Fused in sarcoma (FUS) inhibits milk production efficiency in mammals.

Efficient milk production in mammals confers evolutionary advantages by facilitating the transmission of energy from mother to offspring. However, the regulatory mechanism responsible for the gradual establishment of milk production efficiency in mammals, from marsupials to eutherians, remains elusive. Here, we find that mammary gland of the marsupial sugar glider contained milk components during adolescence, and that mammary gland development is less dynamically cyclic compared to that in placental mammals. Furthermore, fused in sarcoma (FUS) is found to be partially responsible for this establishment of low efficiency. In mouse model, FUS inhibit mammary epithelial cell differentiation through the cyclin-dependent kinase inhibitor p57Kip2, leading to lactation failure and pup starvation. Clinically, FUS levels are negatively correlated with milk production in lactating women. Overall, our results shed light on FUS as a negative regulator of milk production, providing a potential mechanism for the establishment of milk production from marsupial to eutherian mammals.

Imprinted gene alterations in the kidneys of growth restricted offspring may be mediated by a long non-coding RNA.

Altered epigenetic mechanisms have been previously reported in growth restricted offspring whose mothers experienced environmental insults during pregnancy in both human and rodent studies. We previously reported changes in the expression of the DNA methyltransferase Dnmt3a and the imprinted genes Cdkn1c (Cyclin-dependent kinase inhibitor 1C) and Kcnq1 (Potassium voltage-gated channel subfamily Q member 1) in the kidney tissue of growth restricted rats whose mothers had uteroplacental insufficiency induced on day 18 of gestation, at both embryonic day 20 (E20) and postnatal day 1 (PN1). To determine the mechanisms responsible for changes in the expression of these imprinted genes, we investigated DNA methylation of KvDMR1, an imprinting control region (ICR) that includes the promoter of the antisense long non-coding RNA Kcnq1ot1 (Kcnq1 opposite strand/antisense transcript 1). Kcnq1ot1 expression decreased by 51% in growth restricted offspring compared to sham at PN1. Interestingly, there was a negative correlation between Kcnq1ot1 and Kcnq1 in the E20 growth restricted group (Spearman's ρ = 0.014). No correlation was observed between Kcnq1ot1 and Cdkn1c expression in either group at any time point. Additionally, there was a 11.25% decrease in the methylation level at one CpG site within KvDMR1 ICR. This study, together with others in the literature, supports that long non-coding RNAs may mediate changes seen in tissues of growth restricted offspring.

Structure-Function Analysis of p57KIP2 in the Human Pancreatic Beta Cell Reveals a Bipartite Nuclear Localization Signal.

Mutations in CDKN1C, encoding p57KIP2, a canonical cell cycle inhibitor, underlie multiple pediatric endocrine syndromes. Despite this central role in disease, little is known about the structure and function of p57KIP2 in the human pancreatic beta cell. Since p57KIP2 is predominantly nuclear in human beta cells, we hypothesized that disease-causing mutations in its nuclear localization sequence (NLS) may correlate with abnormal phenotypes. We prepared RIP1 insulin promoter-driven adenoviruses encoding deletions of multiple disease-associated but unexplored regions of p57KIP2 and performed a comprehensive structure-function analysis of CDKN1C/p57KIP2. Real-time polymerase chain reaction and immunoblot analyses confirmed p57KIP2 overexpression, construct size, and beta cell specificity. By immunocytochemistry, wild-type (WT) p57KIP2 displayed nuclear localization. In contrast, deletion of a putative NLS at amino acids 278-281 failed to access the nucleus. Unexpectedly, we identified a second downstream NLS at amino acids 312-316. Further analysis showed that each individual NLS is required for nuclear localization, but neither alone is sufficient. In summary, p57KIP2 contains a classical bipartite NLS characterized by 2 clusters of positively charged amino acids separated by a proline-rich linker region. Variants in the sequences encoding these 2 NLS sequences account for functional p57KIP2 loss and beta cell expansion seen in human disease.

Beckwith-Wiedemann Syndrome in Newborn of Mother with HELLP Syndrome/Preeclampsia: An Analysis of Literature and Case Report with Fetal Growth Restriction and Absence of CDKN1C Typical Pathogenic Genetic Variation.

