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.

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.

Generation and Application of Mouse-Rat Allodiploid Embryonic Stem Cells.

Mammalian interspecific hybrids provide unique advantages for mechanistic studies of speciation, gene expression regulation, and X chromosome inactivation (XCI) but are constrained by their limited natural resources. Previous artificially generated mammalian interspecific hybrid cells are usually tetraploids with unstable genomes and limited developmental abilities. Here, we report the generation of mouse-rat allodiploid embryonic stem cells (AdESCs) by fusing haploid ESCs of the two species. The AdESCs have a stable allodiploid genome and are capable of differentiating into all three germ layers and early-stage germ cells. Both the mouse and rat alleles have comparable contributions to the expression of most genes. We have proven AdESCs as a powerful tool to study the mechanisms regulating X chromosome inactivation and to identify X inactivation-escaping genes, as well as to efficiently identify genes regulating phenotypic differences between species. A similar method could be used to create hybrid AdESCs of other distantly related species.

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.

SIRT6 deficiency results in developmental retardation in cynomolgus monkeys.

SIRT6 acts as a longevity protein in rodents1,2. However, its biological function in primates remains largely unknown. Here we generate a SIRT6-null cynomolgus monkey (Macaca fascicularis) model using a CRISPR-Cas9-based approach. SIRT6-deficient monkeys die hours after birth and exhibit severe prenatal developmental retardation. SIRT6 loss delays neuronal differentiation by transcriptionally activating the long non-coding RNA H19 (a developmental repressor), and we were able to recapitulate this process in a human neural progenitor cell differentiation system. SIRT6 deficiency results in histone hyperacetylation at the imprinting control region of H19, CTCF recruitment and upregulation of H19. Our results suggest that SIRT6 is involved in regulating development in non-human primates, and may provide mechanistic insight into human perinatal lethality syndrome.

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.

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.

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.

Rat embryonic stem cells produce fertile offspring through tetraploid complementation.

Pluripotency of embryonic stem cells (ESCs) can be functionally assessed according to the developmental potency. Tetraploid complementation, through which an entire organism is produced from the pluripotent donor cells, is taken as the most stringent test for pluripotency. It remains unclear whether ESCs of other species besides mice can pass this test. Here we show that the rat ESCs derived under 2i (two small molecule inhibitors) conditions at very early passages are able to produce fertile offspring by tetraploid complementation. However, they lose this capacity rapidly during culture due to a nearly complete loss of genomic imprinting. Our findings support that the naïve ground state pluripotency can be captured in rat ESCs but also point to the species-specific differences in its regulation and maintenance, which have implications for the derivation and application of naïve pluripotent stem cells in other species including human.

Pre-Expanded Latissimus Dorsi Myocutaneous Flap for Total Scalp Defect Reconstruction.

Scalp defects can be caused by various factors, and reconstruction options for scalp defects include skin grafts, local flaps, tissue expanders, and free flaps. However, currently, it is widely accepted that the use of free flaps is the most feasible method for extensive scalp defect reconstruction. While multiple flaps have been used to reconstruct scalp defects, the reconstruction of total scalp defects still remains challenging. Pre-expansion of free flaps offers several advantages, including increasing flap size and thinning of the tissue for better contour, and is particularly important in scalp reconstruction. This report describes the successful management of total scalp defect reconstruction that involved the entire frontal, parietal, occipital, and temporal regions using a pre-expanded latissimus dorsi myocutaneous flap in a 40-year-old female patient. Over 2 years of follow-up, the transplanted flap survived well and the patient eventually achieved excellent cosmetic appearance, with satisfactory durable coverage. She was able to wear a hairpiece and hat without any wound breakdown. Our report indicates that microsurgery using pre-expanded latissimus dorsi myocutaneous flap transfer is a reliable and safe choice for total scalp reconstruction, allowing reconstruction with a single-flap, an excellent aesthetic effect, and abrasive resistance.

Effects of purified fibre-mixture supplementation of gestation diet on gut microbiota, immunity and reproductive performance of sows.

