A multi-tissue metabolome atlas of primate pregnancy.

Pregnancy induces dramatic metabolic changes in females; yet, the intricacies of this metabolic reprogramming remain poorly understood, especially in primates. Using cynomolgus monkeys, we constructed a comprehensive multi-tissue metabolome atlas, analyzing 273 samples from 23 maternal tissues during pregnancy. We discovered a decline in metabolic coupling between tissues as pregnancy progressed. Core metabolic pathways that were rewired during primate pregnancy included steroidogenesis, fatty acid metabolism, and arachidonic acid metabolism. Our atlas revealed 91 pregnancy-adaptive metabolites changing consistently across 23 tissues, whose roles we verified in human cell models and patient samples. Corticosterone and palmitoyl-carnitine regulated placental maturation and maternal tissue progenitors, respectively, with implications for maternal preeclampsia, diabetes, cardiac hypertrophy, and muscle and liver regeneration. Moreover, we found that corticosterone deficiency induced preeclampsia-like inflammation, indicating the atlas's potential clinical value. Overall, our multi-tissue metabolome atlas serves as a framework for elucidating the role of metabolic regulation in female health during pregnancy.

Effect of PIVKA-II and AFP secretion status on early recurrence of hepatocellular carcinoma after open and laparoscopic surgery.

BACKGROUND: Prothrombin induced by vitamin K absence-II (PIVKA-II) and Alpha-fetoprotein (AFP) have been widely used as diagnostic markers in hepatocellular carcinoma (HCC), but the prognostic values of the two serum markers and their clinical usefulness in patient selection for different surgical approaches remain largely unclear. METHODS: HCC patients received surgical treatment between 2015 and 2019 were included. Patients were divided into four statuses according to the serum PIVKA-II and AFP secretion status: PIVKA-II (-) AFP (-) (status 1); PIVKA-II (+) AFP (-) (status 2); PIVKA-II (-) AFP (+) (status 3); PIVKA-II (+) AFP (+) (status 4). Kaplan-Meier analyses were conducted to compare the survivals of the four groups and the HCC patients received different surgical interventions; time-dependent AUC curves were introduced to evaluate the prognostic value of the PIV-AFP status; Cox regression model was used to identify prognostic indexes for overall survival (OS) and recurrence-free survival (RFS). RESULTS: A total of 518 patients were included. Patients with PIVKA-II (+) and APF (+) presented significantly decreased OS and RFS comparing to the other statuses. The areas under ROC curves of PIV-AFP status in predicting OS and RFS were superior to the PIVKA-II or the AFP alone. The HCC patients in early stages with PIVKA-II (+) and APF (+) had worse RFS when received laparoscopic hepatectomy than those who received open hepatectomy, whereas there was no difference in other secretion statuses. The PIVKA-II (+) and AFP (+) secretion status was an independent risk factor for OS, RFS. CONCLUSIONS: The PIV-AFP secretion status is of favorable clinical utility in predicting the OS and RFS of the HCC patients; extra caution is needed when applicated the laparoscopic approach in the HCC patients with PIVKA-II (+) and AFP (+).

Mitigating the adverse effects of Aflatoxin B1 in LMH, IPEC-J2 and 3D4/21 cells by a novel integrated agent.

This study was to evaluate the efficacy of TOXO-XL (XL), an integrated mycotoxin-mitigating agent, on aflatoxin B1 (AFB1)-induced damage in Leghorn male hepatoma (LMH), porcine jejunum epithelial cell line (IPEC-J2) and porcine alveolar macrophages (3D4/21) cells, and to explore its potential mechanisms. The results showed that 30% inhibition concentration (IC30) of AFB1 in LMH, IPEC-J2 and 3D4/21 cells was 0.5, 15.0, and 2.5 mg/L, respectively. Notably, cell viability, ROS, apoptosis and DNA lesion induced by AFB1 (IC30) could be ameliorated by the supplementation with XL at the dosage of 0.025, 0.025 and 0.005%, respectively. Additionally, the migration and phagocytosis abilities impaired by AFB1 were also restored by XL in 3D4/21. Further experiments revealed that XL supplementation markedly attenuated AFB1-induced inflammatory response by decreasing IL-1ß, IL-6 and IL-10 in LMH, IL-6 in IPEC-J2 and IL-1ß in 3D4/21 cells. Meanwhile, XL supplementation reversed the alterations of BAX, BCL-2 and caspase-3 induced by AFB1 in the three cells, suggesting that AFB1-induced apoptosis may be suppressed via the mitochondria-dependent pathway. Furthermore, XL may have a protective effect on the intestinal barrier through the restoration of occludin protein. Conclusively, these findings indicated that XL could alleviate AFB1-induced cytotoxicity in the three cells, potentially through the regulation of cytokines, ROS, apoptotic and DNA damage signaling.