Beckwith-Wiedemann Syndrome (BWS) is an imprinting disorder, which manifests by overgrowth and predisposition to embryonal tumors. The evidence on the relationship between maternal complications such as HELLP (hemolysis, elevated liver enzymes, and low platelet count) and preeclampsia and the development of BWS in offspring is scarce. A comprehensive clinical evaluation, with genetic testing focused on screening for mutations in the CDKN1C gene, which is commonly associated with BWS, was conducted in a newborn diagnosed with BWS born to a mother with a history of preeclampsia and HELLP syndrome. The case study revealed typical clinical manifestations of BWS in the newborn, including hemihyperplasia, macroglossia, midfacial hypoplasia, omphalocele, and hypoglycemia. Surprisingly, the infant also exhibited fetal growth restriction, a finding less commonly observed in BWS cases. Genetic analysis, however, showed no mutations in the CDKN1C gene, which contrasts with the majority of BWS cases. This case report highlights the complex nature of BWS and its potential association with maternal complications such as preeclampsia and HELLP syndrome. The atypical presence of fetal growth restriction in the newborn and the absence of CDKN1C gene mutations have not been reported to date in BWS.

Androgenetic/biparental mosaicism in a diploid mole-like conceptus: report of a case with triple paternal contribution.

Hydatidiform moles (HMs) are divided into two types: partial hydatidiform mole (PHM) which is most often diandric monogynic triploid and complete hydatidiform mole (CHM) which is most often diploid androgenetic. Morphological features and p57 immunostaining are routinely used to distinguish both entities. Genetic analyses are required in challenging cases to determine the parental origin of the genome and ploidy. Some gestations cannot be accurately classified however. We report a case with atypical pathologic and genetic findings that correspond neither to CHM nor to PHM. Two populations of villi with divergent and discordant p57 expression were observed: morphologically normal p57 + villi and molar-like p57 discordant villi with p57 + stromal cells and p57 - cytotrophoblasts. Genotyping of DNA extracted from microdissected villi demonstrated that the conceptus was an androgenetic/biparental mosaic, originating from a zygote with triple paternal contribution, and that only the p57 - cytotrophoblasts were purely androgenetic, increasing the risk of neoplastic transformation.

Diagnostic Utility of TSSC3 and RB1 Immunohistochemistry in Hydatidiform Mole.

The general notion of complete hydatidiform moles is that most of them consist entirely of paternal DNA; hence, they do not express p57, a paternally imprinted gene. This forms the basis for the diagnosis of hydatidiform moles. There are about 38 paternally imprinted genes. The aim of this study is to determine whether other paternally imprinted genes could also assist in the diagnostic approach of hydatidiform moles. This study comprised of 29 complete moles, 15 partial moles and 17 non-molar abortuses. Immunohistochemical study using the antibodies of paternal-imprinted (RB1, TSSC3 and DOG1) and maternal-imprinted (DNMT1 and GATA3) genes were performed. The antibodies' immunoreactivity was evaluated on various placental cell types, namely cytotrophoblasts, syncytiotrophoblasts, villous stromal cells, extravillous intermediate trophoblasts and decidual cells. TSSC3 and RB1 expression were observed in all cases of partial moles and non-molar abortuses. In contrast, their expression in complete moles was identified in 31% (TSSC3) and 10.3% (RB1), respectively (p < 0.0001). DOG1 was consistently negative in all cell types in all cases. The expressions of maternally imprinted genes were seen in all cases, except for one case of complete mole where GATA3 was negative. Both TSSC3 and RB1 could serve as a useful adjunct to p57 for the discrimination of complete moles from partial moles and non-molar abortuses, especially in laboratories that lack comprehensive molecular service and in cases where p57 staining is equivocal.

PIK3R3 is upregulated in liver cancer and activates Akt signaling to control cancer growth by regulation of CDKN1C and SMC1A.

