Inductive effect of SORT1 on odontoblastic differentiation of human dental pulp-derived stem cells.

This study investigated the expression of sortilin 1 (SORT1) in cultured human dental pulp-derived stem cells (hDPSCs) and its role in their odontoblastic differentiation. Permanent teeth were extracted from five patients, and the dental pulp was harvested for explant culture. Fluorescence-activated cell sorting was used to analyze the outgrowth of adherent cells and cells that had migrated from the tissue margin. SORT1 expression was detected in hDPSCs simultaneously expressing the mesenchymal stem cell markers CD44 and CD90. The odontoblastic differentiation potential of SORT1-positive hDPSCs was examined via staining for alkaline phosphatase (ALP), an early odontoblastic differentiation marker. ALP staining was more intense in SORT1-positive than in SORT1-negative hDPSCs. Consistently, the expression of mRNA encoding SORT1 and p75NTR, a binding partner of SORT1, increased in SORT1-positive hDPSCs during odontoblastic differentiation. In addition, pro-nerve growth factor (NGF), a ligand for SORT1-p75NTR co-receptor, promoted ALP expression in SORT1-positive hDPSCs, and the interaction between SORT1 and p75NTR was detected using a coimmunoprecipitation assay. The function of SORT1 in odontoblastic differentiation was examined via RNA interference using shRNA targeting SORT1. ALP staining intensity in SORT1/shRNA-transfected cells was markedly lower than in control/shRNA-transfected cells. SORT1 knockdown decreased JUN phosphorylation and recruitment of phosphorylated JUN to the ALP promoter. Collectively, these results indicate that SORT1 is involved in the odontoblastic differentiation of hDPSCs through the JUN N-terminal kinases (JNK)/JUN signaling pathway and that the binding of SORT1 and p75NTR plays an important role in this process.

The expression analysis of SerpinB9 in hepatoblastoma microenvironment.

PURPOSE: In this research, we analyzed the expression of serpinB9 in hepatoblastoma and investigated the factors which enhance its expression. METHOD: SerpinB9 expression in hepatoblastoma cell lines and macrophages co-cultured with each other or stimulated by anticancer agents was examined using RT-qPCR and western blotting. Immunohistochemistry for SerpinB9 in hepatoblastoma specimens was performed. Single-cell RNA-sequence data for hepatoblastoma from an online database were analyzed to investigate which types of cells express SerpinB9. RESULT: HepG2, a hepatoblastoma cell line, exhibited increased expression of SerpinB9 when indirectly co-cultured with macrophages. Immunohistochemistry for the specimens demonstrated that serpinB9 is positive not in hepatoblastoma cells but in macrophages. Single-cell RNA sequence analysis in tissues from hepatoblastoma patients showed that macrophages expressed SerpinB9 more than tumor cells did. Co-culture of macrophages with hepatoblastoma cell lines led to the enhanced expression of SerpinB9 in both macrophages and cell lines. Anticancer agents induced an elevation of SerpinB9 in hepatoblastomas cell lines. CONCLUSION: In hepatoblastoma, SerpinB9 is thought to be more highly expressed in macrophages and enhanced by interaction with hepatoblastoma cell.

Hepatic venous outflow obstruction after adult living donor liver transplantation.

Hepatic venous outflow obstruction (HVOO) is a rare but critical vascular complication after adult living donor liver transplantation. We categorized HVOOs according to their morphology (anastomotic stenosis, kinking, and intrahepatic stenosis) and onset (early-onset < 3 mo vs. late-onset ≥ 3 mo). Overall, 16/324 (4.9%) patients developed HVOO between 2000 and 2020. Fifteen patients underwent interventional radiology. Of the 16 hepatic venous anastomoses within these 15 patients, 12 were anastomotic stenosis, 2 were kinking, and 2 were intrahepatic stenoses. All of the kinking and intrahepatic stenoses required stent placement, but most of the anastomotic stenoses (11/12, 92%) were successfully managed with balloon angioplasty, which avoided stent placement. Graft survival tended to be worse for patients with late-onset HVOO than early-onset HVOO (40% vs. 69.3% at 5 y, p = 0.162) despite successful interventional radiology. In conclusion, repeat balloon angioplasty can be considered for simple anastomotic stenosis, but stent placement is recommended for kinking or intrahepatic stenosis. Close follow-up is recommended in patients with late-onset HVOO even after successful treatment.

