Expression analyses of CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS in the one-leaf plant Monophyllaea glabra reveal neoteny evolution of shoot meristem.

The one-leaf plant Monophyllaea glabra exhibits a unique developmental manner in which only one cotyledon continues growing without producing new vegetative organs. This morphology is formed by specific meristems, the groove meristem (GM) and the basal meristem (BM), which are thought to be modified shoot apical meristem (SAM) and leaf meristem. In this study, we analysed the expression of the organ boundary gene CUP-SHAPED COTYLEDON (CUC) and the SAM maintenance gene SHOOT MERISTEMLESS (STM) orthologs by whole-mount in situ hybridisation. We found that CUCs did not show clear border patterns around GM and BM during the vegetative phase. Furthermore, double-colour detection analysis at the cellular level revealed that CUC and STM expression overlapped in the GM region during the vegetative phase. We also found that this overlap is dissolved in the reproductive phase when normal shoot organogenesis is observed. Since co-expression of these genes occurs during SAM initiation under embryogenesis in Arabidopsis, our results demonstrate that GM is a prolonged stage of pre-mature SAM. Therefore, we propose that neotenic meristems could be a novel plant trait acquired by one-leaf plants.

Position of meristems and the angles of the cell division plane regulate the uniqueness of lateral organ shape.

Leaf meristem is a cell proliferative zone present in the lateral organ primordia. In this study, we examined how cell proliferative zones in primordia of planar floral organs and polar auxin transport inhibitor (PATI)-treated leaf organs differ from those of non-treated foliage leaves of Arabidopsis thaliana, with a focus on the accumulation pattern of ANGUSTIFOLIA3 (AN3) protein, a key element for leaf meristem positioning. We found that PATI-induced leaf shape changes were correlated with cell division angle but not with meristem positioning/size or AN3 localisation. In contrast, different shapes between sepals and petals compared with foliage leaves were associated with both altered meristem position, due to altered AN3 expression patterns, and different distributions of cell division angles. A numerical simulation showed that meristem position majorly affected the final shape but biased cell division angles had a minor effect. Taken together, these results suggest that the unique shapes of different lateral organs depend on the position of the meristem in the case of floral organs and cell division angles in the case of leaf organs with different auxin flow.

Auxin and cytokinin control fate determination of cotyledons in the one-leaf plant Monophyllaea glabra.

One-leaf plants in the Gesneriaceae family initially have two cotyledons of identical size; one cotyledon stops growing shortly after germination, whereas the other continues indeterminate growth. Factors involved in the unequal growth have been investigated, and a competitive relationship between the two cotyledons was previously proposed. However, questions regarding the fate determination of the two cotyledons remain: Why does only one cotyledon grow indeterminately while the other stops; is the fate of the cotyledons reversible; and what role does light quality play in the fate determination of the cotyledons? In this study, physiological experiments using the one-leaf plant species Monophyllaea glabra suggest that a biased auxin concentration between the two cotyledons and subsequent cytokinin levels may determine the fate of the cotyledons. In addition, observation of relatively mature individuals without hormone treatment and younger individuals with cytokinin treatment under laboratory growth conditions revealed that the fate determination of the microcotyledon is reversible. Although light quality has been suggested to be important for the determination of cotyledon fate in Streptocarpus rexii, an anisocotylous species, we conclude that light quality is not important in M. glabra.

Expression Profiles of ANGUSTIFOLIA3 and SHOOT MERISTEMLESS, Key Genes for Meristematic Activity in a One-Leaf Plant Monophyllaea glabra, Revealed by Whole-Mount In Situ Hybridization.