BACKGROUND: Liver cancer is a highly malignant disease and the third leading cause of cancer death worldwide. Abnormal activation of PI3K/Akt signaling is common in cancer, but whether phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) plays a role in liver cancer is largely unexplored. METHODS: We determined the expression of PIK3R3 in liver cancer by using TCGA data and our clinical samples and knocked it down by siRNA or overexpressing it by the lentivirus vector system. We also investigated the function of PIK3R3 by colony formation, 5-Ethynyl-2-Deoxyuridine, flow cytometry assay, and subcutaneous xenograft model. The downstream of PIK3R3 was explored by RNA sequence and rescue assays. RESULTS: We found that PIK3R3 was significantly upregulated in liver cancer and correlated with prognosis. PIK3R3 promoted liver cancer growth in vitro and in vivo by controlling cell proliferation and cell cycle. RNA sequence revealed that hundreds of genes were dysregulated upon PIK3R3 knockdown in liver cancer cells. CDKN1C, a cyclin-dependent kinase inhibitor, was significantly upregulated by PIK3R3 knockdown, and CDKN1C siRNA rescued the impaired tumor cell growth. SMC1A was partially responsible for PIK3R3 regulated function, and SMC1A overexpression rescued the impaired tumor cell growth in liver cancer cells. Immunoprecipitation demonstrated there is indirect interaction between PIK3R3 and CNKN1C or SMC1A. Importantly, we verified that PIK3R3-activated Akt signaling determined the expression of CDKN1C and SMC1A, two downstream of PIK3R3 in liver cancer cells. CONCLUSION: PIK3R3 is upregulated in liver cancer and activates Akt signaling to control cancer growth by regulation of CDNK1C and SMC1A. Targeting PIK3R3 could be a promising treatment strategy for liver cancer that deserves further investigation.

Drug-induced loss of imprinting revealed using bioluminescent reporters of Cdkn1c.

Genomic imprinting is an epigenetically mediated mechanism that regulates allelic expression of genes based upon parent-of-origin and provides a paradigm for studying epigenetic silencing and release. Here, bioluminescent reporters for the maternally-expressed imprinted gene Cdkn1c are used to examine the capacity of chromatin-modifying drugs to reverse paternal Cdkn1c silencing. Exposure of reporter mouse embryonic stem cells (mESCs) to 5-Azacytidine, HDAC inhibitors, BET inhibitors or GSK-J4 (KDM6A/B inhibitor) relieved repression of paternal Cdkn1c, either selectively or by inducing biallelic effects. Treatment of reporter fibroblasts with HDAC inhibitors or GSK-J4 resulted in similar paternal Cdkn1c activation, whereas BET inhibitor-induced loss of imprinting was specific to mESCs. Changes in allelic expression were generally not sustained in dividing cultures upon drug removal, indicating that the underlying epigenetic memory of silencing was maintained. In contrast, Cdkn1c de-repression by GSK-J4 was retained in both mESCs and fibroblasts following inhibitor removal, although this impact may be linked to cellular stress and DNA damage. Taken together, these data introduce bioluminescent reporter cells as tools for studying epigenetic silencing and disruption, and demonstrate that Cdkn1c imprinting requires distinct and cell-type specific chromatin features and modifying enzymes to enact and propagate a memory of silencing.

p57Kip2 is an essential regulator of vitamin D receptor-dependent mechanisms.

A cyclin-dependent kinase (CDK) inhibitor, p57Kip2, is an important molecule involved in bone development; p57Kip2-deficient (p57-/-) mice display neonatal lethality resulting from abnormal bone formation and cleft palate. The modulator 1α,25-dihydroxyvitamin D3 (l,25-(OH)2VD3) has shown the potential to suppress the proliferation and induce the differentiation of normal and tumor cells. The current study assessed the role of p57Kip2 in the 1,25-(OH)2VD3-regulated differentiation of osteoblasts because p57Kip2 is associated with the vitamin D receptor (VDR). Additionally, 1,25-(OH)2VD3 treatment increased p57KIP2 expression and induced the colocalization of p57KIP2 with VDR in the osteoblast nucleus. Primary p57-/- osteoblasts exhibited higher proliferation rates with Cdk activation than p57+/+ cells. A lower level of nodule mineralization was observed in p57-/- osteoblasts than in p57+/+ cells. In p57+/+ osteoblasts, 1,25-(OH)2VD3 upregulated the p57Kip2 and opn mRNA expression levels, while the opn expression levels were significantly decreased in p57-/- cells. The osteoclastogenesis assay performed using bone marrow cocultured with 1,25-(OH)2VD3-treated osteoblasts revealed a decreased efficiency of 1,25-(OH)2VD3-stimulated osteoclastogenesis in p57-/- cells. Based on these results, p57Kip2 might function as a mediator of 1,25-(OH)2VD3 signaling, thereby enabling sufficient VDR activation for osteoblast maturation.

IGF2 interacts with the imprinted gene Cdkn1c to promote terminal differentiation of neural stem cells.

