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Abstract Cancer stem cells are associated with aggressive phenotypes of malignant tumors. A prominent feature of uterine endometrial cancer is the activation of the PI3K–Akt–mTOR pathway. In this study, we present variations in sensitivities to a PI3K–Akt–mTORC1 inhibitor among in vitro endometrial cancer stem cell-enriched spheroid cells from clinical specimens. The in vitro sensitivity was consistent with the effects observed in in vivo spheroid-derived xenograft tumor models. Our findings revealed a complementary suppressive effect on endometrial cancer spheroid cell growth with the combined use of aldehyde dehydrogenase (ALDH) and PI3K–Akt inhibitors. In the PI3K–Akt–mTORC1 signaling cascade, the influence of ALDH on mTORC1 was partially channeled through retinoic acid-induced lactate dehydrogenase A (LDHA) activation. LDHA inhibition was found to reduce endometrial cancer cell growth, aligning with the effects of mTORC1 inhibition. Building upon our previous findings highlighting ALDH-driven glycolysis through GLUT1 in uterine endometrial cancer spheroid cells, curbing mTORC1 enhanced glucose transport via GLUT1 activation. Notably, elevated LDHA expression correlated with adverse clinical survival and escalated tumor grade, especially in advanced stages. Collectively, our findings emphasize the pivotal role of ALDH–LDHA–mTORC1 cascade in the proliferation of endometrial cancer. Targeting the interaction between mTORC1 and ALDH-influenced glycolysis holds promise for developing novel strategies to combat this aggressive cancer.

Purpose: The coronal plane alignment of the knee (CPAK) classification has been introduced as a simple system to classify knee phenotypes based on leg alignment [hip–knee–ankle angle (HKA) and joint line obliquity (JLO)]. Differences in post-operative phenotype between mechanical alignment (MA) and kinematic alignment (KA) total knee arthroplasty (TKA) have been reported, but not the phenotypes after unicompartmental knee arthroplasty (UKA). Moreover, most studies have focused upon Western populations. This study compared pre- and post-operative knee phenotypes between MA-TKA, KA-TKA, and UKA in a Japanese cohort. Methods: In this study, 230 knees in 165 patients who underwent UKA, 124 knees in 80 patients who underwent MA-TKA, and 80 knees in 51 patients who underwent restricted KA-TKA in Takatsuki General Hospital between March 2019 and January 2021 were retrospectively investigated. Pre- and post-operative lateral distal femoral angle (LDFA) and medial proximal tibial angle (MPTA) were measured, and JLO (MPTA ​+ ​LDFA) and arithmetic HKA (aHKA) (MPTA – LDFA) were calculated and compared between the surgeries. Results: Pre-operative JLO and aHKA did not significantly differ between the three groups (pre. JLO, p ​= ​0.164; pre. aHKA, p ​= ​0.13). Pre-operatively, 62.0% of knees were categorized as type I (varus leg alignment and medially sloped joint line). After UKA, 59.1% of type I inherited the pre-operative phenotype, whereas most cases were altered in MA-TKA and KA-TKA (4.8% and 30.0%, respectively, inherited the pre-operative phenotype). Conclusions: The pre-operative phenotype was mostly inherited after UKA and to a lesser extent after KA-TKA, whereas it tended to be altered after MA-TKA.

HIV disease remains prevalent in the USA and chronic kidney disease remains a major cause of morbidity in HIV-1-positive patients. Host double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a sensor for viral dsRNA, including HIV-1. We show that PKR inhibition by compound C16 ameliorates the HIV-associated nephropathy (HIVAN) kidney phenotype in the Tg26 transgenic mouse model, with reversal of mitochondrial dysfunction. Combined analysis of single-nucleus RNA-seq and bulk RNA-seq data revealed that oxidative phosphorylation was one of the most downregulated pathways and identified signal transducer and activator of transcription (STAT3) as a potential mediating factor. We identified in Tg26 mice a novel proximal tubular cell cluster enriched in mitochondrial transcripts. Podocytes showed high levels of HIV-1 gene expression and dysregulation of cytoskeleton-related genes, and these cells dedifferentiated. In injured proximal tubules, cell-cell interaction analysis indicated activation of the pro-fibrogenic PKR-STAT3-platelet-derived growth factor (PDGF)-D pathway. These findings suggest that PKR inhibition and mitochondrial rescue are potential novel therapeutic approaches for HIVAN.