This experiment was conducted to investigate the effects of a purified fibre-mixture (FM, 50% guar gum, 50% cellulose) supplementation of gestation diet on the immunity, faecal microbial composition and reproductive performance of sows. A day after breeding, 68 multiparous sows were randomly allocated to receive treatment with a control (CON) diet or a diet containing 3% FM (FM diet). Results showed the FM diet to be associated with a significant increase in the number of live-born piglets relative to CON (13.65 vs. 12.47, p < .05). In addition, this FM diet coincided with significantly increased faecal concentrations of butyrate on day 30 and propionate on day 100 (p < .05), with trends towards increased propionate on day 30 and increased short-chain fatty acids (SCFAs) on days 30 and 110 (p < .1). Meanwhile, FM addition markedly increased the abundance of representative SCFAs producing-related genera as Roseburia on days 30 and 110 (p < .05), Eubacterium-hallii-group on days 30 and 110 (p < .05), and Bacteroides on day 110 of gestation (p < .05). The serotonin concentration on day 110 of gestation had increased (p < .05) and that on day 30 of gestation (p < .1) exhibited a tendency to increase with the FM-supplemented diet in comparison with the CON. Besides, FM supplementation caused an increase in serum interleukin-10 concentrations on days 30 (p < .05) and 110 of gestation (p < .1), and a decrease in interferon-γ concentration on day 30 of gestation (p < .05). Together these results indicated that purified FM was able to improve sow reproductive performance through a mechanism potentially linked with a bias towards type-2 helper T-cell differentiation that supported embryonic survival and thereby improve reproductive yields. Changes in metabolites produced by the intestinal microbiome may thus have an impact on host immunity and reproductive performance.

Ubiquitously expressed genes participate in cell-specific functions via alternative promoter usage.

How do different cell types acquire their specific identities and functions is a fundamental question of biology. Previously significant efforts have been devoted to search for cell-type-specifically expressed genes, especially transcription factors, yet how do ubiquitously expressed genes participate in the formation or maintenance of cell-type-specific features remains largely unknown. Here, we have identified 110 mouse embryonic stem cell (mESC) specifically expressed transcripts with cell-stage-specific alternative transcription start sites (SATS isoforms) from 104 ubiquitously expressed genes, majority of which have active epigenetic modification- or stem cell-related functions. These SATS isoforms are specifically expressed in mESCs, and tend to be transcriptionally regulated by key pluripotency factors through direct promoter binding. Knocking down the SATS isoforms of Nmnat2 or Usp7 leads to differentiation-related phenotype in mESCs. These results demonstrate that cell-type-specific transcription factors are capable to produce cell-type-specific transcripts with alternative transcription start sites from ubiquitously expressed genes, which confer ubiquitously expressed genes novel functions involved in the establishment or maintenance of cell-type-specific features.

Androgenetic haploid embryonic stem cells produce live transgenic mice.

Haploids and double haploids are important resources for studying recessive traits and have large impacts on crop breeding, but natural haploids are rare in animals. Mammalian haploids are restricted to germline cells and are occasionally found in tumours with massive chromosome loss. Recent success in establishing haploid embryonic stem (ES) cells in medaka fish and mice raised the possibility of using engineered mammalian haploid cells in genetic studies. However, the availability and functional characterization of mammalian haploid ES cells are still limited. Here we show that mouse androgenetic haploid ES (ahES) cell lines can be established by transferring sperm into an enucleated oocyte. The ahES cells maintain haploidy and stable growth over 30 passages, express pluripotent markers, possess the ability to differentiate into all three germ layers in vitro and in vivo, and contribute to germlines of chimaeras when injected into blastocysts. Although epigenetically distinct from sperm cells, the ahES cells can produce viable and fertile progenies after intracytoplasmic injection into mature oocytes. The oocyte-injection procedure can also produce viable transgenic mice from genetically engineered ahES cells. Our findings show the developmental pluripotency of androgenentic haploids and provide a new tool to quickly produce genetic models for recessive traits. They may also shed new light on assisted reproduction.

Dietary supplementation with ß-hydroxy-ß-methylbutyrate calcium during the early postnatal period accelerates skeletal muscle fibre growth and maturity in intra-uterine growth-retarded and normal-birth-weight piglets.

Intra-uterine growth restriction (IUGR) impairs postnatal growth and skeletal muscle development in neonatal infants. This study evaluated whether dietary ß-hydroxy-ß-methylbutyrate Ca (HMB-Ca) supplementation during the early postnatal period could improve muscle growth in IUGR neonates using piglets as a model. A total of twelve pairs of IUGR and normal-birth-weight (NBW) male piglets with average initial weights (1·85 (sem 0·36) and 2·51 (sem 0·39) kg, respectively) were randomly allotted to groups that received milk-based diets (CON) or milk-based diets supplemented with 800 mg/kg HMB-Ca (HMB) during days 7-28 after birth. Blood and longissimus dorsi (LD) samples were collected and analysed for plasma amino acid content, fibre morphology and the expression of genes related to muscle development. The results indicate that, regardless of diet, IUGR piglets had a significantly decreased average daily weight gain (ADG) compared with that of NBW piglets (P<0·05). However, IUGR piglets fed HMB-Ca had a net weight and ADG similar to that of NBW piglets fed the CON diet. Irrespective of body weight (BW), HMB-Ca supplementation markedly increased the type II fibre cross-sectional area and the mRNA expression of mammalian target of rapamycin (mTOR), insulin-like growth factor-1 and myosin heavy-chain isoform IIb in the LD of piglets (P<0·05). Moreover, there was a significant interaction between the effects of BW and HMB on mTOR expression in the LD (P<0·05). In conclusion, HMB-Ca supplementation during the early postnatal period could improve skeletal muscle growth and maturity by accelerating fast-twitch glycolytic fibre development in piglets.