Adult neurogenesis is supported by multipotent neural stem cells (NSCs) with unique properties and growth requirements. Adult NSCs constitute a reversibly quiescent cell population that can be activated by extracellular signals from the microenvironment in which they reside in vivo. Although genomic imprinting plays a role in adult neurogenesis through dose regulation of some relevant signals, the roles of many imprinted genes in the process remain elusive. Insulin-like growth factor 2 (IGF2) is encoded by an imprinted gene that contributes to NSC maintenance in the adult subventricular zone through a biallelic expression in only the vascular compartment. We show here that IGF2 additionally promotes terminal differentiation of NSCs into astrocytes, neurons and oligodendrocytes by inducing the expression of the maternally expressed gene cyclin-dependent kinase inhibitor 1c (Cdkn1c), encoding the cell cycle inhibitor p57. Using intraventricular infusion of recombinant IGF2 in a conditional mutant strain with Cdkn1c-deficient NSCs, we confirm that p57 partially mediates the differentiation effects of IGF2 in NSCs and that this occurs independently of its role in cell-cycle progression, balancing the relationship between astrogliogenesis, neurogenesis and oligodendrogenesis.

Dual inhibition of EZH1/2 induces cell cycle arrest of B cell acute lymphoblastic leukemia cells through upregulation of CDKN1C and TP53INP1.

Disease-risk stratification and development of intensified chemotherapy protocols have substantially improved the outcome of acute lymphoblastic leukemia (ALL). However, outcomes of relapsed or refractory cases remain poor. Previous studies have discussed the oncogenic role of enhancer of zeste homolog 1 and 2 (EZH1/2), and the efficacy of dual inhibition of EZH1/2 as a treatment for hematological malignancy. Here, we investigated whether an EZH1/2 dual inhibitor, DS-3201 (valemetostat), has antitumor effects on B cell ALL (B-ALL). DS-3201 inhibited growth of B-ALL cell lines more significantly and strongly than the EZH2-specific inhibitor EPZ-6438, and induced cell cycle arrest and apoptosis in vitro. RNA-seq analysis to determine the effect of DS-3201 on cell cycle arrest-related genes expressed by B-ALL cell lines showed that DS-3201 upregulated CDKN1C and TP53INP1. CRIPSR/Cas9 knockout confirmed that CDKN1C and TP53INP1 are direct targets of EZH1/2 and are responsible for the antitumor effects of DS-3201 against B-ALL. Furthermore, a patient-derived xenograft (PDX) mouse model showed that DS-3201 inhibited the growth of B-ALL harboring MLL-AF4 significantly. Thus, DS-3201 provides another option for treatment of B-ALL.

Epigenetic control of the imprinted growth regulator Cdkn1c in cadmium-induced placental dysfunction.

Cadmium (Cd) is a toxic metal ubiquitous in the environment. In utero, Cd is inefficiently transported to the foetus but causes foetal growth restriction (FGR), likely through impairment of the placenta where Cd accumulates. However, the underlying molecular mechanisms are poorly understood. Cd can modulate the expression of imprinted genes, defined by their transcription from one parental allele, which play critical roles in placental and foetal growth. The expression of imprinted genes is governed by DNA methylation at Imprinting Control Regions (ICRs), which are susceptible to environmental perturbation. The imprinted gene Cdkn1c/CDKN1C is a major regulator of placental development, is implicated in FGR, and shows increased expression in response to Cd exposure in mice. Here, we use a hybrid mouse model of in utero Cd exposure to determine if the increase in placental Cdkn1c expression is caused by changes to ICR DNA methylation and loss of imprinting (LOI). Consistent with prior studies, Cd causes FGR and impacts placental structure and Cdkn1c expression at late gestation. Using polymorphisms to distinguish parental alleles, we demonstrate that increased Cdkn1c expression is not driven by changes to DNA methylation or LOI. We show that Cdkn1c is expressed primarily in the placental labyrinth which is proportionally increased in size in response to Cd. We conclude that the Cd-associated increase in Cdkn1c expression can be fully explained by alterations to placental structure. These results have implications for understanding mechanisms of Cd-induced placental dysfunction and, more broadly, for the study of FGR associated with increased Cdkn1c/CDKN1C expression.

Auxiliary and experimental diagnostic techniques for hydatidiform moles.