Members of the genus Monophyllaea are unique in that they produce no new organ during the vegetative phase in the shoot; instead, one of the cotyledons grows indeterminately. The mechanism of this unique trait is unclear, in part because of the lack of suitable assessment techniques. We therefore established a whole-mount in situ hybridization technique, a powerful means of examining spatial patterns in gene expression, for Monophyllaea glabra. By using this, we examined the expression pattern of a SHOOT MERISTEMLESS (STM) ortholog, which is indispensable for the formation and maintenance of the shoot apical meristem (SAM) in typical angiosperms. Expression was confined to the groove meristem (GM), which corresponds to the SAM. We also assessed the expression pattern of ANGUSTIFOLIA3 (AN3), a key promoter for cell division in the leaf meristem. It was expressed not only in the basal meristem (BM) tissue with active cell division in the basal part of the growing cotyledon but also in the GM. The findings suggest that the unusual gene expression pattern of the GM underpins the fuzzy morphogenesis of Monophyllaea.

One-leaf plants in the Gesneriaceae: Natural mutants of the typical shoot system.

The aerial part of seed plants is called the shoot, which is composed of stems, leaves, and axial buds. These are produced by indeterminate activity in the shoot apical meristem (SAM), whereas the morphogenesis of leaves depends on determinate activity of leaf meristems. However, one-leaf plants in the Gesneriaceae family (eudicots) do not have a typical SAM and do not produce new organs when in the vegetative phase. Instead, they have one cotyledon whose growth is indeterminate. This peculiar development is supported by the groove meristem, which corresponds to the canonical SAM, and the basal meristem, which corresponds to the typical leaf meristem. However, the former does not produce any organ and the latter is active indeterminately. Gene expression and physiological analyses have been conducted in an effort to determine the molecular nature of this peculiar organogenesis. This review summarizes the current understanding of the development of one-leaf plants to provide future perspectives in this field of research.

Up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver.

Although the healthy liver is known to have high regenerative potential, poor liver regeneration under pathological conditions remains a substantial problem. We investigated the key molecules that impair the regeneration of cholestatic liver. C57BL/6 mice were randomly subjected to partial hepatectomy and bile duct ligation (PH+BDL group, n = 16), partial hepatectomy only (PH group, n = 16), or sham operation (Sham group, n = 16). The liver sizes and histological findings were similar in the PH and sham groups 14 days after operation. However, compared with those in the sham group, the livers in mice in the PH+BDL group had a smaller size, a lower cell proliferative activity, and more fibrotic tissue 14 days after the operation, suggesting the insufficient regeneration of the cholestatic liver. Pathway-focused array analysis showed that many genes were up- or down-regulated over 1.5-fold in both PH+BDL and PH groups at 1, 3, 7, and 14 days after treatment. Interestingly, more genes that were functionally related to the extracellular matrix and inflammatory chemokines were found in the PH+BDL group than in the PH group at 7 and 14 days after treatment. Our data suggest that up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver.

Does the development of chronic kidney disease and acute kidney injury affect the prognosis after living donor liver transplantation?

BACKGROUND AND AIMS: Chronic kidney disease (CKD) and acute kidney injury (AKI) have been discussed as complications following living donor liver transplantation (LDLT). The aim of this study was to clarify the relationships among CKD, AKI, and the prognosis after LDLT. METHODS: This study included 118 patients who underwent LDLT in our department. A low eGFR (<60 mL/min/1.73 m(2) ) was regarded to indicate CKD. AKI 1 and AKI 2 were characterized by an increase in the serum creatinine level of 0.5 and 1.0 mg/dL, respectively, within one wk after LDLT. We investigated the risk factors for and the relevance of CKD and AKI on the prognosis. RESULTS: AKI 1 was associated with sepsis and intra-operative bleeding (p = 0.0032, p = 0.001). AKI 2 was associated with sepsis and hepatitis C infection (p < 0.001, p = 0.027). A pre-operative eGFR of 60-89 and diabetes were the risk factors for the development of CKD in POY 2 (p = 0.018, p = 0.002). AKI 2, sepsis, and diabetes were the risk factors for the patient death within one yr after LDLT (p = 0.010, p = 0.002, p = 0.022). AKI 2 and sepsis were the risk factors for death within two yr after LDLT (p = 0.005, p = 0.018). CONCLUSIONS: Recognizing the risk factors and careful management for preventing both AKI and CKD may improve the prognosis of patients following LDLT.

