The Prognostic Value of Alpha-Fetoprotein Ratio in Patients With Resectable Alpha-Fetoprotein-Negative Hepatocellular Carcinoma.

PURPOSE: This study aimed to investigate the prognostic value of alpha-fetoprotein (AFP) ratio in patients with AFP-negative hepatocellular carcinoma (HCC). PATIENTS AND METHODS: We retrospectively analyzed 600 AFP-negative HCC patients who underwent hepatectomy. The AFP ratio was calculated as the ratio of AFP level 1 week before surgery to the level 20-40 days after hepatectomy. Immunohistochemistry assay was used to assess protein expression in HCC tissue. The primary outcome measures were overall survival (OS) and disease-free survival (DFS). RESULTS: The study found that a cutoff value of 1.6 ng/ml for AFP ratio, determined using X-tile software, was optimal for predicting prognosis. Patients with a high AFP ratio had a worse prognosis compare to those with a low AFP ratio (DFS, P = .026; OS, P = .034). Patients with a high AFP ratio had a worse prognosis compared to those with a low AFP ratio. Multivariate analysis revealed that AFP ratio >1.6, negative HepPar-1 expression, and vascular invasion were independent predictors of both DFS and OS. Vascular invasion had a higher area under the curve (AUC) than AFP ratio and HepPar-1 expression in predicting recurrence and death. The combination of AFP ratio, HepPar-1 expression, and vascular invasion provided better predictive accuracy for DFS and OS. CONCLUSION: The AFP ratio is a potential prognostic marker for AFP-negative HCC patients after hepatectomy. Combining the analysis of AFP ratio with HepPar-1 expression and vascular invasion can enhance the accuracy of predicting prognosis in these patients.

REGγ promotes mantle cell lymphoma cell apoptosis by downregulating NF-κB signaling.

Background: Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma (NHL). REGγ is important for tumor occurrence and development, but understanding of the specific role of REGγ in MCL is lacking. We aimed to identify REGγ effects on the proliferation and apoptosis of MCL cells and clarify the underlying mechanisms. Methods: JEKO-1 cells stably transfected with a doxycycline-inducible Tet-On system expressed high levels of REGγ. JEKO-1 cells stably expressing shRNA-REGγ to reduce REGγ levels were constructed. Cell proliferation, apoptosis, and p-NF-κB, NF-κB, IkB, REGγ, p-STAT3, STAT3, and PSMB5 levels in transfected cells and in transfected cells treated with Stattic, that is a nonpeptidic small molecule exhibited to selectively inhibit signal transducer and activator of transcription factor 3 through blocking the function of its SH2 domain, were analyzed using western blotting. Results: The proliferation of JEKO-1 cells was inhibited, and apoptosis was enhanced by increased expression of REGγ (P<0.01). REGγ inhibited MCL cell proliferation in a mouse tumor xenograft model by promoting apoptosis, increased the expression of the three IκB subunits and inhibited NF-κB signaling. Overexpressed REGγ inhibited STAT3 and downregulated PSMB5 expression in MCL cells. Stattic downregulated PSMB5 and nuclear factor-kappa B (NF-κB) expressions and upregulated IκBε expression in JEKO-1 cells. Conclusions: We found that REGγ regulated p-STAT3 expression by accelerating its half-life and downregulated the NF-κB signaling pathway to promote MCL cell apoptosis by negatively regulating STAT3-mediated PSMB5 expression and subsequently upregulating IκB expression.

Prenatal environmental antibiotics and fetal and postnatal growth: A biomonitoring-based prospective study in Eastern China.