Hydatidiform moles are classified into complete hydatidiform moles (CHMs), which are androgenetic and diploid, and partial hydatidiform moles (PHM), which are triploid with two paternal chromosomes and one maternal chromosome. The incidence of gestational trophoblastic neoplasia differs substantially between CHM and PHM. However, they are occasionally difficult to diagnose. In this review, auxiliary and experimental methods based on cytogenetic features and advanced molecular detection techniques applied to the diagnosis and analysis of hydatidiform moles are summarized, including basic principles, characteristics, and clinical implications. Short tandem repeat polymorphism analysis is considered the gold standard for the genetic diagnosis of hydatidiform moles. In clinical settings, immunohistochemical analyses of p57KIP2 , an imprinted gene product, are widely used to differentiate CHMs from other conceptuses, including PHMs. Recently, new molecular genetic techniques, such as single nucleotide polymorphism arrays, have been applied to research on hydatidiform moles. In addition to insights from classical methods, such as chromosome analysis, recently developed approaches have yielded novel findings related to the mechanism underlying the development of androgenetic CHMs.

Fbxo22 promotes cervical cancer progression via targeting p57Kip2 for ubiquitination and degradation.

F-box only protein 22 (FBXO22) is a key subunit of the Skp1-Cullin 1-F-box protein (SCF) E3 ubiquitin ligase complex. Little is known regarding its biological function and underlying molecular mechanisms in regulating cervical cancer (CC) progression. In this study, we aim to explore the role and mechanism of FBXO22 in CC progression. The correlation between FBXO22 and clinicopathological characteristics of CC was analyzed by tissue microarray. MTT, colony formation, flow cytometry, Western blotting, qRT-PCR, protein half-life, co-immunoprecipitation, ubiquitination, and xenograft experiments were performed to assess the functions of FBXO22 and potential molecular mechanisms of FBXO22-mediated malignant progression in CC. The expression of FBXO22 protein in CC tissues was higher than that in adjacent non-tumor cervical tissues. Notably, high expression of FBXO22 was significantly associated with high histology grades, positive lymph node metastasis, and poor outcomes in CC patients. Functionally, ectopic expression of FBXO22 promoted cell viability in vitro and induced tumor growth in vivo, while knockdown of FBXO22 exhibited opposite effects. In addition, overexpression of FBXO22 promoted G1/S phase progression and inhibited apoptosis in CC cells. Mechanistically, FBXO22 physically interacted with the cyclin-dependent kinase inhibitor p57Kip2 and subsequently mediated its ubiquitination and proteasomal degradation leading to tumor progression. FBXO22 protein level was found negatively associated with p57Kip2 protein levels in patient CC samples. FBXO22 promotes CC progression partly through regulating the ubiquitination and proteasomal degradation of p57Kip2. Our study indicates that FBXO22 might be a novel prognostic biomarker and therapeutic target for CC.

Identification and targeting of a HES1-YAP1-CDKN1C functional interaction in fusion-negative rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), a cancer characterized by features of skeletal muscle, is the most common soft-tissue sarcoma of childhood. With 5-year survival rates among high-risk groups at &lt; 30%, new therapeutics are desperately needed. Previously, using a myoblast-based model of fusion-negative RMS (FN-RMS), we found that expression of the Hippo pathway effector transcriptional coactivator YAP1 (YAP1) permitted senescence bypass and subsequent transformation to malignant cells, mimicking FN-RMS. We also found that YAP1 engages in a positive feedback loop with Notch signaling to promote FN-RMS tumorigenesis. However, we could not identify an immediate downstream impact of this Hippo-Notch relationship. Here, we identify a HES1-YAP1-CDKN1C functional interaction, and show that knockdown of the Notch effector HES1 (Hes family BHLH transcription factor 1) impairs growth of multiple FN-RMS cell lines, with knockdown resulting in decreased YAP1 and increased CDKN1C expression. In silico mining of published proteomic and transcriptomic profiles of human RMS patient-derived xenografts revealed the same pattern of HES1-YAP1-CDKN1C expression. Treatment of FN-RMS cells in vitro with the recently described HES1 small-molecule inhibitor, JI130, limited FN-RMS cell growth. Inhibition of HES1 in vivo via conditional expression of a HES1-directed shRNA or JI130 dosing impaired FN-RMS tumor xenograft growth. Lastly, targeted transcriptomic profiling of FN-RMS xenografts in the context of HES1 suppression identified associations between HES1 and RAS-MAPK signaling. In summary, these in vitro and in vivo preclinical studies support the further investigation of HES1 as a therapeutic target in FN-RMS.

Challenges in diagnosing hydatidiform moles: a review of promising molecular biomarkers.

