Comprehensive characterization and resolution of discrepant spectrophotometric bilirubin results in patients on eltrombopag therapy.

Background Eltrombopag is a thrombopoietin receptor agonist used for the treatment of thrombocytopenic conditions. It can cause pH-dependent discoloration of plasma/serum. Eltrombopag is potentially hepatotoxic. It can affect the assessment of hyperbilirubinemia because of its (i) absorbance at ~450 nm (bilirubin), (ii) absorbance at ~550 nm (diazo-bilirubin) and (iii) it can cause yellowish discoloration of the eyes at normal circulating bilirubin levels. Methods We collected 66 samples from patients on a range of eltrombopag dosages up to 150 mg daily. Bilirubin was measured using multiple routine spectrophotometric analyzers, the Doumas reference method and high-performance liquid chromatography (HPLC). Plasma/serum eltrombopag concentrations were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS). Spike-in and admixture experiments delineated the effects of eltrombopag and its metabolites. Results Forty-nine of 52 samples from patients on ≥50 mg daily eltrombopag therapy showed significantly discrepant inter-analyzer total bilirubin results, a difference up to 64 µmol/L (3.7 mg/dL). In one sample, total bilirubin varied from 8 to 65 µmol/L (0.4-3.8 mg/dL) by different routine analyzers, with direct bilirubin ≤4 µmol/L (0.2 mg/dL). There was a positive correlation between total bilirubin difference and plasma eltrombopag concentration (r = 0.679), and spike-in experiments demonstrated that Beckman AU and Doumas reference methods were susceptible to positive interference. HPLC can quantify bilirubin after separating eltrombopag, and results suggest different analyzers are affected to varying degrees by eltrombopag and its metabolites. Conclusions Eltrombopag and its metabolites can cause positive interference to the spectrophotometric measurements of total bilirubin. Accurate measurements of total bilirubin may improve our understanding of the prevalence of hyperbilirubinemia in patients on eltrombopag therapy.

Developmental changes in urinary coproporphyrin ratio in premature infants.

BACKGROUND: Premature infants have a high concentration of conjugated bilirubin in their blood, although they have a poor glucuronide conjugation of bilirubin. This may be due to developmental changes in the function of adenosine triphosphate binding cassette subfamily C member 2, which is involved in the cellular export of conjugated bilirubin. In the present study, we examined the developmental changes in the urinary coproporphyrin I/(urinary coproporphyrin I+ urinary coproporphyrin III) ratio (UCP (I/ [I + III])), a known biomarker for adenosine triphosphate binding cassette subfamily C member 2 function, in premature infants. METHOD: Twenty-one premature infants born between 25 and 32 weeks of gestation were included in the study. Urine samples were collected within 24 h of birth, and at 1 week and 3-4 weeks after birth. The samples were analyzed by high-performance liquid chromatography to calculate UCP (I/ [I + III]) to examine its association with postnatal age and corrected gestational age. Subjects were excluded if they had liver dysfunction, cholestasis, urinary tract infection, or chromosomal abnormalities. RESULTS: The average UCP (I/ [I + III]) within 24 h of birth, at 1 week, and at 3-4 weeks after birth was 0.84, 0.61, and 0.65, respectively. The UCP (I/ [I + III]) within 24 h of birth was significantly higher than that measured at 1 week or 3-4 weeks after birth. There was no significant correlation between UCP (I/ [I + III]) and the corrected gestational age. CONCLUSION: The UCP (I/ [I + III]) was higher within 24 h of birth. It decreased 1 week after birth and remained low without any significant changes for up to 4 weeks after birth.

JMJD2B/KDM4B inactivation in adipose tissues accelerates obesity and systemic metabolic abnormalities.

Obesity is a serious global health issue; however, the roles of genetics and epigenetics in the onset and progression of obesity are still not completely understood. The aim of this study was to determine the role of Kdm4b, which belongs to a subfamily of histone demethylases, in adipogenesis and fat metabolism in vivo. We established conditional Kdm4b knockout mice. Inactivation of Kdm4b in adipocytes (K4bKO) induced profound obesity in mice on a high fat diet (HFD). The HFD-fed K4bKO mice exhibited an increased volume of fat mass and higher expression levels of adipogenesis-related genes. In contrast, the genes involved in energy expenditure and mitochondrial functions were down-regulated. Supporting these findings, the energy expenditure of Kdm4b-deficient cells was markedly decreased. In addition, progression of glucose intolerance and hepatic steatosis with hepatocellular damages was observed. These data indicate that Kdm4b is a critical regulator of systemic metabolism via enhancing energy expenditure in adipocytes.