Abstract Selective degradation of dysfunctional or excess mitochondria is a fundamental process crucial for cell homeostasis in almost all eukaryotes. This process relies on autophagy, an intracellular self-eating system conserved from yeast to humans and is thus called mitophagy. Detailed mechanisms of mitophagy remain to be fully understood. Here we show that mitochondrial degradation in budding yeast, which requires the pro-mitophagic protein Atg32, is strongly reduced in cells lacking Egd1, a beta subunit of the nascent polypeptide-associated complex acting in cytosolic ribosome attachment and protein targeting to mitochondria. By contrast, loss of the sole alpha subunit Egd2 or the beta subunit paralogue Btt1 led to only a partial or slight reduction in mitophagy. We also found that phosphorylation of Atg32, a crucial step for priming mitophagy, is decreased in the absence of Egd1. Forced Atg32 hyperphosphorylation almost completely restored mitophagy in egd1-null cells. Together, we propose that Egd1 acts in Atg32 phosphorylation to facilitate mitophagy.

Abstract A short keel-cortex distance (KCD), especially to the posterior cortex, is a potential risk factor for tibial plateau fracture after Oxford mobile-bearing unicompartmental knee arthroplasty (OUKA). This study aimed to evaluate the effect of tibial component alignment in the coronal plane and tibial proximal morphology on the KCD. Included in this study were 51 patients scheduled for primary Oxford medial unicompartmental knee arthroplasty (UKA). The anterior and posterior KCD were preoperatively assessed using 3D simulation software with the component set perpendicular to the tibial mechanical axis (neutral), 3° valgus, 3° varus, and 6° varus, relative to neutral alignment. We evaluated the existence of overhanging medial tibial condyle where the medial eminence line, the line including the medial tibial eminence parallel to the tibial axis, passes outside of the tibial shaft. In all component alignments, patients with a medial overhanging condyle had significantly shorter posterior KCD than those without. In patients with a medial overhanging condyle, the posterior KCD significantly increased when the tibial component was placed in 3° varus (4.6 ± 1.5 mm, P = 0.003 vs neutral, P

Abstract Gastrointestinal tract organs harbor reserve cells, which are endowed with cellular plasticity and regenerate functional units in response to tissue damage. However, whether the reserve cells in gastrointestinal tract exist as long-term quiescent cells remain incompletely understood. In the present study, we systematically examine H2b-GFP label-retaining cells and identify a long-term slow-cycling population in the gastric corpus but not in other gastrointestinal organs. The label-retaining cells, which reside near the basal layers of the corpus, comprise a subpopulation of chief cells. The identified quiescent cells exhibit induction of Atf4 and its target genes including Atf3, a marker of paligenosis, and activation of the unfolded protein response, but do not show elevated expression of Troy, Lgr5, or Mist. External damage to the gastric mucosa induced by indomethacin treatment triggers proliferation of the quiescent Atf4+ population, indicating that the gastric corpus harbors a specific cell population that is primed to facilitate stomach regeneration.

Abstract Chronic inflammation promotes development and progression of colorectal cancer (CRC). To comprehensively understand the molecular mechanisms underlying the development and progression of inflamed CRC, we perform in vivo screening and identify 142 genes that are frequently mutated in inflammation-associated colon tumors. These genes include senescence and TGFβ-activin signaling genes. We find that TNFα can induce stemness and activate senescence signaling by enhancing cell plasticity in colonic epithelial cells, which could act as a selective pressure to mutate senescence-related genes in inflammation-associated colonic tumors. Furthermore, we show the efficacy of the Cdk4/6 inhibitor in vivo for inflammation-associated colonic tumors. Finally, we functionally validate that Arhgap5 and Mecom are tumor suppressor genes, providing possible therapeutic targets for CRC. Thus, we demonstrate the importance of the inactivation of senescence pathways in CRC development and progression in an inflammatory microenvironment, which can help progress toward precision medicine.