INTRODUCTION: Hydatidiform moles (HMs) are pathologic conceptions with unique genetic bases and abnormal placental villous tissue. Overlapping ultrasonographical and histological manifestations of molar and non-molar (NM) gestations and HMs subtypes makes accurate diagnosis challenging. Currently, immunohistochemical analysis of p57 and molecular genotyping have greatly improved the diagnostic accuracy. AREAS COVERED: The differential expression of molecular biomarkers may be valuable for distinguishing among the subtypes of HMs and their mimics. Thus, biomarkers may be the key to refining HMs diagnosis. In this review, we summarize the current challenges in diagnosing HMs, and provide a critical overview of the recent literature about potential diagnostic biomarkers and their subclassifications. An online search on PubMed, Web of Science, and Google Scholar databases was conducted from the inception to 1 April 2022. EXPERT OPINION: The emerging biomarkers offer new possibilities to refine the diagnosis for HMs and pregnancy loss. Although the additional studies are required to be quantified and investigated in clinical trials to verify their diagnostic utility. It is important to explore, validate, and facilitate the wide adoption of newly developed biomarkers in the coming years.

CDKN1C hyperexpression in two patients with severe growth failure and microdeletions affecting the paternally inherited KCNQ1OT1:TSS-DMR.

BACKGROUND: Two imprinting control centres, H19/IGF2:IG-differentialy methylated region (DMR) and KCNQ1OT1:TSS-DMR, reside on chromosome 11p15.5. Paternal deletions involving the KCNQ1OT1:TSS-DMR result in variable phenotypes, namely, normal phenotype, Silver-Russel syndrome (SRS) and fetal demise. However, expression analyses for CDKN1C in these patients are very limited. CASES: Patient 1 (adult woman) and patient 2 (boy in early childhood) showed prenatal and postnatal growth failure and clinical suspicion of SRS. MOLECULAR ANALYSES: Both patients showed hypermethylation of the KCNQ1OT1:TSS-DMR caused by the paternal heterozygous de novo deletions involving the KCNQ1OT1:TSS-DMR, but not including CDKN1C enhancers. The deletion sizes were 5 kb and 12 kb for patients 1 and 2, respectively. CDKN1C gene expressions in immortalised leucocytes of both patients were increased compared with those of controls. CONCLUSION: Paternal deletions involving the KCNQ1OT1:TSS-DMR, but not including CDKN1C enhancers, disrupt KCNQ1OT1 expression, strongly activate CDKN1C expression and consequently cause severe growth failure.

Twin/Multiple Gestations With a Hydatidiform Mole: Clinicopathologic Analysis of 21 Cases With Emphasis on Molecular Genotyping and Parental Contribution.

Complete hydatidiform moles (CHMs) and partial hydatidiform moles (PHMs) are abnormal gestations characterized by vesicular chorionic villi accompanied by variable trophoblastic hyperplasia, with or without embryonic development. CHMs are purely androgenetic (only paternal [P] chromosome complements), mostly homozygous/monospermic (~85%) but occasionally heterozygous/dispermic, whereas PHMs are overwhelmingly diandric triploid (2 paternal [P] and 1 maternal [M] chromosome complements) and heterozygous/dispermic (>95%). The presence of a fetus in a molar pregnancy usually indicates a PHM rather than a CHM; however, CHMs and PHMs rarely can be associated with a viable fetus or a nonmolar abortus in twin pregnancies and rare multiple gestation molar pregnancies have been reported. A "one-oocyte-model," with diploidization of dispermic triploid zygotes, has been proposed for twin CHM with coexisting fetus, and a "two-oocyte-model" has been proposed for twin PHM with coexisting fetus. Among 2447 products of conception specimens, we identified 21 cases of twin/multiple gestations with a molar component, including 20 CHMs (17 twins, 2 triplets, 1 quintuplet) and 1 PHM (twin). P57 immunohistochemistry was performed on all; DNA genotyping of molar and nonmolar components was performed on 9 twin CHMs, 1 triplet CHM, 1 quintuplet CHM, and 1 twin PHM. All CHM components were p57-negative and those genotyped were purely androgenetic. Twin CHMs had genotypes of P1M1+P2P2 in 5, P1M1+P1P1 in 1, and P1M1+P2P3 in 1, consistent with involvement of 1 oocyte and from 1 to 3 sperm-most commonly a homozygous CHM but involving 2 sperm in the whole conception-and compatible with a "one-oocyte-model." The triplet CHM was P1M1+P1P1+P2M2 and the quintuplet CHM was P1M1+P2M2+P2M2+P3M3+P4P4, consistent with involvement of 2 sperm and at least 2 oocytes for the triplet and 4 sperm and at least 3 oocytes for the quintuplet. The twin PHM had a P1M1+P2P3M2 genotype, consistent with involvement of 2 oocytes and 3 sperm. p57 immunohistochemistry is highly reliable for diagnosis of CHMs in twin/multiple gestations. Refined diagnosis of molar twin/multiple gestations is best accomplished by correlating morphology, p57 immunohistochemistry, and molecular genotyping, with the latter clarifying zygosity/parental chromosome complement contributions to these conceptions.