Thus far, the effect of environmental antibiotics exposure to offspring's growth remains unclear. Here we aimed to evaluate whether and to what extent environmental antibiotics exposure is associated with fetal and postnatal growth. A total of 735 pregnant women and their full-term offspring from the Shanghai Obesity Birth Cohort were involved in the study. Maternal urine specimen was collected during the third trimester, and urinary concentration of fifteen environmental antibiotics was measured by liquid chromatography-tandem mass spectrometry and enzymatic method. Children were followed at birth, 12, 24 and 60 months, and growth parameters of the weight and height of children were recorded. Linear regression model was applied, and it was found that maternal veterinary antibiotic (VA) concentration was negatively associated with birth weight and ponderal index [per natural-logarithm (ln)-unit: adjusted ß (95% confidence interval, CI) = - 42.1 (- 74.0, - 10.3) for birth weight, -0.11 (- 0.19, - 0.02) for birth weight z-score, and - 0.03 (- 0.05, - 0.002) for ponderal index]. Regarding specific VA, each ln-unit increment of florfenicol concentrations was likely to be associate with 39.7 g (95%CI: - 69.3, - 10.1) reduced birth weight, 0.10 (95%CI: - 0.18, - 0.02) reduced birth weight z-score, and 0.02 g/cm3 (95%CI: - 0.04, - 0.00) reduced ponderal index. Ciprofloxacin, a preferred-as-veterinary antibiotic, showed a similar dose-response relationship with neonatal anthropometric parameters to florfenicol. However, these adverse effects diminished as children grew up to 12-, 24- and 60-month-old. Larger prospective cohort studies and animal experiments are warranted to verify the hypothesis that environmental antibiotics exposure in early life, even at low doses, may cause fetal growth restriction.

A Dominant Heterozygous Mutation in COG4 Causes Saul-Wilson Syndrome, a Primordial Dwarfism, and Disrupts Zebrafish Development via Wnt Signaling.

Saul-Wilson syndrome (SWS) is a rare, skeletal dysplasia with progeroid appearance and primordial dwarfism. It is caused by a heterozygous, dominant variant (p.G516R) in COG4, a subunit of the conserved oligomeric Golgi (COG) complex involved in intracellular vesicular transport. Our previous work has shown the intracellular disturbances caused by this mutation; however, the pathological mechanism of SWS needs further investigation. We sought to understand the molecular mechanism of specific aspects of the SWS phenotype by analyzing SWS-derived fibroblasts and zebrafish embryos expressing this dominant variant. SWS fibroblasts accumulate glypicans, a group of heparan sulfate proteoglycans (HSPGs) critical for growth and bone development through multiple signaling pathways. Consistently, we find that glypicans are increased in zebrafish embryos expressing the COG4 p.G516R variant. These animals show phenotypes consistent with convergent extension (CE) defects during gastrulation, shortened body length, and malformed jaw cartilage chondrocyte intercalation at larval stages. Since non-canonical Wnt signaling was shown in zebrafish to be related to the regulation of these processes by glypican 4, we assessed wnt levels and found a selective increase of wnt4 transcripts in the presence of COG4 p.G516R . Moreover, overexpression of wnt4 mRNA phenocopies these developmental defects. LGK974, an inhibitor of Wnt signaling, corrects the shortened body length at low concentrations but amplifies it at slightly higher concentrations. WNT4 and the non-canonical Wnt signaling component phospho-JNK are also elevated in cultured SWS-derived fibroblasts. Similar results from SWS cell lines and zebrafish point to altered non-canonical Wnt signaling as one possible mechanism underlying SWS pathology.

Identification of Transcriptional Variation in Aortic Remodeling Using a Murine Transverse Aortic Constriction (TAC) Model.