Abstract Introduction This retrospective study aims to clarify if there are benefits of performing unicompartmental knee arthroplasty (UKA) on just one indicated side in patients who undergo simultaneous bilateral knee arthroplasty. Materials and methods We compared 33 cases of simultaneous bilateral UKA/total knee arthroplasty (TKA) (S-UT) with 99 cases of simultaneous bilateral TKA (S-TT). Comparison included blood tests [C-reactive protein (CRP), albumin, and D-dimer], the incidence of deep vein thrombosis (DVT), range of motion (ROM), and clinical scores before and 1 year after surgery. Results Clinical scores were not significantly different between the groups. The postoperative flexion angle was significantly better in UKA sides. Blood tests showed that the S-UT had a significantly higher albumin value at 4 and 7 days after surgery. The CRP value at 4 and 7 days, and the D-dimer value at 7 and 14 days after surgery were significantly lower in the S-UT. The S-UT had significantly lower incidence of DVT. Conclusions In cases of bilateral arthroplasty, if there is an indication on only one side, a better flexion angle can be obtained by UKA on that side, and with less surgical invasion. Moreover, the incidence of DVT is low, which is considered to be a benefit of performing UKA on just one side.

Based on the classical Gursone-Tvergaarde-Needleman (GTN) method, a theoretical model was developed and applied to predict the mechanical behavior and fracture characteristics of hydrogenated zirconium cladding. With full consideration of the influence of hydrogen on plastic stain and void volume in materials, the damage parameters in the classical GTN model were modified. Through benchmarking with experimental data, it was proved that the hydrogen embrittlement phenomenon in zirconium materials can be properly simulated by the modified GTN model proposed in this study. By integrating this modified model into ABAQUS software, numerical simulation of a cladding tensile test was further conducted at different temperature, hydrogen concentration and oxidation conditions. Influence of hydrogen embrittlement on the mechanical properties and fracture characteristics of zirconium cladding was analyzed. The results suggest that the effect of hydrogenation on the cladding mechanical performance varies in magnitude at different temperatures, because hydrogen exists in different forms and also varies significantly at different temperatures. At a relatively low temperature, a considerable amount of hydrogen is presented in the form of precipitates, which is more likely to cause hydrogen embrittlement failure. In addition, the numerical simulation of a cladding tube burst test was carried out under a loss of coolant accident. It was concluded that the influence of hydrogen embrittlement on the burst stress and burst strain was still considerable, despite of the rather high temperature at the burst position.

Abstract Background This study aims to assess the influence of intramedullary rods on the implantation positions of femoral components using Microplasty instrumentation in Oxford unicompartmental knee arthroplasty. We hypothesized that femoral components can be laterally implanted incorrectly when using intramedullary rods. Methods This prospective study included all 45 consecutive patients (53 knees) who underwent Oxford unicompartmental knee arthroplasty surgery for anteromedial osteoarthritis or spontaneous osteonecrosis of the knee at our hospital during the study period. A custom-made toolset comprising a triangular caliper and circular trial bearings was used to evaluate the distance between the bearing and the vertical wall of the tibia implant (wall-bearing space) using the caliper at 90° flexion both with and without intramedullary rods. Results The wall-bearing space was significantly larger when the intramedullary rod was used than when intramedullary rod was not used (1.8 ± 1.1 mm versus 3.4 ± 1.2 mm, P

The kinematic alignment (KA) approach to total knee arthroplasty (TKA) has recently increased in popularity. Accordingly, a number of derivatives have arisen and have caused confusion. Clarification is therefore needed for a better understanding of KA-TKA. Calipered (or true, pure) KA is performed by cutting the bone parallel to the articular surface, compensating for cartilage wear. In soft-tissue respecting KA, the tibial cutting surface is decided parallel to the femoral cutting surface (or trial component) with in-line traction. These approaches are categorized as unrestricted KA because there is no consideration of leg alignment or component orientation. Restricted KA is an approach where the periarthritic joint surface is replicated within a safe range, due to concerns about extreme alignments that have been considered ‘alignment outliers’ in the neutral mechanical alignment approach. More recently, functional alignment and inverse kinematic alignment have been advocated, where bone cuts are made following intraoperative planning, using intraoperative measurements acquired with computer assistance to fulfill good coordination of soft-tissue balance and alignment. The KA-TKA approach aims to restore the patients’ own harmony of three knee elements (morphology, soft-tissue balance, and alignment) and eventually the patients’ own kinematics. The respective approaches start from different points corresponding to one of the elements, yet each aim for the same goal, although the existing implants and techniques have not yet perfectly fulfilled that goal.

