Crystal structures of multidrug efflux transporters from Burkholderia pseudomallei suggest details of transport mechanism.

BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from Klebsiella pneumoniae, where both are symmetric trimers composed of three "binding"-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.

Transient Methionine Deprivation Triggers Histone Modification and Potentiates Differentiation of Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) require high levels of methionine (Met). Met deprivation results in a rapid decrease in intracellular S-adenosyl-methionine (SAM), poising human iPSCs for differentiation and leading to the apoptosis of undifferentiated cells. Met deprivation triggers rapid metabolic changes, including SAM, followed by reversible epigenetic modifications. Here, we show that short-term Met deprivation impairs the pluripotency network through epigenetic modification in a 3D suspension culture. The trimethylation of lysine 4 on histone H3 (H3K4me3) was drastically affected compared with other histone modifications. Short-term Met deprivation specifically affects the transcription start site (TSS) region of genes, such as those involved in the transforming growth factor ß pathway and cholesterol biosynthetic process, besides key pluripotent genes such as NANOG and POU5F1. The expression levels of these genes decreased, correlating with the loss of H3K4me3 marks. Upon differentiation, Met deprivation triggers the upregulation of various lineage-specific genes, including key definitive endoderm genes, such as GATA6. Upon differentiation, loss of H3K27me3 occurs in many endodermal genes, switching from a bivalent to a monovalent (H3K4me3) state. In conclusion, Met metabolism maintains the pluripotent network with histone marks, and their loss potentiates differentiation.

Association of Nrf2 expression and mutation with Weiss and Helsinki scores in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignant tumor. Genetic abnormalities that may represent therapeutic targets and prognostic factors in ACC remain unclear. Besides being one of the main cellular defense mechanisms that regulates antioxidant pathways for detoxifying reactive oxygen species (ROS), the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) promotes tumor proliferation by increasing metabolic activity. In surgical specimens from 12 cases of nonmetastatic ACCs and nine cases of benign adrenocortical adenoma (ACA), we investigated gene mutation and protein expressions for Nrf2 and the preoperative maximum standard glucose uptake (SUVmax) on [18 F]fluorodeoxy-glucose positron emission tomography. Three of five ACCs with a Weiss score of 7 to 9 were Nrf2 mutants; these ACCs had higher expression of Nrf2 and higher preoperative SUVmax. The other seven ACCs had a Weiss score of 3 to 6; these seven ACCs and all the ACAs were non-Nrf2 gene mutants. Patients with a Weiss score of 7 to 9 and Nrf2 mutant ACC had shorter overall survival. Based on Helsinki scoring, three ACCs with a Helsinki score greater than 17 had Nrf2 mutants, higher expression of Nrf2, higher preoperative SUVmax, and shorter overall survival. Our findings indicate that Nrf2 activation and the associated increase in metabolism play roles in ACC, in particular in ACC with a Weiss score of 7 to 9 and a Helsinki score of greater than 17.

Structural and functional characteristics of the tripartite ABC transporter.

ATP-binding cassette (ABC) transporters are one of the largest protein superfamilies and are found in all living organisms. These transporters use the energy from ATP binding and hydrolysis to transport various substrates. In this review, we focus on the structural and functional aspects of ABC transporters, with special emphasis on type VII ABC transporters, a newly defined class possessing characteristic structures. A notable feature of type VII ABC transporters is that they assemble into tripartite complexes that span both the inner and outer membranes of Gram-negative bacteria. One of the original type VII ABC transporters, which possesses all characteristic features of this class, is the macrolide efflux transporter MacB. Recent structural analyses of MacB and homologue proteins revealed the unique mechanisms of substrate translocation by type VII ABC transporters.

Increased expression of Nrf2 and elevated glucose uptake in pheochromocytoma and paraganglioma with SDHB gene mutation.