Factors predicting surgical difficulties in congenital biliary dilatation in pediatric patients.

BACKGROUND: The effects of disease classification and the patient's preoperative condition on the difficulty of performing a laparotomy for pediatric congenital biliary dilatation (CBD) have not been fully elucidated. METHODS: The present study retrospectively analyzed 46 pediatric CBD laparotomies performed at the study center between March 2010 and December 2021 and predictors of operative time. The patients were separated into a short operative time group (SOT) (≤ 360 min, n = 27) and a long operative time group (LOT) (> 360 min, n = 19). RESULTS: The preoperative AST and ALT values were higher, and the bile duct anastomosis diameter was larger, in the LOT. Correlation analysis demonstrated that the maximum cyst diameter, preoperative neutrophil-to-lymphocyte ratio, AST, ALT, AMY, and bile duct anastomosis diameter correlated positively with operative time. Multivariate analysis identified the maximal cyst diameter, preoperative AST, and bile duct anastomosis diameter as significant factors affecting surgical time. Postoperatively, intrapancreatic stones and paralytic ileus were observed in one patient each in the SOT, and mild bile leakage was observed in one patient in the LOT. CONCLUSIONS: The maximum cyst diameter, preoperative AST, and bile duct anastomosis diameter have the potential to predict the difficulty of performing a pediatric CBD laparotomy.

Potential involvement of IL-32 in cell-to-cell communication between macrophages and hepatoblastoma.

PURPOSE: This study investigated the expression of interleukin 32 (IL-32) in hepatoblastoma, the most common primary pediatric liver tumor, and its possible roles in tumorigenesis. METHODS: IL-32 expression was investigated in two hepatoblastoma cell lines (Hep G2 and HuH 6) in the steady state and after co-culture with macrophages by RNA-seq analysis and RT-qPCR, and after stimulation with chemotherapy. Cultured macrophages were stimulated by IL-32 isoforms followed by RT-qPCR and western blot analysis. IL-32 immunohistochemical staining (IHC) was performed using specimens from 21 hepatoblastoma patients. Clustering analysis was also performed using scRNA-seq data downloaded from Gene Expression Omnibus. RESULTS: The IL-32 gene is expressed by hepatoblastoma cell lines; expression is upregulated by paracrine cell-cell communication with macrophages, also by carboplatin and etoposide. IL-32 causes protumor activation of macrophages with upregulation of PD-L1, IDO-1, IL-6, and IL-10. In the patient pool, IHC was positive only in 48% of cases. However, in the downloaded dataset, IL-32 gene expression was negative. CONCLUSION: IL-32 was detected in hepatoblastoma cell lines, but not in all hepatoblastoma patients. We hypothesized that stimulation such as chemotherapy might induce expression of IL-32, which might be a critical mediator of chemoresistance in hepatoblastoma through inducing protumor activation in macrophages.

Surgical Outcomes of Domino Liver Transplantation Using Grafts From Living Donors With Familial Amyloid Polyneuropathy.

Domino liver transplantation (DLT) using grafts from donors with familial amyloid polyneuropathy is an acceptable procedure for expanding the donor pool. The vascular and biliary reconstructions in living donor DLT (LDDLT) are technically demanding, and data on the short-term and long-term surgical outcomes of domino donors and recipients in LDDLT are limited. In this study, we identified 25 domino recipients from our liver transplantation program (1999-2018), analyzed the vascular and biliary reconstructions performed, and evaluated the surgical outcomes, including graft survival. Piggyback technique was adopted in all 25 domino donors. The only surgical complication in domino donors was hepatic vein (HV) stenosis with an incidence rate of 4%. In 22 domino recipients, right HV and middle/left HV were reconstructed separately. A total of 10 recipients had 2 arteries anastomosed, and 18 underwent duct-to-duct biliary anastomosis. HV stenosis and biliary stricture had incidence rates of 8% and 24%, respectively, in the recipients, but none of them developed hepatic artery thrombosis. The 1-year and 5-year graft survival rates were 100% each in the domino donors, and 84.0% and 67.3% in the domino recipients, respectively. In conclusion, LDDLT has acceptable outcomes without increasing the operative risk in donors despite the demanding surgical technique involved.