Relationship between immune function recovery and infectious complications in patients following living donor liver transplantation.

AIM: The ImmuKnow (IK) assay enables the evaluation of peripheral blood CD4(+) adenosine triphosphate activity to facilitate an objective assessment of the cellular immune function in immunosuppressed patients. However, it is unclear whether the IK assay is utilized during the acute postoperative periods following living donor liver transplantation (LDLT). METHODS: The IK values of 43 LDLT recipients were measured during the month following LDLT to evaluate the relationship between the measured IK values and infectious events. RESULTS: The IK values after LDLT were significantly increased compared with the IK values before LDLT ( P < 0.01). During the month following transplantation, the rate of bacterial infection in the recipients with IK values of more than 225 ng/mL was significantly lower than that in the recipients with IK values of 225 ng/mL or less ( 42.1% vs 91.7%, respectively; P < 0.01). The rate of severe infections among the recipients who maintained IK values of more than 150 ng/mL was significantly lower than that among the recipients with IK values of 150 ng/mL or less during the month following transplantation ( 3.7% vs 56.3%, respectively; P < 0.01). CONCLUSION: The immune system of LDLT recipients dramatically improved following transplantation. The IK values of LDLT recipients were associated with the incidence of infectious events during the perioperative period after LDLT. Monitoring IK values was useful during both the acute and long-term postoperative periods.

Efficiency of herbal medicine Dai-kenchu-to on portal blood flow in rat models.

INTRODUCTION: To clarify the influence of Dai-Kenchu-To (DKT) on portal blood flow (PBF), PBF was continuously measured with Doppler ultrasound. METHODS: Normal liver rats were divided into a DKT 90 mg/kg, DKT 270 mg/kg administered group, and control, while cirrhotic liver rats were divided into a DKT-LC 90 mg/kg administered group and Control-LC. The PBF was measured after the administration of either DKT or water for 60 min by laser Doppler flowmetry system. RESULTS: The PBF in the DKT 90 increased approximately 10 min after DKT was administrated, and elevated levels were maintained for approximately 10 min. A comparison of the increase in PBF by the calculating the area under the curve (AUC) revealed that flow was significantly higher in the DKT 90 compared to either the control or the DKT 270 (p < 0.05). The cirrhotic liver group showed stable PBF in both the DKT-LC and Control-LC. The AUC, revealed no significant difference between the DKT-LC and Control-LC. DISCUSSION: DKT induced an increase in PBF in normal livers; however, its effects were insufficient to increase PBF in the cirrhotic livers. No increase in the portal blood flow in the cirrhotic liver rats was probably the result of the cirrhotic liver, which had fibrotic change, and, therefore, may not have had sufficient compliance to accept the increasing blood flow volume from the intestinal tract. CONCLUSION: We suggested DKT has the potential to protect the liver by increasing PBF when the liver has either normal or mild to moderate dysfunction.

Technical refinement of hepatic vein reconstruction in living donor liver transplantation using left liver graft.

BACKGROUND: In adult living donor liver transplantation (LDLT), left liver graft is generally safer for the donor. The aim of this study was to demonstrate a technical refinement for achieving sufficient outflow using left liver graft. MATERIAL AND METHODS: Forty-seven cases using left liver were divided into 2 groups according to the procedures of hepatic vein reconstruction: the side-clamp group (21 cases), and the cross-clamp group (26 cases), to sufficiently enlarge the diameter of the hepatic vein with excising the inferior vena cava (IVC). RESULTS: The liver function tests at 7 days after LDLT were not significantly different between the 2 groups, but the median amount of ascites was significantly greater in the side-clamp group (1250 ml; range, 484-3690) than in the cross-clamp group (582 ml; 190-2785). When we selected the patients with the ratio of graft weight to recipient standard liver volume less than 30%, the 1-year patient survival after transplantation was significantly better in the cross-clamp group than in the side-clamp group (90% in cross-clamp group vs. 71% in side-clamp group, P<0.05). CONCLUSIONS: In conclusion, hepatic vein reconstruction with cross-clamping of the IVC can secure a sufficient outflow in LDLT using left liver graft.