BACKGROUND: Pheochromocytomas (PCC) and paragangliomas (PGL) are catecholamine-producing neuroendocrine tumors. According to the World Health Organization Classification 2017, all PCC/PGL are considered to have malignant potential. There is growing evidence that PCC/PGL represent a metabolic disease that leads to aerobic glycolysis. Cellular energy metabolism involves both transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and succinate dehydrogenase (SDH) subtypes, but the association of these substances with PCC/PGL is largely unknown. METHODS: We investigated SDHB gene mutation and protein expressions for SDHB and Nrf2 in surgical specimens from 29 PCC/PGL. We also assessed preoperative maximum standard glucose uptake (SUVmax) on [18F]fluorodeoxy-glucose positron emission tomography and mRNA levels for Nrf2. RESULTS: Among 5 PCC/PGL with a PASS Score ≥ 4 or with a moderately to poorly differentiated type in the GAPP Score, 4 were metastatic and found to be SDHB mutants with homogeneous deletion of SDHB protein. SDHB mutants showed a higher expression of Nrf2 protein and a higher preoperative SUVmax than non-SDHB mutants with a PASS < 4 or a well-differentiated GAPP type. Furthermore, protein expression of Nrf2 was positively associated with preoperative SUVmax. The Nrf2 mRNA level positively correlated with malignant phenotype, higher expression for Nrf2 protein and SDHB gene mutant, but negatively correlated with expression for SDHB protein. There was also a positive correlation between Nrf2 mRNA level and SUVmax. CONCLUSION: These results suggest that activation of Nrf2 and elevated metabolism play roles in PCC/PGL with malignant potential that have SDHB gene mutation and SDHB deficiency.

Single nucleotide variants of succinate dehydrogenase A gene in renal cell carcinoma.

Succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC) is mainly associated with a mutation in the SDHB gene and sometimes with mutations in the SDHC or SDHD genes. However, only three cases of succinate dehydrogenase A (SDHA)-deficient RCC have been reported, and the relation between SDHA mutations and RCC has not been clarified. This study assessed the role of SDHA gene mutations in human RCC. We investigated SDHA/B/C/D gene mutations in 129 human RCCs. Targeted next-generation sequencing and direct Sanger sequencing revealed single nucleotide variants (SNVs) of the SDHA gene with amino acid sequence variations in 11/129 tumors, while no SDHB/C/D gene mutations were found. Tumor cells with SNVs of the SDHA gene were characterized by eosinophilic cytoplasm and various patterns of proliferation. Immunohistochemistry examination found that the 11 tumors with SNVs of the SDHA gene showed significant reduction of SDHA protein and SDHB protein expression compared to the 19 tumors without SDHA or SDHB mutations (both P < .0001). Western blotting showed a greater decrease in the expression of SDHA and SDHB proteins in the 11 tumors with SNVs of the SDHA gene than in the 19 tumors without (both P < .0001). There was a positive correlation between SDHA and SDHB protein levels (P < .0001). On immunohistochemistry and Western blotting, the 11 tumors with SNVs of the SDHA gene had higher protein expression for nuclear factor E2-related factor 2 (Nrf2) compared to the 19 tumors without the mutation (P < .01). These observations suggest that SDHA gene mutations might be associated with a subset of RCC.

Contribution of transglutaminases and their substrate proteins to the formation of cornified cell envelope in oral mucosal epithelium.

Cornified envelope formation is crucial for the final differentiation of keratinized epithelium. However, the mechanisms of cornified envelope formation in the oral epithelium remain unclear. The aim of this study was to clarify the differences in the distribution and expression of cornified envelope related proteins and genes between keratinized and non-keratinized oral epithelia. We immunohistochemically investigated the distribution patterns of transglutaminase 1 (TG1), transglutaminase 3 (TG3), and their substrate proteins involucrin (IVL), loricrin (LOR), and small proline rich proteins (SPRs), in 19 keratinized and 14 non-keratinized oral epithelium samples. TG1 and TG3 mRNA levels were investigated in both types of epithelium by real time reverse transcription polymerase chain reaction (RT-PCR) using paraffin-embedded specimens. Data were analyzed to identify factors involved in cornified envelope formation. We demonstrate that 11 localization patterns show statistically significant differences between keratinized and non-keratinized oral epithelia. These factors clearly drove the separation of the two groups during cluster analysis. TG1 mRNA levels in keratinized oral epithelium were significantly higher than those in non-keratinized oral epithelium. In conclusion, the characteristic distribution of transglutaminases and their substrates and the mRNA levels of TG1 can regulate cornified envelope formation in keratinized oral epithelium, together with the contribution of TG3 first reported in this paper.

Factors Associated with Dose Modification of Lenalidomide Plus Dexamethasone Therapy in Multiple Myeloma.