Neurulation of the cynomolgus monkey embryo achieved from 3D blastocyst culture.

Neural tube (NT) defects arise from abnormal neurulation and result in the most common birth defects worldwide. Yet, mechanisms of primate neurulation remain largely unknown due to prohibitions on human embryo research and limitations of available model systems. Here, we establish a three-dimensional (3D) prolonged in vitro culture (pIVC) system supporting cynomolgus monkey embryo development from 7 to 25 days post-fertilization. Through single-cell multi-omics analyses, we demonstrate that pIVC embryos form three germ layers, including primordial germ cells, and establish proper DNA methylation and chromatin accessibility through advanced gastrulation stages. In addition, pIVC embryo immunofluorescence confirms neural crest formation, NT closure, and neural progenitor regionalization. Finally, we demonstrate that the transcriptional profiles and morphogenetics of pIVC embryos resemble key features of similarly staged in vivo cynomolgus and human embryos. This work therefore describes a system to study non-human primate embryogenesis through advanced gastrulation and early neurulation.

Identifying a dynamic transcriptomic landscape of the cynomolgus macaque placenta during pregnancy at single-cell resolution.

Supporting healthy pregnancy outcomes requires a comprehensive understanding of the cellular hierarchy and underlying molecular mechanisms in the primate placenta during gestation. Here, we present a single-cell transcriptome-wide view of the cynomolgus macaque placenta throughout gestation. Bioinformatics analyses and multiple validation experiments suggested that placental trophoblast cells exhibited stage-specific differences across gestation. Interactions between trophoblast cells and decidual cells also showed gestational stage-dependent differences. The trajectories of the villous core cells indicated that placental mesenchymal cells were derived from extraembryonic mesoderm (ExE.Meso) 1, whereas placental Hofbauer cells, erythrocytes, and endothelial cells were derived from ExE.Meso2. Comparative analyses of human and macaque placentas uncovered conserved features of placentation across species, and the discrepancies of extravillous trophoblast cells (EVTs) between human and macaque correlated to their differences in invasion patterns and maternal-fetal interactions. Our study provides a groundwork for elucidating the cellular basis of primate placentation.

Parenchymal Sparing Laparoscopic Segmentectomy III and IV with Indocyanine Green Fluorescence Negative Stain Method Using Glisson Pedicle Approach.

BACKGROUND: To minimize the loss of functional liver volume in cases of severe cirrhosis and repeat hepatectomy for recurrence of hepatocellular carcinoma (HCC), anatomical hepatectomy is gradually extended from major to minor hepatectomy (Miyama et al. in Cancers (Basel):13, 2021; Ishizawa et al. in Ann Surg 256:959-964, 2012). For local located HCC, (sub)segmentectomy can yet be regarded as a choice instead of hemihepatectomy. Indocyanine green (ICG) has been used for tumor location, navigation of resected margin and liver segment, and identification of bile leakage. Negative stain that ICG dye was administered intravenously after occluding the target portal pedicle is more applicable to sectionectomy or hemihepatectomy, especially in cases where multiple target pedicles exist or portal vein puncture is difficult to carry out to achieve anatomic resection. Herein, we present a video of laparoscopic segmentectomy III and IV with ICG fluorescence negative stain using Glisson Pedicle approach. METHOD: A 49-year-old woman with hepatitis B related cirrhosis for 2 years was referred for treatment of a single nodule in segment IV invading the umbilical portion of left portal vein. The preoperative alpha-fetoprotein (AFP) was 442 ng/ml and protein induced by vitamin K absence or antagonist-II (PIVKA-II) was 122 mAu/ml. Liver function was Child-Pugh A and indocyanine green retention test at 15 min (ICG-R15) was 9.2%. The surgical procedure involved the following steps: (1) Extrahepatic Glisson pedicle dissection based on Laennec's s capsule (Sugioka et al. in J Hepatobiliary Pancreat Sci 24:17-23, 2017) was performed for isolation of the pedicles towards segments III and IV in the umbilical fossa. (2) Demarcation line was revealed and ICG (1 ml, 5 mg/l) was administered intravenously for the negative stain after dividing the target pedicles. (3) Parenchyma transection was performed along the border of the negative staining area in the cranial and caudal direction. RESULTS: Operative time was 220 min and blood loss was 150 ml with no transfusion. HCC sized 2.5 cm*1.7 cm*1.2 cm was confirmed in histopathology with a free margin and no microvascular invasion. The fibrosis of the liver parenchyma was S4 based on Ishak system. The patient was discharged on the postoperative day 6 without any complications. No recurrence in residual liver was noted on the CT scan at 9 months during follow-up. CONCLUSION: Laparoscopic segmentectomy III and IV is an effective procedure for HCC especially in cases with demands of hepatic parenchymal preservation. ICG navigation and Glisson Pedicle approach may be particularly helpful.

