Multimodal decoding of human liver regeneration.

The liver has a unique ability to regenerate1,2; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option3-5. Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may advance new areas of therapeutic discovery in regenerative medicine.

Resolving the fibrotic niche of human liver cirrhosis at single-cell level.

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis.

An anatomical hypothesis: a "concentric-structured model" for the theoretical understanding of the surgical anatomy in the upper mediastinum required for esophagectomy with radical mediastinal lymph node dissection.

Understanding the surgical anatomy is the key to reducing surgical invasiveness especially in the upper mediastinal dissection for esophageal cancer, which is supposed to have a significant impact on curability and morbidity. However, there is no theoretical recognition regarding the surgical anatomy required for esophagectomy, although the surgical anatomy in abdominal digestive surgery has been developed on the basis of embryological findings of intestinal rotation and fusion fascia. Therefore, we developed a hypothesis of a 'concentric-structured model' of the surgical anatomy in the upper mediastinum based on human embryonic development. This model was characterized by three factors: (1) a concentric and symmetric three-layer structure, (2) bilateral vascular distribution, and (3) an 'inter-layer potential space' composed of loose connective tissue. The concentric three-layer structure consists of the 'visceral layer', the 'vascular layer', and the 'parietal layer': the visceral layer containing the esophagus, trachea, and recurrent laryngeal nerves as the central core, the vascular layer of major blood vessels surrounding the visceral core to maintain the circulation, and the parietal layer as the outer frame of the body. The bilateral vascular distribution consists of the inferior thyroid arteries and bronchial arteries originating from the bilateral dorsal aortae in an embryo. This bilateral vascular distribution may be related to the formation of the proper mesentery of the esophagus and frequent lymph node metastasis observed in the visceral layer around recurrent laryngeal nerves. The three concentric layers are bordered by loose connective tissue called the 'inter-layer potential space'. This inter-layer potential space is the fundamental factor of our concentric-structured model as the appropriate surgical plane of dissection. The peripheral blood vessels, nerves, and lymphatics transition between each layer, thereby penetrating this loose connective tissue forming the inter-layer potential space. Recurrent laryngeal nerves also transition from the vascular layer after branching off from the vagal nerves and then ascend consistently in the visceral layer. We investigated the validity of this concentric-structured model, confirming the intraoperative images and the surgical outcomes of thoracoscopic esophagectomy in a prone position (TSEP) before and after the introduction of this hypothetical anatomy model. A total of 226 patients with esophageal cancer underwent TSEP from January 2015 to December 2016. After the introduction of this model, the surgical outcomes in 105 patients clearly improved for the operation time of the thoracoscopic procedure (160 min vs. 182 min, P = 0.01) and the incidence of recurrent laryngeal nerve palsy (19.0% vs. 36.4%, P = 0.004). Moreover, we were able to identify the concentric and symmetric layer structure through surgical dissection along the inter-layer potential space between the visceral and vascular layers ('viscero-vascular space') in all 105 cases after introduction of the hypothetical model. The concentric-structured model based on embryonic development is clinically beneficial for achieving less-invasive esophagectomy by ensuring a theoretical understanding of the surgical anatomy in the upper mediastinum.

Thermogenesis induced by amino acid administration prevents intraoperative hypothermia and reduces postoperative infectious complications after thoracoscopic esophagectomy.

Minimally invasive thoracoscopic esophagectomy has potential advantages in minimizing the impairment of respiratory function and reducing surgical stress. However, thoracoscopic esophagectomy occasionally results in anesthesia-induced hypothermia, particularly in cases involving artificial pneumothorax with CO2. Thermogenesis induced by amino acid administration has been reported during anesthesia. Here, we tested the efficacy of amino acid treatment for the prevention of hypothermia, and we investigated the potential of this treatment to reduce postoperative infectious complications after thoracoscopic esophagectomy. We conducted a randomized trial in patients with esophageal cancer who underwent thoracoscopic esophagectomy in the prone position in two groups and analyzed the incidences of hypothermia and surgical complications. One-hundred and thirty patients were randomized. Administration of amino acids resulted in a significant increase in core body temperature. In the saline (n = 60) and amino acid (n = 70) administration groups, 30% and 14.2% of patients, respectively, experienced infectious surgical complications (P = 0.029), and 21.6% and 22.8% of patients, respectively, experienced noninfectious surgical complications (P = 0.86). Univariate analysis revealed that blood loss and amino acid administration were significant factors for infectious surgical complications. Multivariate analysis revealed that amino acid administration was an independent factor reducing infectious surgical complications (P = 0.025, 95% confidence interval: 0.105-0.864). Administration of amino acids prevents hypothermia and reduces postoperative infectious complications after thoracoscopic esophagectomy.

Characterization of Bacillus sp. endo-beta-N-acetylglucosaminidase and its application to deglycosylation of hen ovomucoid.

A bacterial strain isolated from soil and identified as a Bacillus species produced two endo-beta-N-acetylglucosaminidases in the culture broth when it was cultivated on medium containing only hen ovomucoid. Almost no production of the enzymes occurred when the bacterium was grown on glucose medium. The two endo-beta-N-acetylglucosaminidases, named Endo-BI and Endo-BII, were separated and purified to homogeneity by preparative gel electrophoresis after partial purification by column chromatography on DEAE-resins. Endo-BI hydrolysed oligosaccharides of both hen ovalbumin and ovomucoid. In contrast, Endo-BII could act only on oligosaccharides of hen ovalbumin and showed almost no activity towards those of hen ovomucoid. Deglycosylation of hen ovomucoid was performed with the partly purified endo-beta-N-acetylglucosaminidase preparation, with the aid of contaminating beta-N-acetylhexosaminidase. The deglycosylated ovomucoid exhibited no changes in trypsin inhibitory activity but was very unstable to heat treatment in comparison with native ovomucoid. These results suggest that oligosaccharides of ovomucoid have an important role in the stabilization of the protein against heat.

Improvement of the Off-flavor of Soy Protein Isolate by Removing Oil-body Associated Proteins and Polar Lipids.

The precipitate formed by ultracentrifuging a defatted soybean extract at 200,000×g for 50 min at pH 7.5 was composed of particles of 100-200 nm in diameter and enriched with 34-kDa, 24-kDa and 18-kDa proteins. An SDS-PAGE analysis showed these proteins to migrate to a position identical to that of oil-body-associated proteins (OBAPs; Herman, Planta, 172, 336-345, 1987).(1)) They were recovered in the precipitate of soy protein with 30-40% saturated ammonium sulfate in the presence of 10 mM 2-ME. The lipid composition of the precipitate by a TLC analysis showed that most of the polar lipids in the soybean extract had been condensed in the fraction, suggesting the association between OBAP and the polar lipids. Removal of OBAP and the polar lipids from the soybean extract by conventional centrifugation (10,000×g for 10 min) in the presence of 30 mM Na2SO4 and 30 mM CaCl2 at pH 2.8 was achieved with concomitant improvement of the volatile off-flavor. A soy protein isolate (SPI) prepared from such a soybean extract contained far fewer volatile off-flavor compounds than normal SPI did.