Green light-emitting diode phototherapy for neonatal hyperbilirubinemia: Randomized controlled trial.

BACKGROUND: The main photochemical pathway in phototherapy for neonatal hyperbilirubinemia is the production and elimination (in bile or urine) of cyclobilirubin, which is a structural photoisomer of bilirubin, and which is most efficiently produced by green light. Green light-emitting diode (LED) phototherapy, however, has not been evaluated in the clinical setting because it is not recommended in American Academy of Pediatrics guidelines. We therefore compared the efficacy of green LED phototherapy and blue LED phototherapy in patients with neonatal hyperbilirubinemia. METHODS: In this prospective randomized controlled trial, neonates with hyperbilirubinemia were randomly allocated to a green LED or blue LED phototherapy group. Both groups underwent 24 h of phototherapy, and blood was sampled before and after 24 h of phototherapy. Total serum bilirubin (TSB) was measured using enzymatic methods and bilirubin photoisomers were measured on high-performance liquid chromatography. RESULTS: Thirty-four infants were randomized (green, n = 16; blue, n = 18). TSB decreased significantly from 15.3 ± 1.5 to 13.9 ± 1.5 mg/dL in the green LED group (P < 0.01) and from 16.2 ± 1.3 to 14.5 ± 1.7 mg/dL in the blue LED group (P < 0.01) after 24 h of phototherapy. No significant difference was found in TSB reduction after phototherapy between the groups. CONCLUSIONS: Both light sources produced a significant reduction in TSB, indicating clinical effectiveness.

Identification of a novel fusion gene HMGA2-EGFR in glioblastoma.

Glioblastoma is one of the most malignant forms of cancer, for which no effective targeted therapy has been found. Although The Cancer Genome Atlas has provided a list of fusion genes in glioblastoma, their role in progression of glioblastoma remains largely unknown. To search for novel fusion genes, we obtained RNA-seq data from TGS-01 human glioma-initiating cells, and identified a novel fusion gene (HMGA2-EGFR), encoding a protein comprising the N-terminal region of the high-mobility group AT-hook protein 2 (HMGA2) fused to the C-terminal region of epidermal growth factor receptor (EGFR), which retained the transmembrane and kinase domains of the EGFR. This fusion gene product showed transforming potential and a high tumor-forming capacity in cell culture and in vivo. Mechanistically, HMGA2-EGFR constitutively induced a higher level of phosphorylated STAT5B than EGFRvIII, an in-frame exon deletion product of the EGFR gene that is commonly found in primary glioblastoma. Forced expression of HMGA2-EGFR enhanced orthotopic tumor formation of the U87MG human glioma cell line. Furthermore, the EGFR kinase inhibitor erlotinib blocked sphere formation of TGS-01 cells in culture and inhibited tumor formation in vivo. These findings suggest that, in addition to gene amplification and in-frame exon deletion, EGFR signaling can also be activated by gene fusion, suggesting a possible avenue for treatment of glioblastoma.

Phototherapy for neonatal hyperbilirubinemia.

Approximately 60 years ago in England, phototherapy for neonatal hyperbilirubinemia was used in clinical practice. It was introduced in Japan approximately 50 years ago. At that time, the mechanism underlying the serum bilirubin concentration decrease by phototherapy was still unknown. The mechanism was identified by chemists, biochemists, and pediatricians. Clarification started with the report that unconjugated bilirubin was excreted into bile after photoirradiation in Gunn rats. After confirmation of the molecular structure of bilirubin on X-ray analysis, the mechanism for bile excretion of unconjugated bilirubin was verified based on geometric configurational photoisomers in the Gunn rat. Finally, the reaction and excretion of structural bilirubin photoisomers was proved to be the main mechanism for the decrease in serum bilirubin during phototherapy for neonatal hyperbilirubinemia, which differs from the mechanism in the Gunn rat. The most effective and safest light source and the optimal method to evaluate phototherapy, however, remain unknown. Moreover, as for bronze baby syndrome, which is a well-known adverse reaction to phototherapy, the etiology is unclear. Hence, we review phototherapy for hyperbilirubinemia including a fundamental understanding of the bilirubin photochemical reactions, and discuss the subclinical carcinogenic risk of phototherapy and the increased mortality rate of extremely low-birthweight infants due to aggressive phototherapy, which is becoming an increasing problem.