Long-term combination treatment with lenalidomide and low-dose dexamethasone is important to achieve a curative effect in patients with multiple myeloma (MM). In this study, the plasma concentration of lenalidomide was measured at 3 h after oral administration, when the drug is in the elimination phase and can be easily measured in outpatients, to identify factors that may lead to the discontinuation of this combination therapy. Patients were assigned to continuation or discontinuation of therapy groups, and the baseline characteristics of patients, lenalidomide concentration, and concentration/dose (C/D) ratios reflecting oral clearance were compared between the two groups. The efficacy and severity of adverse events were also compared. The results showed that patients who discontinued or modified treatment had low plasma concentrations of lenalidomide and C/D ratios, indicating high oral clearance of lenalidomide. The estimated creatinine clearance rate was negatively correlated with the C/D ratio. The plasma concentrations of lenalidomide were independent from kidney function and differed significantly among patients. Taken together, the results indicate that low plasma concentrations of lenalidomide and low C/D ratios may lead to discontinuation of combination therapy in patients with MM. This suggests that early measurement of lenalidomide plasma continuation would help to prevent discontinuation of therapy or a delay in modifying the dose of lenalidomide.

The ß-hairpin region of the cyanobacterial F1-ATPase γ-subunit plays a regulatory role in the enzyme activity.

The γ-subunit of cyanobacterial and chloroplast ATP synthase, the rotary shaft of F1-ATPase, equips a specific insertion region that is only observed in photosynthetic organisms. This region plays a physiologically pivotal role in enzyme regulation, such as in ADP inhibition and redox response. Recently solved crystal structures of the γ-subunit of F1-ATPase from photosynthetic organisms revealed that the insertion region forms a ß-hairpin structure, which is positioned along the central stalk. The structure-function relationship of this specific region was studied by constraining the expected conformational change in this region caused by the formation of a disulfide bond between Cys residues introduced on the central stalk and this ß-hairpin structure. This fixation of the ß-hairpin region in the α3ß3γ complex affects both ADP inhibition and the binding of the ε-subunit to the complex, indicating the critical role that the ß-hairpin region plays as a regulator of the enzyme. This role must be important for the maintenance of the intracellular ATP levels in photosynthetic organisms.

Molecular analyses of G3A/G3B and G14 equine group A rotaviruses detected between 2012 and 2018 in Japan.

Equine group A rotaviruses (RVAs) cause diarrhoea in foals. We investigated the G genotypes of 360 RVA-positive samples obtained from diarrhoeic foals between 2012 and 2018 in the Hidaka district of Hokkaido, Japan, through sequence analysis of VP7. All samples were classified into genotypes G3A, G3B and G14. G3B RVAs were detected until 2016, and G3A RVAs were detected from 2016 to 2018. G14 RVAs were detected from 2012 to 2018. Although G3B RVAs had been circulating in Japan for a long time, G3A RVAs suddenly emerged in 2016, and have replaced G3B RVAs since 2017. Molecular analyses of VP7 and VP4 showed that these Japanese G3A RVAs are closely related to North American G3A RVAs detected in 2017. Additionally, whole-genome analyses suggested that genetic reassortments occurred between G3A and G14 RVAs in NSP1, NSP2, NSP4 and NSP5.

Nrf2 gene mutation and single nucleotide polymorphism rs6721961 of the Nrf2 promoter region in renal cell cancer.

BACKGROUND: Nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in cell proliferation by promotion of metabolic activity. It is also the major regulator of antioxidants and has a pivotal role in tumor cell proliferation and resistance to chemotherapy. Accordingly, we investigated the role of Nrf2 in renal cell carcinoma (RCC). METHODS: In 50 patients who had metastatic RCC and received cytoreductive nephrectomy, we performed Nrf2 gene mutation analysis using targeted next-generation sequencing, as well as investigating a specific single nucleotide polymorphism (SNP; rs6721961) in the Nrf2 promoter region and Nrf2 protein expression. RESULTS: Targeted next-generation sequencing revealed that five tumors had SNPs of Nrf2 associated with amino acid sequence variation, while 11 tumors had SNPs of Kelch-like ECH-associated protein 1 gene, 35 had SNPs of von Hippel-Lindau gene, and none had SNPs of fumarate hydratase gene. The three genotypes of rs6721961 showed the following frequencies: 60% for C/C, 34% for C/A, and 6% for A/A. Nrf2 mutation and the C/A or A/A genotypes were significantly associated with increased Nrf2 protein expression (p = 0.0184 and p = 0.0005, respectively). When the primary tumor showed Nrf2 gene mutation, the C/A or A/A genotype, or elevated Nrf2 protein expression, the response of metastases to vascular endothelial growth factor-targeting therapy was significantly worse (p = 0.0142, p = 0.0018, and p <  0.0001, respectively), and overall survival was significantly reduced (p = 0.0343, p = 0.0421, and p <  0.0001, respectively). Elevated Nrf2 protein expression was also associated with shorter survival according to multivariate Cox proportional analysis. CONCLUSION: These findings suggest an associated between progression of RCC and Nrf2 signaling.