Oral Supplementation with Z-Isomer-Rich Astaxanthin Inhibits Ultraviolet Light-Induced Skin Damage in Guinea Pigs.

The effect of oral supplementation with astaxanthin of different Z-isomer ratios on ultraviolet (UV) light-induced skin damage in guinea pigs was investigated. Astaxanthin with a high Z-isomer content was prepared from the all-E-isomer via thermal isomerization. Intact (all-E)-astaxanthin and the prepared Z-isomer-rich astaxanthin were suspended in soybean oil and fed to guinea pigs for three weeks. The UV-light irradiation was applied to the dorsal skin on the seventh day after the start of the test diet supplementation, and skin parameters, such as elasticity, transepidermal water loss (TEWL), and pigmentation (melanin and erythema values), were evaluated. The accumulation of astaxanthin in the dorsal skin was almost the same after consumption of the all-E-isomer-rich astaxanthin diet (E-AST-D; total Z-isomer ratio = 3.2%) and the Z-isomer-rich astaxanthin diet (Z-AST-D; total Z-isomer ratio = 84.4%); however, the total Z-isomer ratio of astaxanthin in the skin was higher in the case of the Z-AST-D supplementation. Both diets inhibited UV light-induced skin-damaging effects, such as the reduction in elasticity and the increase in TEWL level. Between E-AST-D and Z-AST-D, Z-AST-D showed better skin-protective ability against UV-light exposure than E-AST-D, which might be because of the greater UV-light-shielding ability of astaxanthin Z-isomers than the all-E-isomer. Furthermore, supplementation with Z-AST-D resulted in a greater reduction in skin pigmentation caused by astaxanthin accumulation compared to that of E-AST-D. This study indicates that dietary astaxanthin accumulates in the skin and appears to prevent UV light-induced skin damage, and the Z-isomers are more potent oral sunscreen agents than the all-E-isomer.

Simulated slide tracheoplasty for congenital tracheal stenosis using three-dimensional printed models.

PURPOSE: The slide tracheoplasty (STP) is the standard treatment for severe congenital tracheal stenosis (CTS). Understanding the features of the tracheal stenosis in each case and choosing an appropriate incision design are very important for successfully executing the procedure. The present study aimed to evaluate the advantages of three-dimensional (3D) printed models of the trachea for improving CTS. METHODS: Three-D tracheal models were created using computed tomography (CT) data from ten patients undergoing STP for CTS. Simulated surgery was performed using the hollow models after reinforcing with them with a coating of gum spray. Clinical outcomes, including patient survival, postoperative surgical interventions, and time required for STP, were compared with the corresponding values in the last ten patients before the introduction of 3D model simulations. RESULTS: All ten patients for whom simulated surgery using a 3D tracheal model were conducted achieved good airway patency after their STP. The surgeons reported feeling that the 3D model simulations were highly effective although there was no significant difference in the clinical outcomes of the groups with or without simulated STP. The models were useful not only for surgical planning but also for sharing important information among the multidisciplinary team and the patients' family. CONCLUSION: Our experience using 3D tracheal models demonstrated several features enabling improvement in the surgical treatment of CTS.

Intellectual development of patients with biliary atresia who underwent living donor liver transplantation in infancy.

BACKGROUND: The impact of pediatric liver transplantation on intellectual development has yet to be determined. We investigated the intellectual outcomes of school-aged patients after living donor liver transplantation for biliary atresia in infancy. METHODS: The Wechsler Intelligence Scale for Children-fourth edition test was administered to 20 patients who survived [Formula: see text] 5 years after living donor liver transplantation. Borderline full scale intelligence quotient was defined as ≤ 85. Pre-, peri-, and postoperative data were compared between patients with > 85 and ≤ 85 to identify predictive factors of borderline performance. RESULTS: The one-sample t test demonstrated that the mean full scale intelligence quotient of patients after transplantation for biliary atresia was significantly lower than that of the general population (91.8 vs. 100.0, p = 0.026) and 7 (35%) were classified as intellectual borderline functioning. Multivariable logistic regression models were unable to identify any factors predictive of full scale intelligence quotients of ≤ 85. CONCLUSION: This is the first study to indicate that the mean full scale intelligence quotient among school-aged patients who underwent living donor liver transplantation for biliary atresia in infancy is significantly lower than that of the general population.