Arterial remodeling is a major pathological consequence of hypertension, which is recognized as the most common chronic non-communicable disease. However, the detailed mechanism of how arterial remodeling is induced by hypertension has not yet been fully elucidated. Evaluating the transcriptional changes in arterial tissue in response to elevated blood pressure at an early stage may provide new insights and identify novel therapeutic candidates in preventing arterial remodeling. Here, we used the ascending aorta of the transverse aortic constriction (TAC) model to induce arterial remodeling in C57BL/6 male mice. Age-matched mice were subjected to sham surgery as controls. The TAC model was only considered successful if the mice conformed to the criteria (RC/LC blood flow velocity with 5-10-fold change) 1 week after the surgery. Two weeks after surgery, the ascending aorta developed severe remodeling in TAC mice as compared to the sham group. High throughput sequencing was then applied to identify differentially expressed (DE) transcripts. In silicon analysis were then performed to systematically network transcriptional changes. A total of 1,019 mRNAs were significantly changed between TAC and the sham group at the transcriptional level. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed that stress/stimulus/immune-related biological processes played a crucial role during arterial remodeling. Our data provide a comprehensive understanding of global gene expression changes in the TAC model, which suggests that targeting inflammation and vascular smooth cell transformation are potential therapeutic strategies to interfere with the aortic remodeling at an early stage in the development of hypertension.

Patient-specific genomics and cross-species functional analysis implicate LRP2 in hypoplastic left heart syndrome.

Congenital heart diseases (CHDs), including hypoplastic left heart syndrome (HLHS), are genetically complex and poorly understood. Here, a multidisciplinary platform was established to functionally evaluate novel CHD gene candidates, based on whole-genome and iPSC RNA sequencing of a HLHS family-trio. Filtering for rare variants and altered expression in proband iPSCs prioritized 10 candidates. siRNA/RNAi-mediated knockdown in healthy human iPSC-derived cardiomyocytes (hiPSC-CM) and in developing Drosophila and zebrafish hearts revealed that LDL receptor-related protein LRP2 is required for cardiomyocyte proliferation and differentiation. Consistent with hypoplastic heart defects, compared to patents the proband's iPSC-CMs exhibited reduced proliferation. Interestingly, rare, predicted-damaging LRP2 variants were enriched in a HLHS cohort; however, understanding their contribution to HLHS requires further investigation. Collectively, we have established a multi-species high-throughput platform to rapidly evaluate candidate genes and their interactions during heart development, which are crucial first steps toward deciphering oligogenic underpinnings of CHDs, including hypoplastic left hearts.

Wnt/ß-catenin signaling pathway regulates asthma airway remodeling by influencing the expression of c-Myc and cyclin D1 via the p38 MAPK-dependent pathway.

Airway remodeling is the main characteristic of asthma; however, the mechanisms underlying this pathophysiological change have not been fully elucidated. Previous studies have indicated that the Wnt/ß-catenin and mitogen-activated protein kinase (MAPK) signaling pathway are involved in the development of airway remodeling during asthma. Therefore, the present study established an airway remodeling rat model, after which ß-catenin, cyclin D1 and c-Myc protein expressions were analyzed via western blotting in the lung tissue and airway smooth muscle cells (ASMCs) of rats. The mRNA expression of the aforementioned proteins were evaluated via reverse transcription-quantitative PCR. ß-catenin, cyclin D1 and c-Myc are core transcription factors and target genes of the Wnt/ß-catenin and MAPK signaling pathways. Furthermore, ß-catenin, c-Myc and cyclin D1 protein expression were determined following blocking of the p38 MAPK signaling pathway in vitro. The results demonstrated that higher expressions of ß-catenin, cyclin D1 and c-Myc were detected in lung tissues and ASMCs in the asthma group compared with the control. Blocking the p38 MAPK signaling pathway with a specific inhibitor SB203580 also downregulated the expressions of ß-catenin, cyclin D1 and c-Myc in vitro. Taken together, these results indicated that the Wnt/ß-catenin signaling pathway may regulate the process of airway remodeling via the p38 MAPK-dependent pathway.

Canonical Wnt5b Signaling Directs Outlying Nkx2.5+ Mesoderm into Pacemaker Cardiomyocytes.