Abstract FMS-like tyrosine kinase 3 (FLT3) in hematopoietic cells binds to its ligand at the plasma membrane (PM), then transduces growth signals. FLT3 gene alterations that lead the kinase to assume its permanently active form, such as internal tandem duplication (ITD) and D835Y substitution, are found in 30–40% of acute myelogenous leukemia (AML) patients. Thus, drugs for molecular targeting of FLT3 mutants have been developed for the treatment of AML. Several groups have reported that compared with wild-type FLT3 (FLT3-wt), FLT3 mutants are retained in organelles, resulting in low levels of PM localization of the receptor. However, the precise subcellular localization of mutant FLT3 remains unclear, and the relationship between oncogenic signaling and the mislocalization is not completely understood. In this study, we show that in cell lines established from leukemia patients, endogenous FLT3-ITD but not FLT3-wt clearly accumulates in the perinuclear region. Our co-immunofluorescence assays demonstrate that Golgi markers are co-localized with the perinuclear region, indicating that FLT3-ITD mainly localizes to the Golgi region in AML cells. FLT3-ITD biosynthetically traffics to the Golgi apparatus and remains there in a manner dependent on its tyrosine kinase activity. Tyrosine kinase inhibitors, such as quizartinib (AC220) and midostaurin (PKC412), markedly decrease FLT3-ITD retention and increase PM levels of the mutant. FLT3-ITD activates downstream in the endoplasmic reticulum (ER) and the Golgi apparatus during its biosynthetic trafficking. Results of our trafficking inhibitor treatment assays show that FLT3-ITD in the ER activates STAT5, whereas that in the Golgi can cause the activation of AKT and ERK. We provide evidence that FLT3-ITD signals from the early secretory compartments before reaching the PM in AML cells.

In this study, experiments of downward-facing flow boiling were conducted to investigate material effects on CHF. Experiments were conducted using aluminum, copper, and carbon steel. It was found that different materials had different CHFs. Aluminum has the biggest CHF while copper has the lowest CHF for each mass flux. After experiment, surface wettability increased and surface became rougher, which was probably due to the oxidation process during nucleate boiling. The CHF difference is likely to be related to the surface wettability, roughness and thermal effusivity, which influences the bubble behavior and in turn affects CHF. Further studies are needed to determine which factor is dominant.

Abstract Background Intra-tumor heterogeneity (ITH) encompasses cellular differences in tumors and is related to clinical outcomes such as drug resistance. However, little is known about the dynamics of ITH, owing to the lack of time-series analysis at the single-cell level. Mouse models that recapitulate cancer development are useful for controlled serial time sampling. Results We performed single-cell exome and transcriptome sequencing of 200 cells to investigate how ITH is generated in a mouse colorectal cancer model. In the model, a single normal intestinal cell is grown into organoids that mimic the intestinal crypt structure. Upon RNAi-mediated downregulation of a tumor suppressor gene APC, the transduced organoids were serially transplanted into mice to allow exposure to in vivo microenvironments, which play relevant roles in cancer development. The ITH of the transcriptome increased after the transplantation, while that of the exome decreased. Mutations generated during organoid culture did not greatly change at the bulk-cell level upon the transplantation. The RNA ITH increase was due to the emergence of new transcriptional subpopulations. In contrast to the initial cells expressing mesenchymal-marker genes, new subpopulations repressed these genes after the transplantation. Analyses of colorectal cancer data from The Cancer Genome Atlas revealed a high proportion of metastatic cases in human subjects with expression patterns similar to the new cell subpopulations in mouse. These results suggest that the birth of transcriptional subpopulations may be a key for adaptation to drastic micro-environmental changes when cancer cells have sufficient genetic alterations at later tumor stages. Conclusions This study revealed an evolutionary dynamics of single-cell RNA and DNA heterogeneity in tumor progression, giving insights into the mesenchymal-epithelial transformation of tumor cells at metastasis in colorectal cancer.