M-CSFR expression in the embryonal component of hepatoblastoma and cell-to-cell interaction between macrophages and hepatoblastoma.

Tumor-associated macrophages (TAMs) have protumor functions in various cancers. However, their significance in hepatoblastoma, the most common liver tumor in children, remains unclear. The aim of this study was to explore the potential roles of TAMs in hepatoblastoma. Immunohistochemical analysis revealed that the density of CD204-positive TAMs was significantly higher in the embryonal component than in other histological subtypes of hepatoblastoma. An in vitro co-culture study with Huh6 cells and human monocyte-derived macrophages (HMDMs) showed that macrophage-colony-stimulating factor receptor (M-CSFR) was strongly up-regulated in the Huh6 cells that were directly co-cultured with HMDMs. The expressions of M-CSFR ligands (interleukin-34 and M-CSF) were also increased by co-culture with HMDMs. The proliferation of HepG2 cells (another hepatoblastoma cell line expressing M-CSFR) was inhibited by an M-CSFR inhibitor. M-CSFR was found to be highly expressed in the embryonal component and in recurrent lesions. The number of CD204-positive macrophages was also higher in the M-CSFR-positive areas than in the M-CSFR-negative areas. Thus, M-CSFR expression appeared to be induced by cell-cell contact with macrophages in hepatoblastoma cells, and M-CSFR inhibitor is potentially effective against M-CSFR-positive hepatoblastoma, especially recurrent cases.

Portal Vein Complications After Adult Living Donor Liver Transplantation: Time of Onset and Deformity Patterns Affect Long-Term Outcomes.

Portal vein complications (PVCs) after adult living donor liver transplantation (LDLT) are potentially lethal. We categorized PVCs by the time of onset (early versus late, <1 month versus ≥1 month, respectively) and deformity patterns (portal vein stenosis [PVS], portal vein thrombosis [PVT], and portal vein occlusion [PVO]) to establish optimal treatment strategies. Overall, 35/322 (10.9%) recipients developed PVCs between 2000 and 2019. Pretransplant PVT (odds ratio [OR], 15.20; 95% confidence interval [CI], 3.70-62.40; P < 0.001) was the only independent risk factor for PVS. In contrast, male sex (OR, 5.57; 95% CI, 1.71-18.20; P = 0.004), pretransplant PVT (OR, 4.79; 95% CI, 1.64-14.00; P = 0.004), and splenectomy (OR, 3.24; 95% CI, 1.23-8.57; P = 0.018) were independent risk factors for PVT. PVS was successfully treated with interventional radiology regardless of its time of onset. On the other hand, late PVT and PVO had significantly lower treatment success rates (2/15, 13%) compared with those that occurred in the early period (10/11, 91%) despite aggressive intervention (P < 0.001). Deformity patterns had a significant impact on the 5-year cumulative incidence of graft loss as a result of PVC (PVO + Yerdel grades 2-4 PVT group [n = 16], 41% versus PVS + Yerdel grade 1 PVT group [n = 19], 0%; P = 0.02). In conclusion, late grades 2 to 4 PVT and PVO are refractory to treatment and associated with poor prognoses, whereas PVS has a good prognosis regardless of time of onset. A tailored approach according to the time of onset and deformity patterns of PVC is essential.

Micro-computed tomography analysis of the relationship between root canal number and root concavity in maxillary first and second molars in a Japanese population.

PURPOSE: The aim of this study was to investigate root canal configurations in maxillary first and second molars from a Japanese population, as well as the relationship between the root canal number and root concavity depth, using micro-computed tomography. METHODS: Maxillary first (N = 74) and second (N = 70) molars from a Japanese population were used. Virtual horizontal sections were created, and the number of root canals and the degree of root concavity were measured. Mesiobuccal (MB) roots were categorized into Group 1 (with a single root canal) or Group 2 (with two or more root canals). The relationship between the root canal number and root concavity depth was evaluated using the Mann-Whitney U test. RESULTS: Approximately 51% and 35% of the MB roots in the three-rooted maxillary first and second molars, respectively, had two or more root canals. The depths of the root concavities in maxillary first molars differed significantly between Groups 1 and 2 at the mesial and distal sides of the MB root, in the section 2 mm apical to the furcation area (p < 0.05). CONCLUSIONS: A significant relationship between the number of root canals and the depth of root concavity in the MB root of the maxillary first molars was observed. Close clinical monitoring is needed during the removal of calculus and cleaning of the root surfaces of maxillary first molars: specifically, excessive removal of cementum and dentin should be avoided, particularly when the molar has already undergone root canal treatment.