Mammary inflammatory gene expression was associated with reproductive stage and regulated by docosahexenoic acid: in vitro and in vivo studies.

BACKGROUND: Periparturient mastitis is the most prevalent disease affecting lactating animals. However, it has long been relied on antibiotics to deal with mastitis, leading to a potential threat to food safety. This study was aimed to investigate the expression of pro-inflammatory cytokines in mammary glands of sows around parturition when mastitis and oxidative stress usually occur, and evaluate the anti-inflammatory effect of docosahexenoic acid (DHA) in porcine mammary epithelial cells (PMEC) challenged by lipopolysaccharide (LPS). METHODS: Mammary tissues and blood samples were collected from seven pregnant sows at different reproductive stages. Primarily cultured PMEC at passage 4 were assigned to four treatments: basal medium (control), basal medium with LPS (10 µg/mL) (LPS treatment), basal medium with LPS (10 µg/mL) and DHA (100 or 200 µM) (LPS + DHA treatments), and cell samples were harvested after 24 h incubation. The measurements included oxidative stress markers in blood samples and gene expression in mammary tissues and PMEC samples. RESULTS: Serum α-tocopherol concentration was lower at parturition than at day 90 of gestation and day 28 post parturition, while serum malondialdehyde concentration was higher at day 28 post parturition than at day 90 of gestation. Higher interleukin (IL)-1ß mRNA abundance while lower LPS binding protein mRNA abundance in mammary tissues were observed at day 90 of gestation compared with that at parturition and at day 28 and 35 post parturition. Mammary tumor necrosis factor (TNF)-α mRNA abundance were lower at parturition than at day 90 of gestation and day 28 and 35 post parturition, whereas mammary IL-8 mRNA abundance were lower at parturition than at day 35 post parturition. In the PMEC experiment, compared with the control, increased mRNA abundances of Toll-like receptor (TLR)-4 downstream target, myeloid differentiation factor 88 (MyD88), IL-6 and IL-8 were observed in LPS treatment, whereas DHA appeared to decrease mRNA abundances of MyD88, IL-6 and IL-8 induced by LPS. CONCLUSIONS: The down-regulated expression of pro-inflammatory cytokines in mammary tissues and aggravated systemic oxidative stress at parturition suggest that sows are in a vulnerable status during periparturient period. DHA appears to attenuate inflammatory responses in LPS-challenged PMEC through modulation of TLR4 signalling pathway.

Differences in plasma metabolomics between sows fed DL-methionine and its hydroxy analogue reveal a strong association of milk composition and neonatal growth with maternal methionine nutrition.

The aim of the present study was to determine whether increased consumption of methionine as DL-methionine (DLM) or its hydroxy analogue DL-2-hydroxy-4-methylthiobutanoic acid (HMTBA) could benefit milk synthesis and neonatal growth. For this purpose, eighteen cross-bred (Landrace × Yorkshire) primiparous sows were fed a control (CON), DLM or HMTBA diet (n 6 per diet) from 0 to 14 d post-partum. At postnatal day 14, piglets in the HMTBA group had higher body weight (P= 0·02) than those in the CON group, tended (P= 0·07) to be higher than those in the DLM group, and had higher (P< 0·05) mRNA abundance of jejunal fatty acid-binding protein 2, intestinal than those in the CON and DLM groups. Compared with the CON diet-fed sows, milk protein, non-fat solid, and lysine, histidine and ornithine concentrations decreased in the DLM diet-fed sows (P< 0·05), and milk fat, lactose, and cysteine and taurine concentrations increased in the HMTBA diet-fed sows (P< 0·05). Plasma homocysteine and urea N concentrations that averaged across time were increased (P< 0·05) in sows fed the DLM diet compared with those fed the CON diet. Metabolomic results based on ¹H NMR spectroscopy revealed that consumption of the HMTBA and DLM diets increased (P< 0·05) both sow plasma methionine and valine levels; however, consumption of the DLM diet led to lower (P< 0·05) plasma levels of lysine, tyrosine, glucose and acetate and higher (P< 0·05) plasma levels of citrate, lactate, formate, glycerol, myo-inositol and N-acetyl glycoprotein in sows. Collectively, neonatal growth and milk synthesis were regulated by dietary methionine levels and sources, which resulted in marked alterations in amino acid, lipid and glycogen metabolism.