For a sodium-cooled fast reactor, the capability for stable cooling and avoiding re-criticality on the debris bed is essential for achieving in-vessel retention when severe accidents occur. However, an unexploited uncertainty still existed regarding the compound effect of the heterogeneous configuration and dynamic particle redistribution for the debris bed’s criticality and cooling safety assessment. Therefore, this research aims to develop a numerical tool for investigating the effects of the different transformations of the heterogeneous configurations on the debris bed’s criticality/cooling assessment. Based on the newly proposed methodology in this research, via integrating the Discrete Element Method (DEM) with Computational Fluid Dynamics (CFD) and Monte-Carlo-based Neutronics (MCN), the coupled CFD–DEM–MCN solver was constructed with the originally created interface to integrate two existing codes. The effects of the different bed configurations’ transformations on the bed safety assessments were also quantitively confirmed, indicating that the effect of the particle-centralized fissile material had the dominant negative effect on the safety margin of avoiding re-criticality and particle re-melting accidents and had a more evident impact than the net bed-centralized effect. This coupled solver can serve to further assess the debris bed’s safety via a multi-physics simulation approach, leading to safer SFR design concepts.

Abstract Patients with chronic kidney disease (CKD) commonly exhibit hypercoagulability. Increased levels of uremic toxins cause thrombogenicity by increasing tissue factor (TF) expression and activating the extrinsic coagulation cascade. TF is induced in monocytes and macrophages under pathological conditions, such as inflammatory diseases. However, the role of monocyte myeloid cell TF in CKD progression remains unclear. We aimed to clarify this issue, and the present study found that patients with CKD had elevated levels of D-dimer, a marker of fibrin degradation, which was associated with decreased estimated glomerular filtration rate and increased serum levels of uremic toxins, such as indoxyl sulfate. In vitro studies showed that several uremic toxins increased cellular TF levels in monocytic THP-1 cells. Mice with TF specifically deleted in myeloid cells were fed an adenine diet to cause uremic kidney injury. Myeloid TF deletion reduced tubular injury and pro-inflammatory gene expression in the kidneys of adenine-induced CKD but did not improve renal function as measured by plasma creatinine or blood urea nitrogen. Collectively, our findings suggest a novel concept of pathogenesis of coagulation-mediated kidney injury, in which elevated TF levels in monocytes under uremic conditions is partly involved in the development of CKD.

The spreading-solidification behaviour of the stratified molten metals was investigated. This is important in understanding the practical fuel debris distribution spread and solidified in the primary containment vessel (PCV) of Fukushima Daiichi Nuclear Power Plants (1F NPPs). In this study, the effect of outflow diameter on the material distribution before discharging was studied both experimentally and numerically. The two simulant metals were chosen so that the density ratio could be similar to the practical fuel and structure elements of the plant. They were arranged in a vessel and discharged on a receiving plate. The spreading experiments were performed using various outlet diameters with a density and reverse density stratification arrangement. After the experiment, X-ray analysis was performed to evaluate the material distribution in the solidified material. Moreover, a numerical analysis was performed to reveal the mechanisms that affect the material distribution after solidification. As a result, the low-density metal accumulated at the centre region regardless of the outlet diameters in the density stratification. In contrast, the outlet diameters affected the material distribution in the reverse density stratification because they affected the material outflow order. These findings may help increase our understanding of the fuel debris distribution in 1F NPPs.