A correlation between the graft volume evaluation and the prognosis in consideration of hepatic "compliance" in living donor liver transplantation.

BACKGROUND/AIMS: The aim is to clarify the correlation between the hepatic compliance and prognosis of the patients who underwent LDLT. METHODOLOGY: Volumetry was performed using a 3D volume analyzer. The hepatic compliance was evaluated based on the difference between the estimated total liver volume in the arterial phase and venous phase (n=66). The correlations among the hepatic compliance, donor background factors and the recipient's prognosis were evaluated. RESULTS: Fourteen cases (21%) presented with a more than 5% increase in volume during the venous phase than in the arterial phase, and 12 of these recipients were still alive. There was a significant increase in death among recipients from donors age 60 years old and older (5/7, 71.4%, p<0.01). In these cases, the hepatic compliance was significantly different between the deceased cases and the surviving cases; while there was no hepatic compliance in the five deceased cases, favorable hepatic compliance was observed in two of the surviving cases (p<0.01). CONCLUSIONS: Hepatic compliance appears to exist. The poor prognosis of liver grafts in recipients from elderly donors may be attributable to hepatic compliance, and assessing the hepatic compliance may be useful for preoperative liver graft evaluation.

Peritoneal recurrence of initially controlled hepatocellular carcinoma after living donor liver transplantation.

It is well known that the presence of end-stage liver disease increases the risk of developing hepatocellular carcinoma (HCC). Liver transplantation (LT) for patients within the Milan criteria has become a standard treatment for HCC in most developed centers worldwide. However, a major cause of death in cirrhotic patients with HCC after transplantation is tumor recurrence, including peritoneal recurrences, which develops rarely but presents a significant problem with regard to their management. Our experience includes two cases with HCC within the Milan criteria of peritoneal recurrences after living donor LT. Both patients had interventions for HCC in their medical history before LT, and we propose that these might have been a possible cause of the HCC peritoneal recurrence.

Prospective study of the safety and efficacy of intermittent inflow occlusion (Pringle maneuver) in living donor left hepatectomy.

AIM: The impact of intermittent inflow occlusion (Pringle maneuver) in living donor hepatectomy on the outcome of both the donor and the recipient is unknown. The aim of this study is to elucidate the safety and efficacy of Pringle maneuver in living donor hepatectomy. METHODS: Twenty consecutive cases of living donors who underwent left hepatectomy were prospectively divided into two groups, with (Group A, n = 10) or without (Group B, n = 10) the Pringle maneuver during hepatectomy. Intraoperative blood loss, postoperative liver functions in the donors and recipient outcome were reviewed. RESULTS: Median blood loss was significantly less in group A than in group B. Median alanine aminotransferase was significantly higher on postoperative day 1 in group A than in group B, but the difference was not significant at 7 days after surgery. Eight of 10 recipients in each group survived with good graft function with a median follow-up period of 20 months in group A and 19 months in group B. CONCLUSION: The Pringle maneuver was safely applied in living donor hepatectomy, but the only benefit was the reduction of blood loss during the donor surgery, and no positive impact on the recipient outcome.

The impact of treated bacterial infections within one month before living donor liver transplantation in adults.