Developmental characteristics of urinary coproporphyrin I/(I + III) ratio.

BACKGROUND: The ratio of urinary coproporphyrin (UCP) I to total urinary coproporphyrin I and III [UCP {I/(I + III)]] serves as a biomarker of the ATP-binding cassette, sub-family C, member 2 (ABCC2) function. The aim of this study was to clarify the characteristics of the developmental pattern of UCP [I/(I + III)] in order to estimate ABCC2 function in children, especially in the neonatal period, by measuring it throughout the entirety of childhood. METHOD: Measurement of UCP [I/(I + III)] was done high-performance liquid chromatography, using urine samples collected from children from 1 day to 15 years old, involving one sample per child. Urine samples from children with liver and kidney disease and urinary tract infection were excluded. RESULTS: UCP [I/(I + III)] varied widely in infants younger than 6 months old, and was ≥0.3 in 80% of the infants. In contrast, it decreased to <0.30, the lowest, at 1-2 years old. In the 0-6-month-old group, no significant correlation was noted between postnatal age and UCP [I/(I + III)], but a moderate inverse correlation was noted between corrected gestational age and UCP [I/(I + III)]. CONCLUSION: UCP [I/(I + III)] is inversely correlated with corrected gestational age and is lowest at 1-2 years old. This suggests that ABCC2 activity is correlated with corrected gestational age and is highest at 1-2 years old.

Evans syndrome after autologous peripheral blood stem cell transplantation for recurrent Hodgkin lymphoma.

There have been a number of recent reports on the occurrence of autoimmune conditions after autologous hematopoietic stem cell transplantation. We describe a rare case of Evans syndrome (ES) that developed in a 16-year-old patient >1 year after autologous peripheral blood stem cell transplantation for recurrent Hodgkin lymphoma. ES is a rare and frequently refractory condition. No therapy for the condition has been established, and it can often be fatal. In the present case, i.v. cyclosporine A injection was significantly effective against the ES, which has not recurred.

Is yolk sac tumor related to the pathophysiology of low birthweight?

An 8-year-old Japanese girl was admitted with an ovarian yolk sac tumor. Regarding birth history, the patient had been delivered by cesarean section at 25 weeks of gestation with a birthweight of 711g. She had required neonatal intensive care including oxygenation, various medications, and tests. After surgery and chemotherapy, there was no recurrence for 2 years, at the time of writing. Yolk sac tumor, which is a malignant germ cell tumor, is rare in children. Although the cause and risk factors are unclear, it has been reported that malignant germ cell tumors in childhood have been associated with pathophysiology at birth. Given that premature infants are more likely to survive due to advances in perinatal care, it is expected that such cases will increase in the near future. We suggest that children born prematurely require careful follow up.

Structures of KIX domain of CBP in complex with two FOXO3a transactivation domains reveal promiscuity and plasticity in coactivator recruitment.

Forkhead box class O 3a (FOXO3a) is a transcription factor and tumor suppressor linked to longevity that determines cell fate through activating transcription of cell differentiation, survival, and apoptotic genes. Recruitment of the coactivator CBP/p300 is a crucial step in transcription, and we revealed that in addition to conserved region 3 (CR3) of FOXO3a, the C-terminal segment of CR2 (CR2C) binds CBP/p300 and contributes to transcriptional activity. CR2C and CR3 of FOXO3a interact with the KIX domain of CBP/p300 at both "MLL" and "c-Myb" binding sites simultaneously. A FOXO3a CR2C-CR3 peptide in complex with KIX exists in equilibrium between two equally populated conformational states, one of which has CR2C bound to the MLL site and CR3 bound to the c-Myb site, whereas in the other, CR2C and CR3 bind the c-Myb and MLL sites, respectively. This promiscuous interaction between FOXO3a and CBP/p300 is further supported by additional binding sites on CBP/p300, namely, the TAZ1 and TAZ2 domains. In functional studies, our structure-guided mutagenesis showed that both CR2C and CR3 are involved in the activation of certain endogenous FOXO3a target genes. Further, phosphorylation of S626, a known AMP-dependent protein kinase target in CR3, increased affinity for CBP/p300 and the phosphomimetic mutation enhanced transactivation of luciferase. These findings underscore the significance of promiscuous multivalent interactions and posttranslational modification in the recruitment of transcriptional coactivators, which may allow transcription factors to adapt to various gene-specific genomic and chromatin structures and respond to cell signals.