The role of specialized cell cycles during erythroid lineage development: insights from single-cell RNA sequencing.

Early erythroid progenitors known as CFU-e undergo multiple self-renewal cell cycles. The CFU-e developmental stage ends with the onset of erythroid terminal differentiation (ETD). The transition from CFU-e to ETD is a critical cell fate decision that determines erythropoietic rate. Here we review recent insights into the regulation of this transition, garnered from flow cytometric and single-cell RNA sequencing studies. We find that the CFU-e/ETD transition is a rapid S phase-dependent transcriptional switch. It takes place during an S phase that is much shorter than in preceding or subsequent cycles, as a result of globally faster replication forks. Furthermore, it is preceded by cycles in which G1 becomes gradually shorter. These dramatic cell cycle and S phase remodeling events are directly linked to regulation of the CFU-e/ETD switch. Moreover, regulators of erythropoietic rate exert their effects by modulating cell cycle duration and S phase speed. Glucocorticoids increase erythropoietic rate by inducing the CDK inhibitor p57KIP2, which slows replication forks, inhibiting the CFU-e/ETD switch. Conversely, erythropoietin promotes induction of ETD by shortening the cycle. S phase shortening was reported during cell fate decisions in non-erythroid lineages, suggesting a fundamentally new developmental role for cell cycle speed.

Multimodal Imaging under Artificial Intelligence Algorithm for the Diagnosis of Liver Cancer and Its Relationship with Expressions of EZH2 and p57.

Objective: It aimed to explore the diagnostic efficacy of multimodal ultrasound images based on mask region with convolutional neural network (M-RCNN) segmentation algorithm for small liver cancer and analyze the expression of zeste gene enhancer homolog 2 (EZH2) and p57 (P57 Kip2) genes in cancer cells. Methods: A total of 100 patients suspected of small liver cancer were randomly divided into Doppler group (color Doppler ultrasound examination), contrast group (contrast ultrasound examination), elastic group (ultrasound elastography examination), and multimodal group (combined examination of the three methods), with 25 patients in each group. Images were processed by the M-RCNN segmentation algorithm. The results of the pathological biopsy were used to evaluate the diagnostic efficacy of the four methods. The liver tissues were then extracted and divided into observation group 1 (lesion tissue specimen), observation group 2 (liver tissue around cancer lesion), and control group (normal liver tissue), and the expression activities of EZH2 and p57 genes in the three groups were analyzed. Results: The accuracy of M-RCNN (97.23%) and average precision (AP) (71.90%) were higher than other methods (P < 0.05). Sensitivity (88.87%), specific degree of consistency (90.91%), accuracy (89.47%), and consistence (0.68) of the multimodal group were better than the other three groups (P < 0.05). Low and medium differentiated cancer tissues had an irregular shape, unclear boundary, uneven internal echo, unchanged/enhanced posterior echo, blood flow level 1∼2, elastic score 4∼5, and enhancement mode fast in and fast out. The positive expression rate of EZH2 in observation group 1 (75.95%) was higher than that in the other two groups, the positive expression rate of p57 in observation group 1 (80.79%) was lower than that in the other two groups, and the positive expression rate of p57 in the highly differentiated cancer foci (80.79%) was significantly lower than that in the middle and low differentiated cancer foci (P < 0.05). Conclusions: M-RCNN segmentation algorithm had a better segmentation effect. Multimodal ultrasound had a good effect on the benign and malignant diagnosis of small liver cancer and had a high clinical application value. The high expression of EZH2 and the decreased expression of p57 can promote the occurrence of small hepatocellular carcinoma, and the deficiency of the P57 gene was related to the low differentiation of cancer cells.