Systemic therapies in hepatocellular carcinoma: Existing and emerging biomarkers for treatment response.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third most common cause of cancer-related death worldwide. Due to asymptomatic patients in the early stage, most patients are diagnosed at an advanced stage and lose the opportunity for radical resection. In addition, for patients who underwent procedures with curative intent for early-stage HCC, up to 70% of patients may have disease recurrence within 5 years. With the advent of an increasing number of systemic therapy medications, we now have more options for the treatment of HCC. However, data from clinical studies show that with different combinations of regimens, the objective response rate is approximately 40%, and most patients will not respond to treatment. In this setting, biomarkers for predicting treatment response are of great significance for precise treatment, reducing drug side effects and saving medical resources. In this review, we summarized the existing and emerging biomarkers in the literature, with special emphasis on the pathways and mechanism underlying the prediction value of those biomarkers for systemic treatment response.

Primate gastrulation and early organogenesis at single-cell resolution.

Our understanding of human early development is severely hampered by limited access to embryonic tissues. Due to their close evolutionary relationship with humans, nonhuman primates are often used as surrogates to understand human development but currently suffer from a lack of in vivo datasets, especially from gastrulation to early organogenesis during which the major embryonic cell types are dynamically specified. To fill this gap, we collected six Carnegie stage 8-11 cynomolgus monkey (Macaca fascicularis) embryos and performed in-depth transcriptomic analyses of 56,636 single cells. Our analyses show transcriptomic features of major perigastrulation cell types, which help shed light on morphogenetic events including primitive streak development, somitogenesis, gut tube formation, neural tube patterning and neural crest differentiation in primates. In addition, comparative analyses with mouse embryos and human embryoids uncovered conserved and divergent features of perigastrulation development across species-for example, species-specific dependency on Hippo signalling during presomitic mesoderm differentiation-and provide an initial assessment of relevant stem cell models of human early organogenesis. This comprehensive single-cell transcriptome atlas not only fills the knowledge gap in the nonhuman primate research field but also serves as an invaluable resource for understanding human embryogenesis and developmental disorders.

Immune-Related Biomarkers Improve Performance of Risk Prediction Models for Survival in Patients With Hepatocellular Carcinoma.