Comparison between autologous and allogeneic stem cell transplantation as salvage therapy for multiple myeloma relapsing/progressing after autologous stem cell transplantation.

Allogeneic stem cell transplantation (allo-SCT) offers a clinical option to young patients with multiple myeloma (MM) relapsing/progressing after autologous SCT (ASCT); however, this claim remains debatable. Thus, in this retrospective study, we analyzed 526 patients with MM who underwent SCT for MM relapsing/progressing after the prior ASCT using the registry data of the Japan Society for Hematopoietic Cell Transplantation (2001-2015) and compared overall survival (OS) between allo-SCT (n = 192) and autologous stem cell retransplantation groups (ReASCT; n = 334) based on risk factor points. Significant adverse factors for OS in all patients were (1) male sex, (2) less than partial response to SCT, (3) performance status of 2 to 4, and (4) short duration from the prior ASCT. We scored factor 2 as 1 point, factor 3 as 2 points, and factor 4 as 0, 1, or 2 points for more than 30, 9 to 30, or less than 9 months, respectively. We categorized patients into three risk subgroups based on their total points (0, 1-3, and 4-5 points), indicating the usefulness of this scoring system for prognosis prediction and treatment selection. Subgroup comparison revealed OS after ReASCT to be higher than that after allo-SCT in the intermediate-risk subgroup comprising the largest population (28.2% vs 21.5%, P < .004). We observed no significant advantages of allo-SCT over ReASCT in the low- and high-risk subgroups. These findings suggest that ReASCT is more advantageous than allo-SCT in many patients with MM relapsing/progressing after the prior ASCT. However, long-term survival patients were noted only in the allo-SCT group, and allo-SCT could exhibit clinical efficacy, particularly in the low-risk group. While further examination is warranted, allo-SCT could be a potential tool for a specific population with MM relapsing/progressing after the prior ASCT.

Isolation of Salmonella enterica serovar Agona strains and their similarities to strains derived from a clone caused a serovar shift in broilers.

Salmonella enterica serovar Agona strains isolated from human cases were compared to strains that were derived from a clone caused a serovar shift in broilers. Pulsed field gel electrophoresis (PFGE) analysis with XbaI or BlnI digestion showed that three of seven strains from human case strains and most of the 81 strains from broilers were clustered in single complex in a minimum spanning tree (MST) reconstructed from the PFGE data. All the strains from human cases and 22 randomly selected strains from broilers were also analyzed by whole genome sequencing (WGS). Analysis of single nucleotide polymorphism (SNP) in the S. Agona core genes showed that four strains from human cases and all the strains from broilers were clustered in a maximum likelihood phylogenetic tree (ML tree) and an MST. These results indicated that the strains derived from the clone caused the serovar shift had already spread to humans. PFGE analysis with XbaI showed that four strains from broilers did not cluster with the other strains in an MST, though all those strains clustered in an ML tree and an MST reconstructed from SNP data. Moreover, three strains from broilers did not cluster in an MST reconstructed from PFGE with BlnI digestion, though those strains clustered in an ML tree and an MST reconstructed from SNP data. Therefore, it was suggested that S. Agona strains derived from a particular clone could not be traced by PFGE analysis but can be investigated by WGS analysis.

Structure of the γ-ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs.