Asymptomatic hepatic artery dissection early after living-donor liver transplantation with simultaneous splenectomy: two case reports.

BACKGROUND: Hepatic artery dissection after liver transplantation is an uncommon morbidity. The onset mechanism and management for this disorder remain unclear. The present report describes the cases of two patients with hepatic artery dissection after living-donor liver transplantation (LDLT) with simultaneous splenectomy and provides new insight into the onset mechanism of this disorder. CASE PRESENTATION: CASE 1: A 51-year-old man with liver cirrhosis caused by hepatitis B virus underwent LDLT with a right lobe graft and splenectomy simultaneously. The recipient's right hepatic artery had partial dissection at the anastomosis site; therefore, his left hepatic artery was anastomosed. Contrast-enhanced computed tomography (CT) on postoperative day (POD) 27 showed dissection from his celiac artery to his left hepatic artery with bleeding in the false lumen. There was a risk of rupture of the false lumen; therefore, emergency interventional radiology and coil embolization of the false lumen were performed. The patient was doing well at 6 months after LDLT. CASE 2: A 58-year-old woman with liver cirrhosis caused by primary biliary cholangitis underwent LDLT with a left lobe graft and splenectomy simultaneously. Her hepatic artery had a dissection that extended from her left hepatic artery to the proper hepatic artery. The gastroduodenal artery was anastomosed. Contrast-enhanced CT on POD 8 revealed dissection from the celiac artery to the common hepatic artery as well as a pseudoaneurysm at the celiac artery. We managed the patient with conservative treatment and performed daily follow-ups with Doppler ultrasonography examination and serial contrast-enhanced CT. At the time of writing this report, the patient was doing well at 34 months after LDLT. CONCLUSIONS: Patients who have an intimal dissection at the anastomosis site and/or simultaneous splenectomy are at a higher risk of hepatic artery dissection. Most patients with asymptomatic hepatic artery dissections can be treated conservatively. Blood flow in the intrahepatic artery should be checked frequently using Doppler ultrasonography or contrast-enhanced CT soon after diagnosis.

Evaluation of root morphology of maxillary and mandibular second molars lost due to periodontitis.

BACKGROUND AND OBJECTIVE: Little is known about the anatomical characteristics of root morphology in molars lost due to periodontal reason. The aim of this study was to study root morphology in maxillary and mandibular molars lost due to periodontitis by investigating the frequency of root fusion, classifying fusion types, and measuring radicular groove depth by micro-computed tomography. MATERIAL AND METHODS: Ninety-eight posterior teeth were collected from 87 Japanese patients during the study period. Of these, maxillary (N = 36) and mandibular (N = 22) second molars lost most frequently were assessed for root fusion and morphology. RESULTS: The 36 maxillary second molars included nine (25%) teeth with a single root, 14 (39%) with two roots, and 13 (36%) with three roots. Of the 23 maxillary second molars with fused (1 and 2) roots, there were 11 (48%), 2 (9%), 1 (4%), 1 (4%), 3 (13%), and 5 (22%) teeth with root types 1-6, respectively. The 22 mandibular second molars comprised 14 (64%) teeth with a single root, 7 (32%) with two roots, and one (4%) with three roots. Of the 14 mandibular second molars with a fused (1) root, 12 (86%) had a C-shaped root and two (14%) had a non-C-shaped root. CONCLUSION: We observed a higher frequency of root fusion in the present study compared with that reported by previous studies using randomly selected second molars.

Morphological plasticity of hyperelongated cells caused by overexpression of translation elongation factor P in Synechococcus elongatus PCC7942.