Current incidence of clinical kernicterus in preterm infants in Japan.

Clinical kernicterus in preterm infants has recently been reported in Japan, diagnosed on the basis of clinical findings during the neonatal and infancy periods. We investigated the incidence of clinical kernicterus in preterm infants <30 weeks gestational age (GA) based on a nationwide survey conducted in 233 certified educational facilities for neonatologists. The numbers of infants admitted and infants who died within 14 days after birth during 2011, and the number of infants who subsequently developed clinical kernicterus, were recorded. A total of 2720 infants were analyzed, representing 59% (2720/4623) of all preterm live births <30 weeks GA in Japan in 2011. Of these, 159 (5.8%) died within 14 days after birth, similar to the national rate. Five infants developed clinical kernicterus in infancy (5/2720, 0.18%). The current incidence of clinical kernicterus in Japan is therefore estimated at 1.8 per 1000 live births <30 weeks GA.

Neonatal Dubin-Johnson syndrome: novel compound heterozygous mutation in the ABCC2 gene.

Dubin-Johnson syndrome (DJS) is an autosomal recessive inherited disorder characterized by conjugated hyperbilirubinemia. Neonatal-onset DJS is rare. It is caused by dysfunction of adenosine triphosphate-binding cassette, sub-family C, member 2 (ABCC2). We found a novel compound heterozygous mutation of DJS-related gene: W709R (T2145C): a missense mutation in exon 17, and R768W (C2302T), a missense mutation in exon 18. Serum diglucuronosyl bilirubin/monoglucuronosyl bilirubin ratio was high. ABCC2 may excrete diglucuronosyl bilirubin preferentially over monoglucuronosyl bilirubin.

Deoxycytidine kinase inactivation enhances gemcitabine resistance and sensitizes mitochondrial metabolism interference in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most lethal forms of cancer. Although in the last decade, an increase in 5-year patient survival has been observed, the mortality rate remains high. As a first-line treatment for PDAC, gemcitabine alone or in combination (gemcitabine plus paclitaxel) has been used; however, drug resistance to this regimen is a growing issue. In our previous study, we reported MYC/glutamine dependency as a therapeutic target in gemcitabine-resistant PDAC secondary to deoxycytidine kinase (DCK) inactivation. Moreover, enrichment of oxidative phosphorylation (OXPHOS)-associated genes was a common property shared by PDAC cell lines, and patient clinical samples coupled with low DCK expression was also demonstrated, which implicates DCK in cancer metabolism. In this article, we reveal that the expression of most genes encoding mitochondrial complexes is remarkably upregulated in PDAC patients with low DCK expression. The DCK-knockout (DCK KO) CFPAC-1 PDAC cell line model reiterated this observation. Particularly, OXPHOS was functionally enhanced in DCK KO cells as shown by a higher oxygen consumption rate and mitochondrial ATP production. Electron microscopic observations revealed abnormal mitochondrial morphology in DCK KO cells. Furthermore, DCK inactivation exhibited reactive oxygen species (ROS) reduction accompanied with ROS-scavenging gene activation, such as SOD1 and SOD2. SOD2 inhibition in DCK KO cells clearly induced cell growth suppression. In combination with increased anti-apoptotic gene BCL2 expression in DCK KO cells, we finally reveal that venetoclax and a mitochondrial complex I inhibitor are therapeutically efficacious for DCK-inactivated CFPAC-1 cells in in vitro and xenograft models. Hence, our work provides insight into inhibition of mitochondrial metabolism as a novel therapeutic approach to overcome DCK inactivation-mediated gemcitabine resistance in PDAC patient treatment.

Caffeine Concentrations in Human Milk Donated to a Human Milk Bank in Japan.

BACKGROUND: Human milk banks have been established to provide human milk to preterm infants who are unable to obtain milk from their mothers. Donor screening methods vary, and prospective donors are commonly screened for drug and recreational substance use through behavioral screening. Although the risk of illegal drug consumption in Japan is extremely low, caffeine may be consumed unknowingly and can be found in human milk. To date, only a few reports have been conducted on the concentration of caffeine in donor milk. RESEARCH AIM: This study aimed to examine the pre-pasteurization levels of caffeine in human milk donated to a milk bank in Japan. METHODS: This was a cross-sectional, observational study of caffeine concentrations in human milk donated to a human milk bank in Japan. Caffeine concentration in the donor milk was measured using high-performance liquid chromatography. RESULTS: Caffeine was detected in 70% of the donor milk samples (N = 350). The median (range) of caffeine concentration was 0.46 [< 0.10, 7.54] mg/L, and 64.0% of the samples had less than 1 mg/L of caffeine. The caffeine concentration varied widely among as well as within individuals. CONCLUSION: The average caffeine concentration in Japanese donor milk samples was higher than that previously reported in samples from Spain, but the range was similar. Donors should be informed that caffeine intake should be within a moderate range, to further increase the safety of donor milk.