F1-ATPase forms the membrane-associated segment of F0F1-ATP synthase - the fundamental enzyme complex in cellular bioenergetics for ATP hydrolysis and synthesis. Here, we report a crystal structure of the central F1 subcomplex, consisting of the rotary shaft γ subunit and the inhibitory ε subunit, from the photosynthetic cyanobacterium Thermosynechococcus elongatus BP-1, at 1.98 Šresolution. In contrast with their homologous bacterial and mitochondrial counterparts, the γ subunits of photosynthetic organisms harbour a unique insertion of 35-40 amino acids. Our structural data reveal that this region forms a ß-hairpin structure along the central stalk. We identified numerous critical hydrogen bonds and electrostatic interactions between residues in the hairpin and the rest of the γ subunit. To elaborate the critical function of this ß-hairpin in inhibiting ATP hydrolysis, the corresponding domain was deleted in the cyanobacterial F1 subcomplex. Biochemical analyses of the corresponding α3ß3γ complex confirm that the clinch of the hairpin structure plays a critical role and accounts for a significant interaction in the α3ß3 complex to induce ADP inhibition during ATP hydrolysis. In addition, we found that truncating the ß-hairpin insertion structure resulted in a marked impairment of the interaction with the ε subunit, which binds to the opposite side of the γ subunit from the ß-hairpin structure. Combined with structural analyses, our work provides experimental evidence supporting the molecular principle of how the insertion region of the γ subunit suppresses F1 rotation during ATP hydrolysis.

[Spontaneous regression of primary gastric EBV-positive diffuse large B-cell lymphoma].

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) in the elderly was revised from the category EBV-positive DLBCL not otherwise specified in WHO 2017. The prognosis of this lymphoma is very poor. We report a case of an 82-year-old woman diagnosed with gastric EBV-positive DLBCL (WHO 2008). Gastroduodenoscopy revealed multiple ulcers and fold thickening. She was followed-up without any treatment because of her old age. Repeat endoscopy one year and eight months later revealed a single ulcer with no lymphoma cells found in a biopsy specimen. Two years later, the lesion had spontaneously disappeared.

[Successful treatment of relapse/refractory multiple myeloma with carfilzomib, lenalidomide, and dexamethasone combination therapy following allogeneic bone marrow transplantation].

A 50-year-old male was diagnosed with multiple myeloma (MM) and treated by high-dose melphalan followed by autologous stem cell transplantation in April 2014. However, he relapsed and received non-myeloablative bone marrow transplantation from an unrelated HLA-matched donor (UR-BMT) in July 2016. After 100 days of UR-BMT, the disease remained stable disease and the patient was treated with carfilzomib, lenalidomide, and dexamethaonse (KRd) therapy. After 10 cycles of KRd, he obtained stringent complete response without exacerbation of graft-versus-host disease. We concluded that KRd after allogeneic stem cell transplantation is one of the useful treatment regimens for relapsed refractory MM.

[Latent essential thrombocythemia becoming perceptible after splenectomy].

A 47-year-old male was admitted to our hospital because of left hypochondrium part pain and was diagnosed with splenomegaly with splenic infarctions in May 2016. His complete blood cell count was almost within normal limits, and a bone marrow biopsy revealed normal cellularity with no fibrosis. In addition, no abnormal uptake was noted on FDG PET/CT. In August 2016, he underwent splenectomy for splenomegaly. The histological examination revealed fibrotic stenosis of the blood vessels in the spleen. After splenectomy, his platelet count elevated and remained at >1,000×109/l 3 months later. Finally, he was diagnosed with latent essential thrombocythemia (ET) because the JAK2V617F mutation was positive. Accordingly, oral hydroxyurea was initiated. Thrombosis could be a complication in myeloproliferative neoplasms (MPN). In our case, ET was masked, perhaps, because of hypersplenism and splenomegaly because of splenic vein thrombosis. Hence, examination of the JAK2V617F mutation in patients with splanchnic vein thrombosis is recommended because of the possibility of latent MPN.

Two Components of Aversive Memory in Drosophila, Anesthesia-Sensitive and Anesthesia-Resistant Memory, Require Distinct Domains Within the Rgk1 Small GTPase.

Multiple components have been identified that exhibit different stabilities for aversive olfactory memory in Drosophila These components have been defined by behavioral and genetic studies and genes specifically required for a specific component have also been identified. Intermediate-term memory generated after single cycle conditioning is divided into anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM), with the latter being more stable. We determined that the ASM and ARM pathways converged on the Rgk1 small GTPase and that the N-terminal domain-deleted Rgk1 was sufficient for ASM formation, whereas the full-length form was required for ARM formation. Rgk1 is specifically accumulated at the synaptic site of the Kenyon cells (KCs), the intrinsic neurons of the mushroom bodies, which play a pivotal role in olfactory memory formation. A higher than normal Rgk1 level enhanced memory retention, which is consistent with the result that Rgk1 suppressed Rac-dependent memory decay; these findings suggest that rgk1 bolsters ASM via the suppression of forgetting. We propose that Rgk1 plays a pivotal role in the regulation of memory stabilization by serving as a molecular node that resides at KC synapses, where the ASM and ARM pathway may interact.SIGNIFICANCE STATEMENT Memory consists of multiple components. Drosophila olfactory memory serves as a fundamental model with which to investigate the mechanisms that underlie memory formation and has provided genetic and molecular means to identify the components of memory, namely short-term, intermediate-term, and long-term memory, depending on how long the memory lasts. Intermediate memory is further divided into anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM), with the latter being more stable. We have identified a small GTPase in Drosophila, Rgk1, which plays a pivotal role in the regulation of olfactory memory stability. Rgk1 is required for both ASM and ARM. Moreover, N-terminal domain-deleted Rgk1 was sufficient for ASM formation, whereas the full-length form was required for ARM formation.