Pacemaker cardiomyocytes that create the sinoatrial node are essential for the initiation and maintenance of proper heart rhythm. However, illuminating developmental cues that direct their differentiation has remained particularly challenging due to the unclear cellular origins of these specialized cardiomyocytes. By discovering the origins of pacemaker cardiomyocytes, we reveal an evolutionarily conserved Wnt signaling mechanism that coordinates gene regulatory changes directing mesoderm cell fate decisions, which lead to the differentiation of pacemaker cardiomyocytes. We show that in zebrafish, pacemaker cardiomyocytes derive from a subset of Nkx2.5+ mesoderm that responds to canonical Wnt5b signaling to initiate the cardiac pacemaker program, including activation of pacemaker cell differentiation transcription factors Isl1 and Tbx18 and silencing of Nkx2.5. Moreover, applying these developmental findings to human pluripotent stem cells (hPSCs) notably results in the creation of hPSC-pacemaker cardiomyocytes, which successfully pace three-dimensional bioprinted hPSC-cardiomyocytes, thus providing potential strategies for biological cardiac pacemaker therapy.

Endoderm Jagged induces liver and pancreas duct lineage in zebrafish.

Liver duct paucity is characteristic of children born with Alagille Syndrome (ALGS), a disease associated with JAGGED1 mutations. Here, we report that zebrafish embryos with compound homozygous mutations in two Notch ligand genes, jagged1b (jag1b) and jagged2b (jag2b) exhibit a complete loss of canonical Notch activity and duct cells within the liver and exocrine pancreas, whereas hepatocyte and acinar pancreas development is not affected. Further, animal chimera studies demonstrate that wild-type endoderm cells within the liver and pancreas can rescue Notch activity and duct lineage specification in adjacent cells lacking jag1b and jag2b expression. We conclude that these two Notch ligands are directly and solely responsible for all duct lineage specification in these organs in zebrafish. Our study uncovers genes required for lineage specification of the intrahepatopancreatic duct cells, challenges the role of duct cells as progenitors, and suggests a genetic mechanism for ALGS ductal paucity.The hepatopancreatic duct cells connect liver hepatocytes and pancreatic acinar cells to the intestine, but the mechanism for their lineage specification is unclear. Here, the authors reveal that Notch ligands Jagged1b and Jagged2b induce duct cell lineage in the liver and pancreas of the zebrafish.

FGF signaling enforces cardiac chamber identity in the developing ventricle.

Atrial and ventricular cardiac chambers behave as distinct subunits with unique morphological, electrophysiological and contractile properties. Despite the importance of chamber-specific features, chamber fate assignments remain relatively plastic, even after differentiation is underway. In zebrafish, Nkx transcription factors are essential for the maintenance of ventricular characteristics, but the signaling pathways that operate upstream of Nkx factors in this context are not well understood. Here, we show that FGF signaling plays an essential part in enforcing ventricular identity. Loss of FGF signaling results in a gradual accumulation of atrial cells, a corresponding loss of ventricular cells, and the appearance of ectopic atrial gene expression within the ventricle. These phenotypes reflect important roles for FGF signaling in promoting ventricular traits, both in early-differentiating cells that form the initial ventricle and in late-differentiating cells that append to its arterial pole. Moreover, we find that FGF signaling functions upstream of Nkx genes to inhibit ectopic atrial gene expression. Together, our data suggest a model in which sustained FGF signaling acts to suppress cardiomyocyte plasticity and to preserve the integrity of the ventricular chamber.

Cadm4 restricts the production of cardiac outflow tract progenitor cells.

Heart assembly requires input from two populations of progenitor cells, the first and second heart fields (FHF and SHF), that differentiate at distinct times and create different cardiac components. The cardiac outflow tract (OFT) is built through recruitment of late-differentiating, SHF-derived cardiomyocytes to the arterial pole of the heart. The mechanisms responsible for selection of an appropriate number of OFT cells from the SHF remain unclear. Here, we find that cell adhesion molecule 4 (cadm4) is essential for restricting the size of the zebrafish OFT. Knockdown of cadm4 causes dramatic OFT expansion, and overexpression of cadm4 results in a greatly diminished OFT. Moreover, cadm4 activity limits the production of OFT progenitor cells and the duration of their accumulation at the arterial pole. Together, our data suggest a role for cell adhesion in restraining SHF deployment to the OFT, perturbation of which could cause congenital OFT defects.