Abstract Background Zinc is an essential micronutrient for maintaining biological activity. The level of zinc in the blood is known to decrease with age, especially in those over 75 years of age. In older adults patients with impaired functional status, aspiration pneumonia based on dysphagia often becomes problematic. However, the relationship between zinc deficiency and swallowing function has not been studied before. Methods A total of 52 older adults subjects (15 males and 37 females) living in a nursing home were enrolled for this study. At the time of enrollment, data of gender, age, body weight, serum zinc levels, serum albumin levels, and the time in a simple 2-step swallowing provocation test (S-SPT) were collected. In patients with serum zinc levels

ABSTRACT Objective: Although the mobile-bearing Oxford unicompartmental knee arthroplasty (OUKA) seems an appropriate procedure to treat spontaneous osteonecrosis of the knee (SONK), aseptic tibial component loosening was the leading cause for medial UKA failure. This study aimed to observe short-term and midterm clinical outcomes following OUKA and determine whether tibial lesion affects the procedure clinical and radiographic outcomes. Methods: Sixty patients (mean age 73.1 ± 6.6 years) diagnosed with SONK in the medial femoral condyle and treated with OUKA were separated into two groups using T1-weighted preoperative magnetic resonance imaging (MRI): group F (necrotic lesion confined to the femur) and group T (necrotic lesion spread to the tibia). The Oxford Knee Score (OKS), maximum flexion angle (MFA), and radiographic findings (radiolucent line and subsidence) were compared between the two groups using unpaired t-test. Results: Both groups showed significant improvement in OKS and MFA values at the final follow-up, but without significant differences in the clinical and radiographic outcomes. Conclusion: OUKA is a reliable treatment procedure for SONK in the short and midterm. The presence of tibial lesions on preoperative MRI does not affect postoperative radiographic and clinical outcomes. Level of Evidence IV, Case Series.

HIV-associated nephropathy (HIVAN) impairs functions of both glomeruli and tubules. Attention has been previously focused on the HIVAN glomerulopathy. Tubular injury has drawn increased attention because sodium wasting is common in hospitalized HIV/AIDS patients. We used viral protein R (Vpr)-transgenic mice to investigate the mechanisms whereby Vpr contributes to urinary sodium wasting. In phosphoenolpyruvate carboxykinase promoter-driven Vpr-transgenic mice, in situ hybridization showed that Vpr mRNA was expressed in all nephron segments, including the distal convoluted tubule. Vpr-transgenic mice, compared with wild-type littermates, markedly increased urinary sodium excretion, despite similar plasma renin activity and aldosterone levels. Kidneys from Vpr-transgenic mice also markedly reduced protein abundance of the Na+-Cl- cotransporter (NCC), while mineralocorticoid receptor (MR) protein expression level was unchanged. In African green monkey kidney cells, Vpr abrogated the aldosterone-mediated stimulation of MR transcriptional activity. Gene expression of Slc12a3 (NCC) in Vpr-transgenic mice was significantly lower compared with wild-type mice, assessed by both qRT-PCR and RNAScope in situ hybridization analysis. Chromatin immunoprecipitation assays identified multiple MR response elements (MRE), located from 5 kb upstream of the transcription start site and extending to the third exon of the SLC12A3 gene. Mutation of MRE and SP1 sites in the SLC12A3 promoter region abrogated the transcriptional responses to aldosterone and Vpr, indicating that functional MRE and SP1 are required for the SLC12A3 gene suppression in response to Vpr. Thus, Vpr attenuates MR transcriptional activity and inhibits Slc12a3 transcription in the distal convoluted tubule and contributes to salt wasting in Vpr-transgenic mice.

Background: The purpose of this study was to use second-look arthroscopic findings and clinical assessment to determine outcome in two cases of knee osteoarthritis treated by intra-articular knee injection of adipose-derived regenerative cells (ADRCs). Case presentation: This study involved two patients who received ADRC therapy for knee osteoarthritis and completed the six-month post-treatment follow-up period. For each treatment, 130 mL of subcutaneous adipose tissue was harvested using tumescent liposuction technique and manual aspiration of tissue from the thigh using a suction cannula under local anesthesia in the operating room. The adipose tissue harvested was processed using the Celution® Centrifuge in a dedicated cell processing room. The ADRCs were injected into the articular cavity of both knees for one patient and into a single affected knee in the second patient (three joints). Pain and knee function were assessed using a Visual Analogue Scale (VAS) and the Knee Outcome in Osteoarthritis Score (KOOS) respectively. The cartilage defect was assessed by direct visualization (arthroscopy). No serious adverse events were reported throughout follow-up. Pain and knee function were significantly improved from baseline in all treated knees at one, three and six months after ADRCs. At six-months after ADRCs treatment, the second-look arthroscopy showed that almost all the cartilage defect areas were covered by regenerated cartilage, some cartilage fibrillation area was reduced, and meniscus tear areas were repaired. Conclusions: Cartilage and meniscus repair were observed six-months after ADRCs therapy under second-look arthroscopy. It was shown that a single administration of ADRCs might be effective as a treatment for knee osteoarthritis. Keywords: Adipose-derived regenerative cells, Second-look arthroscopy, Knee osteoarthritis, Cartilage, Meniscus