Doublecortin marks a new population of transiently amplifying muscle progenitor cells and is required for myofiber maturation during skeletal muscle regeneration.

Muscle satellite cells are indispensable for muscle regeneration, but the functional diversity of their daughter cells is unknown. Here, we show that many Pax7(+)MyoD(-) cells locate both beneath and outside the basal lamina during myofiber maturation. A large majority of these Pax7(+)MyoD(-) cells are not self-renewed satellite cells, but have different potentials for both proliferation and differentiation from Pax7(+)MyoD(+) myoblasts (classical daughter cells), and are specifically marked by expression of the doublecortin (Dcx) gene. Transplantation and lineage-tracing experiments demonstrated that Dcx-expressing cells originate from quiescent satellite cells and that the microenvironment induces Dcx in myoblasts. Expression of Dcx seems to be necessary for myofiber maturation because Dcx-deficient mice exhibited impaired myofiber maturation resulting from a decrease in the number of myonuclei. Furthermore, in vitro and in vivo studies suggest that one function of Dcx in myogenic cells is acceleration of cell motility. These results indicate that Dcx is a new marker for the Pax7(+)MyoD(-) subpopulation, which contributes to myofiber maturation during muscle regeneration.

Changes in MicroRNA Expression Level of Circulating Platelets Contribute to Platelet Defect After Cardiopulmonary Bypass.

OBJECTIVES: Platelet defect mechanisms after cardiopulmonary bypass remain unclear. Our hypothesis microRNA expressions in circulating platelets significantly change between pre and post cardiopulmonary bypass, and consequent messenger RNA and protein expression level alterations cause postcardiopulmonary bypass platelet defect. DESIGN: Single-center prospective observational study. SETTING: Operating room of Kyoto Prefectural University of Medicine. PATIENTS: Twenty-five adult patients scheduled for elective cardiac surgeries under cardiopulmonary bypass. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In the initial phase, changes in microRNA expression between pre and post cardiopulmonary bypass underwent next generation sequencing analysis (10 patients). Based on the results, we focused on changes in mir-10b and mir-96, which regulate glycoprotein 1b and vesicle-associated membrane protein 8, respectively, and followed them until messenger RNA and protein syntheses (15 patients) using quantitative polymerase chain reaction and Western blotting. Seven microRNAs including mir-10b and mir-96 exhibited significant differences in the initial phase. In the subsequent phase, mir-10b-5p and mir-96-5p overexpressions were confirmed, and glycoprotein 1b and vesicle-associated membrane protein 8 messenger RNA levels were significantly decreased after cardiopulmonary bypass: fold differences (95% CI): mir-10b-5p: 1.35 (1.05-2.85), p value equals to 0.01; mir-96-5p: 1.59 (1.06-2.13), p value equals to 0.03; glycoprotein 1b messenger RNA: 0.46 (0.32-0.60), p value of less than 0.001; and vesicle-associated membrane protein messenger RNA: 0.70 (0.56-0.84), p value of less than 0.001. Glycoprotein 1b and vesicle-associated membrane protein 8 were also significantly decreased after cardiopulmonary bypass: glycoprotein 1b: 82.6% (71.3-93.8%), p value equals to 0.005; vesicle-associated membrane protein 8: 79.0% (70.7-82.3%), p value of less than 0.001. CONCLUSIONS: Expressions of several microRNAs in circulating platelets significantly changed between pre and post cardiopulmonary bypass. Overexpressions of mir-10b and mir-96 decreased glycoprotein 1b and vesicle-associated membrane protein 8 messenger RNA as well as protein, possibly causing platelet defect after cardiopulmonary bypass.