The BMP pathway acts to directly regulate Tbx20 in the developing heart.

TBX20 has been shown to be essential for vertebrate heart development. Mutations within the TBX20 coding region are associated with human congenital heart disease, and the loss of Tbx20 in a wide variety of model systems leads to cardiac defects and eventually heart failure. Despite the crucial role of TBX20 in a range of cardiac cellular processes, the signal transduction pathways that act upstream of Tbx20 remain unknown. Here, we have identified and characterized a conserved 334 bp Tbx20 cardiac regulatory element that is directly activated by the BMP/SMAD1 signaling pathway. We demonstrate that this element is both necessary and sufficient to drive cardiac-specific expression of Tbx20 in Xenopus, and that blocking SMAD1 signaling in vivo specifically abolishes transcription of Tbx20, but not that of other cardiac factors, such as Tbx5 and MHC, in the developing heart. We further demonstrate that activation of Tbx20 by SMAD1 is mediated by a set of novel, non-canonical, high-affinity SMAD-binding sites located within this regulatory element and that phospho-SMAD1 directly binds a non-canonical SMAD1 site in vivo. Finally, we show that these non-canonical sites are necessary and sufficient for Tbx20 expression in Xenopus, and that reporter constructs containing these sites are expressed in a cardiac-specific manner in zebrafish and mouse. Collectively, our findings define Tbx20 as a direct transcriptional target of the BMP/SMAD1 signaling pathway during cardiac maturation.

Phospholipase D1 is required for angiogenesis of intersegmental blood vessels in zebrafish.

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid and choline. Studies in cultured cells and Drosophila melanogaster have implicated PLD in the regulation of many cellular functions, including intracellular vesicle trafficking, cell proliferation and differentiation. However, the function of PLD in vertebrate development has not been explored. Here we report cloning and characterization of a zebrafish PLD1 (pld1) homolog. Like mammalian PLDs, zebrafish Pld1 contains two conservative HKD motifs. Maternally contributed pld1 transcripts are uniformly distributed in early embryo. Localized expression of pld1 is observed in the notochord during early segmentation, in the somites during later segmentation and in the liver at the larval stages. Studies in intact and cell-free preparations demonstrate evolutionary conservation of regulation. Inhibition of Pld1 expression using antisense morpholino oligonucleotides (MO) interfering with the translation or splicing of pld1 impaired intersegmental vessel (ISV) development. Incubating embryos with 1-butanol, which diverts production of phosphatidic acid to a phosphatidylalcohol, caused similar ISV defects. To determine where Pld1 is required for ISV development we performed transplantation experiments. Analyses of the mosaic Pld1 deficient embryos showed partial suppression of ISV defects in the segments containing transplanted wild-type notochord cells but not in the ones containing wild-type somitic cells. These results provide the first evidence that function of Pld1 in the developing notochord is essential for vascular development in vertebrates.

Apelin and its receptor control heart field formation during zebrafish gastrulation.

The vertebrate heart arises during gastrulation as cardiac precursors converge from the lateral plate mesoderm territories toward the embryonic midline and extend rostrally to form bilateral heart fields. G protein-coupled receptors (GPCRs) mediate functions of the nervous and immune systems; however, their roles in gastrulation remain largely unexplored. Here, we show that the zebrafish homologs of the Agtrl1b receptor and its ligand, Apelin, implicated in physiology and angiogenesis, control heart field formation. Zebrafish gastrulae express agtrl1b in the lateral plate mesoderm, while apelin expression is confined to the midline. Reduced or excess Agtrl1b or Apelin function caused deficiency of cardiac precursors and, subsequently, the heart. In Apelin-deficient gastrulae, the cardiac precursors converged inefficiently to the heart fields and showed ectopic distribution, whereas cardiac precursors overexpressing Apelin exhibited abnormal morphology and rostral migration. Our results implicate GPCR signaling in movements of discrete cell populations that establish organ rudiments during vertebrate gastrulation.