Generation of transgenic rats through induced pluripotent stem cells.

The rat is an important animal model for human disease research. Using inhibitors of glycogen synthase kinase 3 and MAPK signaling pathways, rat embryonic stem cells and rat induced pluripotent stem cells (riPSCs) have been derived. However, unlike rat embryonic stem cells, germ line competent riPSCs have only been derived from Wistar rats at low efficiency. Here, we found that an optimized induction medium containing knock-out serum replacement and vitamin C improved the rate and efficiency of riPSCs generation from Dark Agouti rat fibroblasts and Sertoli cells. riPSCs maintained an undifferentiated status for >30 passages and could differentiate into various cells types including germ cells when injected into rat blastocysts. Moreover, transgenic riPSCs could be generated through the PiggyBac transposon, which could be used to generate transgenic rats through germ line transmission. riPSCs can be used as a novel tool in genetic and genomic studies of the rat.

Changes in plasma amino acid profiles, growth performance and intestinal antioxidant capacity of piglets following increased consumption of methionine as its hydroxy analogue.

The aim of the present study was to determine whether early weaning-induced growth retardation could be attenuated by increased consumption of methionine as DL-methionine (DLM) or DL-2-hydroxy-4-methylthiobutyrate (HMTBA) in both lactating sows and weaned piglets. Therefore, diets containing DLM and HMTBA at 25% of the total sulphur-containing amino acids (AA) present in the control (CON) diet were fed to lactating sows and weaned piglets and their responses were evaluated. Compared with the CON diet-fed sows, the HMTBA diet-fed sows exhibited a tendency (P<0·10) towards higher plasma taurine concentrations and the DLM diet-fed sows had higher (P<0·05) plasma taurine concentrations, but lower (P<0·05) isoleucine concentrations. Suckling piglets in the HMTBA treatment group had higher (P<0·05) intestinal reduced glutathione (GSH) content, lower (P<0·05) oxidised glutathione (GSSG):GSH ratio, and higher (P<0·05) plasma cysteine and glutathione peroxidase (GPx) activity than those in the CON and DLM treatment groups. The feed intake (P<0·05) and body weight of piglets averaged across post-weaning (PW) days were higher (P< 0·05) in the HMTBA treatment group than in the DLM treatment group and were higher (P<0·05) and tended (P<0·10) to be higher, respectively, in the HMTBA treatment group than in the CON treatment group. Increased (P<0·05) GSSG content and GSSG:GSH ratio and down-regulated (P<0·05) expression of nutrient transport genes were observed in the jejunum of piglets on PW day 7 than on PW day 0. On PW day 14, the HMTBA diet-fed piglets had higher (P<0·05) intestinal GSH content than the CON diet-fed piglets and higher (P<0·05) plasma GPx activity, villus height and goblet cell numbers than the CON diet- and DLM diet-fed piglets. In conclusion, early weaning-induced growth retardation appears to be attenuated through changes in plasma AA profiles and elevation of growth performance and intestinal antioxidant capacity in piglets following increased consumption of methionine as HMTBA.

Early patterning of cloned mouse embryos contributes to post-implantation development.

Several research groups have suggested that the embryonic-abembryonic (Em-Ab) axis in the mouse can be predicted by the first cleavage plane of the early embryo. Currently, it is not known whether this early patterning occurs in cloned embryos produced by nuclear transfer and whether it affects development to term. In this work, the relationship between the first cleavage plane and the Em-Ab axis was determined by the labeling of one blastomere in cloned mouse embryos at the 2-cell stage, followed by ex-vivo tracking until the blastocyst stage. The results demonstrate that approximately half of the cloned blastocysts had an Em-Ab axis perpendicular to the initial cleavage plane of the 2-cell stage. These embryos were classified as "orthogonal" and the remainder as "deviant". Additionally, we report here that cloned embryos were significantly more often orthogonal than their naturally fertilized counterparts and overexpressed Sox2. Orthogonal cloned embryos demonstrated a higher rate of post-implantation embryonic development than deviant embryos, but cloned pups did not all survive. These results reveal that the angular relationship between the Em-Ab axis and the first cleavage plane can influence later development and they support the hypothesis that proper early patterning of mammalian embryos is required after nuclear transfer.

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.