BACKGROUND: The impact of treated preoperative bacterial infections on the outcome of living-donor liver transplantation (LDLT) is not well defined. The aim of this study was to determine the frequency of pre-transplant bacterial infections within one month before LDLT and their impact on the post-transplant morbidity and mortality. MATERIAL AND METHODS: We retrospectively reviewed the records of 50 adult LDLT recipients between January 2009 and October 2011. Patients were divided into two groups based on whether they had episodes of bacterial infections within one month before LDLT. RESULTS: There were 20 patients who required antimicrobial therapy for pre-transplant infections. The pre-transplant infections comprised urinary tract infections (35%), cholangitis (10%), pneumonia (10%), bacteremia (5%), spontaneous bacterial peritonitis (5%), acute sinusitis (5%), subcutaneous abscess (5%), and empirical treatment (25%). Patients with pre-transplant infections had higher Child-Pugh scores [median, 11 vs. 9.5, P<0.05] and model for end-stage liver disease scores [median, 17.5 vs. 14, P<0.05] compared with the other patients. There were no correlations between the pathogens involved in the pre-transplant infections and those involved in post-transplant infections. The incidence of post-transplant infections was higher in the pre-transplant infection group within one week after LDLT, but was almost the same within one month after LDLT. The one-year survival rates were not significantly different between the groups. CONCLUSIONS: Although pre-transplant infections are associated with a high risk of postoperative bacterial infection shortly after LDLT, they did not affect the short-term outcome when they had been appropriately treated before transplantation.

Post-operative complications requiring hospitalization more than one yr after living donor liver transplantation.

BACKGROUND/PURPOSE: The long-term outcomes after living donor liver transplantation (LDLT) have not been clearly established. This retrospective study assessed long-term outcomes after LDLT through reviewing complications requiring hospitalization more than one yr after engraftment. METHODS: Sixty-five LDLT recipients alive more than one yr post-transplantation were enrolled, 37 males and 28 females, with a median age at transplantation of 53 yr (range, 0-68 yr). We reviewed all post-operative complications requiring hospitalization more than one yr after LDLT. RESULTS: There were 61 post-operative complications requiring hospitalization in 43 of the 65 patients (66%), and the majority of these complications were transplantation related (59/61; 97%). Despite this, 43 (78%) of 55 surviving patients had normal liver function at their last follow-up, and 50 patients (91%) achieved normal activity (Karnofsky score 100%). CONCLUSIONS: More than one-half of our LDLT recipients required hospitalization more than one yr post-LDLT to treat a complication. Most were able to maintain their quality of life and liver function with appropriate treatment.

A hybrid method of laparoscopic-assisted open liver resection through a short upper midline laparotomy can be applied for all types of hepatectomies.

BACKGROUND: Although hepatectomy procedures should be designed to provide both curability and safety, minimal invasiveness also should be pursued. METHODS: We analyzed the data related to our method for laparoscopy-assisted open resections (hybrid method) through a short upper midline incision for various types of hepatectomies. Of 215 hepatectomies performed at Nagasaki University Hospital between November 2009 and June 2012, 102 hepatectomies were performed using hybrid methods. RESULTS: A hybrid method was applicable for right trisectionectomy in 1, right hemihepatectomy in 32, left hemihepatectomy in 29, right posterior sectionectomy in 7, right anterior sectionectomy in 1, left lateral sectionectomy in 2, and segmentectomy in 7 patients, and for a minor liver resection in 35 patients (12 combined resections). The median duration of surgery was 366.5 min (range 149-709) min, and the median duration of the laparoscopic procedure was 32 min (range 18-77) min. The median blood loss was 645 g (range 50-5,370) g. Twelve patients (12 %) developed postoperative complications, including bile leakage in three patients, wound infections in two patients, ileus in two patients, and portal venous thrombus, persistent hyperbilirubinemia, incisional hernia, local liver infarction each in one patient. There were no perioperative deaths. CONCLUSIONS: Our method of hybrid hepatectomy through a short upper midline incision is considered to be applicable for all types of hepatectomy and is a reasonable approach with no abdominal muscle disruption, which provides safe management of the hepatic vein and parenchymal resection even for patients with bilobular disease.