Object: The prediction of hepatocellular carcinoma (HCC) prognosis faced great challenge due to tumor heterogeneity. The purpose of this study was to explore the correlation between the immune infiltrate and prognosis. Moreover, we aimed to establish a risk prediction model for survival in HCC patients based on clinicopathological and immune indicators. Methods: In this study, 316 patients with HCC who underwent radical resection in West China Hospital from 2009 to 2014 were included. Clinicopathological data and pathological specimens were collected. H&E staining and immunohistochemical staining were performed on the pathological tissue sections. The evaluation of tumor-infiltrating lymphocyte (TIL) density was based on H&E slices, and the assessment of the expressions of CD8, CD68, Lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin domain and mucin domain-3 (TIM-3), Programmed Cell Death Protein 1 (PD-1), Programmed Cell Death Ligand 1 (PD-L1), OX40, CD66b, and Tryptase. was performed on the immunohistochemical slices. A risk prediction model for survival in HCC patients was established by integrating immune-related biomarkers and clinicopathological indicators. Results: The Barcelona Clinic Liver Cancer (BCLC) stage; the microvascular invasion status; the density of TILs; the expressing levels of CD66b, OX40, and PD-L1 in the immune cell; CD68; and CD8 were the predictors of patients' overall survival (OS). The BCLC stage; the density of TILs; and the expressions of OX40, CD68, and CD8 were associated with disease-free survival (DFS). The expressions of CD66b, CD68, OX40, and CD8 had a cumulative effect on prognosis. The area under the curve of the prediction model for OS based on clinicopathological features was improved from 0.62 to 0.74 by adding to CD8, OX40, CD68, CD66b, and TILs, whereas it was improved from 0.59 to 0.73 for the DFS prediction model. Conclusion: Our results, if confirmed, indicated that immune-related biomarkers should be taken into account or stratified in survival analysis for HCC.

Physicochemical and Colon Cancer Cell Inhibitory Properties of Theabrownins Prepared by Weak Alkali Oxidation of Tea Polyphenols.

Existing studies on the biological activity of theabrownins are not based on their free state but on the complexes of theabrownins, polysaccharides, proteins, and flavonoids. In this study, theabrownins (TBs-C) were prepared by weak alkali oxidation of tea polyphenols. The ultraviolet-visible scanning spectrum of TBs-C showed two characteristic absorption peaks at 203 and 270 nm. The zeta potential of the TBs-C aqueous solution was negative, and the values varied from - 6.26 to -19.55 mV with a solution pH of 3-9. Storage conditions of pH 5.0-7.0 and around 25 °C were beneficial for the physical and chemical stability of the TBS-C solution. Cells were treated with series concentrations and examined by MTT, HE staining, PI immunofluorescence staining, flow cytometry, and real-time PCR to investigate the antiproliferative effect of TBs-C on human colon cancer HT-29 cells. The results showed that TBs-C, particularly at 500 µg/mL, inhibited cell growth. TBs-C induced HT-29 cell apoptosis, as confirmed by morphological changes, nucleus propidium iodide staining, and distributions of the cell cycle. The apoptotic mechanism may be due to the intracellular redox imbalance induced by TBs-C.

Profiling hepatocellular carcinoma aggressiveness with contrast-enhanced ultrasound and gadoxetate disodium-enhanced MRI: An intra-individual comparative study based on the Liver Imaging Reporting and Data System.

PURPOSE: Contrast-enhanced ultrasound (CEUS) and gadoxetate disodium-enhanced MRI (EOB-MRI) may synergize in profiling hepatocellular carcinoma (HCC) aggressiveness considering distinct imaging traits. This study aimed to intra-individually compare CEUS and EOB-MRI with Liver Imaging Reporting and Data System (LI-RADS) in assessing HCC aggressiveness. METHOD: From January 2015 to November 2020, consecutive at-risk patients with surgically-confirmed HCC who underwent both preoperative CEUS and EOB-MRI examinations were retrospectively enrolled. Image analyses were conducted independently by two masked radiologists for CEUS and EOB-MRI, respectively. The diagnostic performance of each modality for macrovascular invasion against pathology was evaluated and compared with the McNemar's test, while Edmondson-Steiner grade and the presence of microvascular invasion (MVI) were compared between patients with and without LR-M features on each modality. RESULTS: A total of 140 patients (mean age, 51.9 years ± 11.0; 116 men) were included. Inter-modality agreement was poor (κ = -0.087 âˆ¼ 0.139) for major LI-RADS features and moderate (κ = 0.449) for overall LI-RADS categorization, and LR-TIV and LR-M were the top sources of inter-modality variations. Although CEUS demonstrated significantly higher specificity for diagnosing macrovascular invasion (96% vs. 89%, P =.02), LR-M features on EOB-MRI were more effective in identifying higher Edmondson-Steiner grades (P =.01) and MVI (P =.02). CONCLUSIONS: Marked discrepancies were found between CEUS and EOB-MRI in evaluating LI-RADS features and categories. Whereas CEUS showed superior diagnostic specificity for macrovascular invasion, LR-M features on EOB-MRI provided more information regarding tumor grade and MVI status in HCC patients.