Translation elongation factors (EFs) are proteins that play important roles during the elongation stage of protein synthesis. In prokaryotes, at least four EFs function in repetitive reactions (EF-Tu, EF-Ts, EF-G, and EF-P). EF-P plays a vital role in the specialized translation of consecutive proline amino acid motifs. It was also recently recognized that EF-P acts throughout translation elongation. Here, we demonstrated for the first time that cell division and morphology are intimately linked to the control of EF-P in the model cyanobacterium Synechococcus elongatus PCC7942. We constructed the overexpression of a wild-type gene product for EF-P (Synpcc7942_2565) as a tool to identify EF-P functionality. The overexpression of EF-P resulted in the morphological plasticity of hyperelongated cells. During the stationary phase, EF-P overexpressors displayed cell lengths of 150 µm or longer, approximately 35 times longer than the control. Total cellular protein and amino acid content were also increased in overexpressors. To explore the molecular mechanisms underlying hyperelongation, gene expression analysis was performed. The results revealed that cell division genes, including ftn6, minD, mreB, mreC, and ftsZ, were modulated in overexpressors. Strikingly, ftn6 was severely down-regulated. Little is known regarding EF-P in prokaryotic photosynthetic organisms. Our results suggest that cyanobacterial EF-P participates in the acceleration of protein synthesis and also regulates cell division processes. These findings suggest new ways to modify translation and metabolism in cyanobacteria. Phenotypic and metabolic alterations caused by overexpressing EF-P may also be beneficial for applications such as low-cost, green molecular factories. KEY POINTS: • Cell division and cell morphology in the cyanobacterium Synechococcus elongatus PCC7942 are closely linked with the control of translation elongation factor P (EF-P). • Overexpression of EF-P leads to morphological plasticity in hyperelongated cells. • Cyanobacterial EF-P is involved in the acceleration of protein synthesis and the regulation of cell division processes.

Z-Isomers of lycopene exhibit greater liver accumulation than the all-E-isomer in mice.

The effect of oral administration of all-E-isomer-rich and Z-isomer-rich lycopene on liver accumulation in mice was investigated. When a diet rich in the Z-isomers was administered for 4 weeks, the total lycopene concentration in the liver was more than 3 times higher than that of all-E-isomer administration. This result clearly indicates that lycopene Z-isomers show greater bioavailability and/or liver accumulation than the all-E-isomer in mice.

The duodenal tube test is more specific than hepatobiliary scintigraphy for identifying bile excretion in the differential diagnosis of biliary atresia.

PURPOSE: Confirmation of bile excretion into the gastrointestinal tract is important to exclude biliary atresia (BA). We compared the duodenal tube test (DTT) with hepatobiliary scintigraphy (HS) for their efficiency in detecting bile secretion. METHODS: The subjects of this retrospective study were 47 infants who underwent both DTT and HS to diagnose or exclude BA between January 2000 and March 2018. RESULTS: BA was diagnosed in 32 of the 47 patients, and 7 of the remaining 15 non-BA patients underwent intraoperative cholangiography. Among the various DTT parameters, the total bile acid in duodenal fluid (DF-TBA)/serum (S) gamma-glutamyl transferase (γGTP) ratio was found to be the most specific for BA, with sensitivity and specificity of 98.0-100%, respectively. One BA patient in whom cut off values were not met was a premature infant. The sensitivity and specificity of HS were 100-56.3%, respectively. The diagnostic accuracy of the DF-TBA/S-γGTP parameter was higher than that of HS (98.6% vs. 85.1%, respectively). CONCLUSIONS: The DTT could be more a specific method than HS to detect bile excretion. Thus, the DTT should be incorporated into the multidisciplinary diagnostic approach for the differential diagnosis of BA to prevent unnecessary intraoperative cholangiography in patients who do not have BA.

A class I fructose-1,6-bisphosphate aldolase is associated with salt stress tolerance in a halotolerant cyanobacterium Halothece sp. PCC 7418.