Correction: Histone Demethylase JMJD2B Functions as a Co-Factor of Estrogen Receptor in Breast Cancer Proliferation and Mammary Gland Development.

[This corrects the article DOI: 10.1371/journal.pone.0017830.].

ACA-28, an ERK MAPK Signaling Modulator, Exerts Anticancer Activity through ROS Induction in Melanoma and Pancreatic Cancer Cells.

The extracellular signal-regulated kinase (ERK) MAPK pathway is dysregulated in various human cancers and is considered an attractive therapeutic target for cancer. Therefore, several inhibitors of this pathway are being developed, and some are already used in the clinic. We have previously identified an anticancer compound, ACA-28, with a unique property to preferentially induce ERK-dependent apoptosis in melanoma cells. To comprehensively understand the biological cellular impact induced by ACA-28, we performed a global gene expression analysis of human melanoma SK-MEL-28 cells exposed to ACA-28 using a DNA microarray. The transcriptome analysis identified nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcription factor that combats oxidative stress, as the most upregulated genetic pathway after ACA-28 treatment. Consistently, ACA-28 showed properties to increase the levels of reactive oxygen species (ROS) as well as Nrf2 protein, which is normally repressed by proteasomal degradation and activated in response to oxidative stresses. Furthermore, the ROS scavenger N-acetyl cysteine significantly attenuated the anticancer activity of ACA-28. Thus, ACA-28 activates Nrf2 signaling and exerts anticancer activity partly via its ROS-stimulating property. Interestingly, human A549 cancer cells with constitutively high levels of Nrf2 protein showed resistance to ACA-28, as compared with SK-MEL-28. Transient overexpression of Nrf2 also increased the resistance of cells to ACA-28, while knockdown of Nrf2 exerted the opposite effect. Thus, upregulation of Nrf2 signaling protects cancer cells from ACA-28-mediated cell death. Notably, the Nrf2 inhibitor ML385 substantially enhanced the cell death-inducing property of ACA-28 in pancreatic cancer cells, T3M4 and PANC-1. Our data suggest that Nrf2 plays a key role in determining cancer cell susceptibility to ACA-28 and provides a novel strategy for cancer therapy to combine the Nrf2 inhibitor and ACA-28.

The effect of blood transfusion on cerebral hemodynamics in preterm infants.

BACKGROUND: Anemia of prematurity commonly occurs in infants with very low birth weight; blood transfusion is an important treatment. However, there is no clear evidence to support the criteria currently widely used, based on blood hemoglobin (bHb) and hematocrit indices. Previous studies showed that overtransfusion or a low threshold for transfusion could induce complications or neurologic sequelae, respectively. We hypothesized that a cerebral hemodynamic index may provide an appropriate criterion for determining the need for transfusion in anemic preterm infants. STUDY DESIGN AND METHODS: We used near-infrared time-resolved spectroscopy to measure cerebral hemoglobin oxygen saturation (ScO2 ) and cerebral blood volume (CBV) before and after transfusion in 19 infants (24 measurements) with anemia of prematurity. The median gestational age was 27 weeks 0 days, median birth weight was 751 g, and median postconceptual age at transfusion was 30 weeks 4 days. RESULTS: bHb levels before and after transfusion (mean ± SD) were 9.3 ± 1.4 and 13.7 ± 1.3 g/dL, respectively. After transfusion, CBV significantly decreased from 2.63 ± 0.60 to 2.13 ± 0.26 mL/100 g of brain, and ScO2 significantly increased from 72.8 ± 4.3% to 74.7 ± 4.2%. CONCLUSION: After transfusion, CBV changes were significantly greater with low compared to high pretransfusion Hb levels. This reflected the physiologic response to severe anemia in premature infants, which is to increase CBV and decrease ScO2 . Therefore, CBV and ScO2 may be useful markers for determining the need for transfusion in very-low-birth-weight infants.