Influence of feeder cells on transcriptomic analysis of pluripotent stem cells.

OBJECTIVES: Human pluripotent stem cells (hPSCs) are of great importance in both scientific research and regenerative medicine. The most classic and widely used culture method for hPSCs is co-culture with feeder cells, usually mouse embryonic fibroblasts. However, whether these feeder cell residues can affect the transcriptomic data analysis of hPSCs, especially gene or miRNA expression quantification, is still largely unknown. METHODS AND RESULTS: In this study, reanalysis of published mRNA-Seq and miRNA-Seq data sets revealed the existence of feeder cell-derived reads in the hPSC transcriptomic samples. We identified potentially influenced human genes and miRNAs due to misalignment of sequencing fragments affected by mouse feeder cells. Furthermore, we developed an optimized miRNA analysis pipeline to avoid quantification bias from different miRNA isoforms in the same family. Finally, by comparing the levels of feeder cell residues in hPSC samples isolated by different methods, we found that fluorescence-activated cell sorting and adhesion methods were more effective in feeder cell removal than the gradient centrifugation method. CONCLUSIONS: Collectively, our results demonstrate that feeder cell residues affect the transcriptomic data analysis of hPSCs. To minimize the impact of feeder cell contamination in hPSC samples, we provide solutions for both data analysis and sample preparation.

The multidisciplinary management of hepatocellular carcinoma with portal vein tumor thrombus.

Portal vein tumor thrombus (PVTT) is one of the most common complications of hepatocellular carcinoma (HCC), which refers to the advanced stage of HCC and indicates an extremely poor prognosis. Monotherapy cannot effectively prolong the survival benefit of patients with HCC-PVTT characterized by a high recurrence rate. With great progress in the area of immune and molecular targeted therapy, there comes a promising era of multidisciplinary management of HCC. Survival benefits can be achieved based on accurate diagnosis, staging, and multidisciplinary management. Additionally, in terms of the presence of controversy about the standard treatment algorithm and the absence of universal treatment guidelines, a multidisciplinary management program may afford the best hope for HCC-PVTT patients via appropriate implement of various treatment protocols.

The density of tumor-infiltrating lymphocytes and prognosis in resectable hepatocellular carcinoma: a two-phase study.

AIM: Previous studies have focused on the subpopulations of tumor-infiltrating lymphocytes (TILs) in tumors. This study focuses only on the concentration of TILs in the tumor irrespective of type and elucidates its prognostic value. METHODS: We used 315 HCC patients as the discovery phase and another 343 HCC patients as the validation phase. By following the standardized guideline, density of TILs were categorized into low (TILs < 10%), intermediate (10% ≦ TILs < 50%), and high (TILs ≧ 50%) levels. Associations of TILs with prognostic, immune-related, and genetic variables were examined. RESULTS: We observed a dose-response relation of TILs with overall survival (intermediate: HR, 0.58; 95% confidence interval (CI), 0.36-0.93; high: HR, 0.37; 95% CI, 0.15-0.93) and disease-free survival (intermediate: HR, 0.35; 95% CI, 0.22-0.58; high: HR, 0.23; 95% CI, 0.09-0.58). The prognostic value of TILs was validated in the TCGA set. Mutation burden or the number of neoantigens were not associated with TILs intensity. However, hepatitis B or C virus infection patients had higher TILs intensity in the para-tumor tissue. CONCLUSIONS: The TILs intensity was associated with patients' survival. If confirmed, this would suggest that clinical routine assessment of TILs could provide prognostic information in HCC.

Long noncoding RNA lnc-NAP sponges mmu-miR-139-5p to modulate Nanog functions in mouse ESCs and embryos.

The pluripotency of embryonic stem cells (ESCs) is controlled by a multilayer regulatory network, of which the key factors include core pluripotency genes Oct4, Sox2 and Nanog, and multiple microRNAs (miRNAs). Recently, long noncoding RNAs (lncRNAs) have been discovered as a class of new regulators for ESCs, and some lncRNAs could function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we identify mmu-miR-139-5p as a new regulator for Nanog by targeting Nanog 3' untranslated region (UTR) to repress Nanog expression in mouse ESCs and embryos. Such regulation could be released by an ESC-specifically expressed ceRNA named lnc-NAP. The expression of lnc-NAP is activated by OCT4, SOX2, as well as NANOG through promoter binding. Downregulation of lnc-NAP reduces Nanog abundance, which leads to decreased pluripotency of mouse ESCs and embryonic lethality. These results reveal lnc-NAP as a new regulator for Nanog in mouse ESCs, and uncover a feed-forward regulatory loop of Nanog through the participation of lnc-NAP.