Self-assessment of postoperative scars in living liver donors.

BACKGROUND: The application of less invasive techniques for liver surgery in patients undergoing living donor hepatectomy (LDH) has been reported. The objective of this study was to evaluate physical status according to type of incision in donors. METHODS: One hundred and forty-seven living liver donors underwent hepatectomy using three types of incisions: (i) Mercedes-Benz incision (M.B.), (ii) right subcostal incision with midline up to xiphoid incision (S.C.), and (iii) short upper midline incision (U.M.). A total of 100 donors answered the questionnaires, and 87 had sufficient data for the analyses. An original questionnaire designed to evaluate the physical status concerning postoperative scars. The questionnaire consisted of three major categories: appearance, sensation, and daily activities. The univariate analysis was performed using the chi-square test. RESULTS: Numbness of the abdominal wall was reported more frequently by the donor with M.B.s and right subcostal incisions up to xiphoid incisions. In terms of appearance, sensation, and daily activities, LDH with a U.M. was found to have a good self-assessment compared with that performed using other types of incisions. CONCLUSIONS: LDH with a U.M. is a preferable procedure in terms of physical status and safety.

Existence of microscopic residual viable lesions in patients with colorectal liver metastases after chemotherapy.

BACKGROUND/AIMS: Hepatic metastases from the colorectal carcinoma frequently recur after resection and microscopic residual cancer lesions (RCL) could be important in the development of recurrence. The aim of our study was to investigate an existence of microscopic RCL after modern chemotherapy and its correlation with the preoperative CT imaging. METHODOLOGY: Thirteen patients who underwent liver resection for colorectal liver metastases (CRM) after preoperative chemotherapy were studied. Resected hepatic specimens were stained with an antibody against carcinoembryonic antigen to detect microscopic RCL, which were defined as discrete microscopic cancerous lesions surrounding the dominant metastases. Also, we evaluated the correlation between the outline of CRM based on CT finding and the detection of microscopic RCLs around CRM. RESULTS: RCL were found immunohistochemically in 10 patients (77.8%). There are one patient whose RCL was most distant from the main tumor more than 10 mm. The presence of microscopic RCL was associated with the irregular outline of CRM (2/13, 15.4%) compared with regular CRM (11/13, 84.6%). CONCLUSIONS: To plan hepatectomy for CRM after chemotherapy, the possible existence of microscopic RCL should be taken into account. In addition, irregular shape tumor on CT should have wider surgical margin than 10 mm.

Evaluation of immune function under conversion from Prograf to Advagraf in living donor liver transplantation.

BACKGROUND: Although some reports have shown the safety and efficacy of conversion from Prograf to Advagraf in liver transplantation, there have been no reports showing the change of immune function after conversion. The aim of this study is not only to analyze the safety and efficacy of conversion from Prograf to Advagraf, but also to evaluate the immune function using the ImmuKnow assay. MATERIAL AND METHODS: Of the 168 living donor liver transplantation (LDLT) patients, 21 recipients whose liver function was stable after discharge in outpatient clinic and who agreed to conversion from Prograf to Advagraf were enrolled in this study. Liver, renal, and immune functions were retrospectively reviewed. RESULTS: There were no significant differences in liver and renal function after conversion from Prograf to Advagraf. The intracellular adenosine triphosphate levels before and after conversion were 263±157 and 256±133 ng/ml, respectively, and there was also no significant difference in immune function. None of the recipients showed adverse effects, rejection, or severe infection during the study. It should be further noted that none of the recipients had to increase the dose of Advagraf, while five of 21 recipients (24%) were able to reduce the dose of Advagraf during this study. CONCLUSIONS: Conversion from Prograf to Advagraf in LDLT can be performed safely and effectively without affecting liver, renal, and immune function.