state for long periods with a very low metabolic rate, and only resume their development with the approach of favorable conditions. The post-diapause development is a very complicated process involving a variety of metabolic and biochemical events. However, the intrinsic mechanisms that regulate this process are unclear. RESULTS: Herein we report the specific activation of an AMP-activated protein kinase (AMPK) in the post-diapause developmental process of Artemia. Using a phospho-AMPKalpha antibody, AMPK was shown to be phosphorylated in the post-diapause developmental process. Results of kinase assay analysis showed that this phosphorylation is essential for AMPK activation. Using whole-mount immunohistochemistry, phosphorylated AMPK was shown to be predominantly located in the ectoderm of the early developed embryos in a ring shape; however, the location and shape of the activation region changed as development proceeded. Additionally, Western blotting analysis on different portions of the cyst extracts showed that phosphorylated AMPKalpha localized to the nuclei and this location was not affected by intracellular pH. Confocal microscopy analysis of immunofluorescent stained cyst nuclei further showed that AMPKalpha localized to the nuclei when activated. Moreover, cellular AMP, ADP, and ATP levels in developing cysts were determined by HPLC, and the results showed that the activation of Artemia AMPK may not be associated with cellular AMP:ATP ratios, suggesting other pathways for regulation of Artemia AMPK activity. CONCLUSION: Together, we report evidence demonstrating the activation of AMPK in Artemia developing cysts and present an argument for its role in the development-related gene expression and energy control in certain cells during post-diapause development of Artemia.

Inhibition of a novel sperm gelatinase in prawn sperm by the male reproduction-related Kazal-type peptidase inhibitor.

Previously, we have identified and characterized a male reproduction-related kazal-type peptidase inhibitor (MRPINK) gene from the prawn, Macrobrachium rosenbergii. In the present study, MRPINK was discovered to have an inhibitory effect on the gelatinolytic activity of M. rosenbergii sperm and immunofluorescence analysis revealed it bound specifically onto the base of sperm. The proteolytic activity of sperm extracts to vitelline coat components was also detected to be interfered by MRPINK. Furthermore, a novel gelatinase on sperm was found to be specifically inhibited by MRPINK and was named M. rosenbergii sperm gelatinase (MSG). MSG was then isolated and purified by reversed-phase high performance liquid chromatography combining with gelatinolytic assay. By amino-terminal amino acid sequence analysis and molecular cloning, the primary structure of MSG was determined. The data presented in this study provided evidence that MRPINK has an inhibitory effect on the gelatinolytic activity as well as proteolytic activity of prawn sperm and specifically blocks the activity of MSG.

Involvement of p90 ribosomal S6 kinase in termination of cell cycle arrest during development of Artemia-encysted embryos.

Artemia has evolved a unique developmental pattern of encysted embryos to cope with various environmental threats. Cell divisions totally cease during the preemergence developmental stage from gastrula to prenauplius. The molecular mechanism of this, however, remains unknown. Our study focuses on the involvement of p90 ribosomal S6 kinase (RSK), a family of serine/threonine kinase-mediating signal transduction downstream of mitogen-activated protein kinase cascades, in the termination of cell cycle arrest during the post-embryonic development of Artemia-encysted gastrula. With immunochemistry, morphology, and cell cycle analysis, the identified Artemia RSK was established to be specifically activated during the post-embryonic and early larval developmental stages when arrested cells of encysted embryos resumed mitoses. In vivo knockdown of RSK activity by RNA interference, kinase inhibition, and antibody neutralization consistently induced defective larvae with distinct gaps between the exoskeleton and internal tissues. In these abnormal individuals, mitoses were detected to be largely inhibited in the affected regions. These results display the requirement of RSK activity during Artemia development and suggest its role in termination of cell cycle (G(2)/M phase) arrest and promotion of mitogenesis. Our findings may, thus, provide insights into the regulation of cell division during Artemia post-embryonic development and reveal further aspects of RSK functions.

A male reproduction-related Kazal-type peptidase inhibitor gene in the prawn, Macrobrachium rosenbergii: molecular characterization and expression patterns.

Peptidase inhibitors in the male reproductive tract are well known in mammals, in which they play roles in protecting the tract epithelium against proteolytic damage or in regulating the fertilization process. By screening the subtracted cDNA clones enriched for male reproductive tract-specific transcripts, one clone encoding a putative protein that showed significant similarity to Kazal-type peptidase inhibitor (KPI) was obtained. This is the first report of an invertebrate in which a male reproductive tract-specific KPI gene has been identified and characterized. The gene contains a 405-bp open reading frame (ORF), a 72 bp 5' untranslated region (UTR), and a 259 bp 3' UTR. The conceptually translated protein consisted of a 21-amino-acid signal peptide and a 113-amino-acid mature polypeptide with two Kazal-type domains (named after the discoverer). Significant levels of the mRNA were observed only in the male reproductive tract, while mRNA expression was not detected in any other tissues tested. The transcription of the gene remained constant during maturation, although not in the postlarval stage. In situ hybridization demonstrated the presence of the mRNA in the secretory epithelial cells of vas deferens and terminal ampullae.