Overcoming Intrinsic H3K27me3 Imprinting Barriers Improves Post-implantation Development after Somatic Cell Nuclear Transfer.

Successful cloning by somatic cell nuclear transfer (SCNT) requires overcoming significant epigenetic barriers. Genomic imprinting is not generally regarded as such a barrier, although H3K27me3-dependent imprinting is differentially distributed in E6.5 epiblast and extraembryonic tissues. Here we report significant enhancement of SCNT efficiency by deriving somatic donor cells carrying simultaneous monoallelic deletion of four H3K27me3-imprinted genes from haploid mouse embryonic stem cells. Quadruple monoallelic deletion of Sfmbt2, Jade1, Gab1, and Smoc1 normalized H3K27me3-imprinted expression patterns and increased fibroblast cloning efficiency to 14% compared with a 0% birth rate from wild-type fibroblasts while preventing the placental and body overgrowth defects frequently observed in cloned animals. Sfmbt2 deletion was the most effective of the four individual gene deletions in improving SCNT. These results show that lack of H3K27me3 imprinting in somatic cells is an epigenetic barrier that impedes post-implantation development of SCNT embryos and can be overcome by monoallelic imprinting gene deletions in donor cells.

A single-cell transcriptomic landscape of primate arterial aging.

Our understanding of how aging affects the cellular and molecular components of the vasculature and contributes to cardiovascular diseases is still limited. Here we report a single-cell transcriptomic survey of aortas and coronary arteries in young and old cynomolgus monkeys. Our data define the molecular signatures of specialized arteries and identify eight markers discriminating aortic and coronary vasculatures. Gene network analyses characterize transcriptional landmarks that regulate vascular senility and position FOXO3A, a longevity-associated transcription factor, as a master regulator gene that is downregulated in six subtypes of monkey vascular cells during aging. Targeted inactivation of FOXO3A in human vascular endothelial cells recapitulates the major phenotypic defects observed in aged monkey arteries, verifying FOXO3A loss as a key driver for arterial endothelial aging. Our study provides a critical resource for understanding the principles underlying primate arterial aging and contributes important clues to future treatment of age-associated vascular disorders.

Laparoscopic Caudate Lobectomy for Cholangiocarcinoma of Caudate Lobe Invading Middle Hepatic Vein.

BACKGROUND: Laparoscopic hepatectomy has gained popularity in the management of malignant liver lesions in the past decade. Its safety and feasibility, with faster recovery and comparable long-term outcomes, have been widely published. Nonetheless, laparoscopic isolated caudate lobectomy is still rare and technically demanding. We herein present a video on laparoscopic total caudate lobectomy for caudate cholangiocarcinoma. METHODS: The patient is a 61-year-old man who presented with epigastric distending discomfort. A contrast-enhanced magnetic resonance imaging was performed, showing a 4.6 × 3.9 cm tumor in the caudate lobe adjacent to the inferior vena cava, middle hepatic vein, right hepatic vein, as well as the bifurcation of the main trunk of the portal pedicle. The carbohydrate antigen was elevated to 54.58 U/ml (normal < 37 U/ml), and his liver function was normal. With the preoperative diagnosis of intrahepatic cholangiocarcinoma, laparoscopic caudate lobectomy was contemplated. RESULTS: The operative time was 300 min. The estimated intraoperative blood loss was 180 ml. The patient was discharged on the seventh postoperative day without any complications. Histopathological examination showed a 4.2 cm cholangiocarcinoma (T2N0M0) with a negative margin. He received a course of adjuvant chemotherapy. No recurrence was noted upon follow-up at 6 months after the operation. CONCLUSIONS: Laparoscopic resection for caudate lobe is a feasible and safe procedure. An experienced hepatobiliary surgeon could perform the procedure in selected cases, even with hepatic vein invasion.