Fructose-1,6-bisphosphate aldolase (FBA) is a key metabolic enzyme, which is involved in glycolysis, gluconeogenesis and the Calvin cycle. The distinct physiological roles of FBAs in various organisms have been reported; however, in cyanobacteria, the functional characterization of FBAs and investigation of the intracellular dynamics of FBAs largely remains unknown. Here, we utilized a two-step chromatographic technique to identify a class I FBA (CI-FBA), which we named H2846. H2846 was induced by salt stress in the halotolerant cyanobacterium Halothece sp. PCC 7418 (hereafter referred to as Halothece 7418). Phylogenetic analysis showed that H2846-like CI-FBAs existed mainly in cyanobacterial species that inhabit hypersaline environments. Subcellular fractionation revealed that H2846 localized in the cytosolic and periplasmic spaces and size-exclusion chromatography suggested that H2846 formed a homohexamer. The CI-FBA activity of recombinant H2846-mediated cleavage of fructose bisphosphate (FBP) was characterized using a coupled enzymatic assay. This analysis allowed us to determine the Km and Vmax values of recombinant H2846, which were then compared to previously reported Km and Vmax values of several FBAs. Our data suggested that H2846 was likely responsible for the salt stress-induced CI-FBA activity from the total soluble protein extracts derived from Halothece 7418 cells. Moreover, heterologous expression of H2846 but not H2847, a class II FBA (CII-FBA), conferred salt stress tolerance to the salt-sensitive freshwater cyanobacterium, Synechococcus elongatus PCC 7942, which only contains the CII-FBA, S1443. S. elongatus PCC 7942 with a S1443 gene deletion was complemented by H2847 expression, but was not complemented by expression of H2846. Taken together, these results indicate the functional differences between two distinct sets of FBAs in cyanobacteria. H2846 is an active CI-FBA that contributes to the mechanism of salt stress tolerance in Halothece 7418.

Effects of Potassium Chloride-Induced Stress on the Carotenoids Canthaxanthin, Astaxanthin, and Lipid Accumulations in the Green Chlorococcal Microalga Strain TISTR 9500.

Microalgae are a diverse group of photosynthetic eukaryotic organisms that are widely distributed globally. They are prolific sources of highly valuable compounds with fascinating chemical structures. Due to their balanced nutritional compositions and health benefits, they are increasingly being used as functional food ingredients. Carotenoid-based pigments and polyunsaturated fatty acids (PUFAs) are examples of high-value nutrients that can be accumulated abundantly in microalgae. Here, the effects of potassium chloride-induced stress on the productions of lipids and carotenoids in the green microalga of the Chlorococcaceae family were investigated. Under normal BG11 medium, this green microalga strain TISTR 9500 accumulated high levels of PUFA and primary carotenoid lutein. Stress tests revealed that KCl enhanced and modulated lipid and carotenoid accumulation levels. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that secondary carotenoids astaxanthin and canthaxanthin were robustly produced under KCl stress with the similar content of lutein. Further, this stress led to a significant increase in the total FA amount with the higher proportion of unsaturated FA than saturated FA. Thus, this green microalga could be an attractive and alternative natural biosource for canthaxanthin and astaxanthin, as well as for functional lipids.

Intravital Imaging of Neutrophil Recruitment Reveals the Efficacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury.

Neutrophils are considered responsible for the pathophysiological changes resulting from hepatic ischemia-reperfusion (I/R) injury, which is a complication of trauma, shock, liver resection, and transplantation. Recently, evidence is accumulating that formyl-peptide receptor (FPR) signaling constitutes an important danger signal that guides neutrophils to sites of inflammation. This study aimed to investigate dynamic neutrophil recruitment using two-photon laser-scanning microscopy (TPLSM) in response to FPR1 blockade during hepatic I/R. LysM-eGFP mice were subjected to partial warm hepatic I/R. They were pretreated with an FPR1 antagonist, cyclosporine H (CsH), or formyl peptide, fMLF. Liver was imaged after hepatic laser irradiation or I/R using the TPLSM technique. CsH treatment alleviated hepatic I/R injury, as evidenced by decreased serum transaminase levels, reduced hepatocyte necrosis/apoptosis, and diminished inflammatory cytokine, chemokine, and oxidative stress. In contrast, systemic administration of fMLF showed few effects. Time-lapse TPLSM showed that FPR1 blockade inhibited the accumulation of neutrophils in the necrotic area induced by laser irradiation in vivo. In the CsH-treated I/R group, the number and crawling velocity of neutrophils in the nonperfused area were lower than those in the control group. Meanwhile, FPR1 blockade did not affect monocyte/macrophage recruitment. Hepatic I/R promoted the retention of neutrophils and their active behavior in the spleen, whereas CsH treatment prevented their changes. Intravital TPLSM revealed that formyl-peptide-FPR1 signaling is responsible for regulating neutrophil chemotaxis to allow migration into the necrotic area in hepatic I/R. Our findings suggest effective approaches for elucidating the mechanisms of immune cell responses in hepatic I/R.