Abstract Background Saccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae. Results The growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. 13C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production. Conclusion These results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.

Abstract Background KIT tyrosine kinase is expressed in mast cells, interstitial cells of Cajal, and hematopoietic cells. Permanently active KIT mutations lead these host cells to tumorigenesis, and to such diseases as mast cell leukemia (MCL), gastrointestinal stromal tumor (GIST), and acute myeloid leukemia (AML). Recently, we reported that in MCL, KIT with mutations (D816V, human; D814Y, mouse) traffics to endolysosomes (EL), where it can then initiate oncogenic signaling. On the other hand, KIT mutants including KITD814Y in GIST accumulate on the Golgi, and from there, activate downstream. KIT mutations, such as N822K, have been found in 30% of core binding factor-AML (CBF-AML) patients. However, how the mutants are tyrosine-phosphorylated and where they activate downstream molecules remain unknown. Moreover, it is unclear whether a KIT mutant other than KITD816V in MCL is able to signal on EL. Methods We used leukemia cell lines, such as Kasumi-1 (KIT N822K , AML), SKNO-1 (KIT N822K , AML), and HMC-1.1 (KIT V560G , MCL), to explore how KIT transduces signals in these cells and to examine the signal platform for the mutants using immunofluorescence microscopy and inhibition of intracellular trafficking. Results In AML cell lines, KITN822K aberrantly localizes to EL. After biosynthesis, KIT traffics to the cell surface via the Golgi and immediately migrates to EL through endocytosis in a manner dependent on its kinase activity. However, results of phosphorylation imaging show that KIT is preferentially activated on the Golgi. Indeed, blockade of KITN822K migration to the Golgi with BFA/M-COPA inhibits the activation of KIT downstream molecules, such as AKT, ERK, and STAT5, indicating that KIT signaling occurs on the Golgi. Moreover, lipid rafts in the Golgi play a role in KIT signaling. Interestingly, KITV560G in HMC-1.1 migrates and activates downstream in a similar manner to KITN822K in Kasumi-1. Conclusions In AML, KITN822K mislocalizes to EL. Our findings, however, suggest that the mutant transduces phosphorylation signals on lipid rafts of the Golgi in leukemia cells. Unexpectedly, the KITV560G signal platform in MCL is similar to that of KITN822K in AML. These observations provide new insights into the pathogenic role of KIT mutants as well as that of other mutant molecules.

Summary: Advanced-stage gynecologic cancer remains a life-threatening disease. Here, we present a protocol for establishment of stable in vitro 3D spheroid cells derived from human uterine endometrial and ovarian cancer tissues. The tumor-derived spheroid cells have cancer stem cell-related characteristics, including tumorigenesis, and can be used for biological and biochemical analyses and drug efficacy assays. Because these cells possess the biological characteristics of original human tumors, spheroid cells and spheroid-derived xenografts will have applications in personalized medicine in the future.For complete details on the use and execution of this protocol, please refer to Ishiguro et al. (2016) and Mori et al. (2019).

Aims: In unicompartmental knee arthroplasty (UKA), tibial components must be correctly sized and positioned so that tibial cut surfaces are well covered without marked under- or overhang with impingement of the surrounding soft tissue. We used morphometric data of both medial and lateral tibial plateaus separately to plan UKA and evaluated the compatibility of the measurement data to the dimensions of six currently available tibial prostheses in a Japanese population. Materials and Methods: Using computed tomography, we preoperatively examined 60 patients (30 medial and 30 lateral osteoarthritis (OA)) scheduled for primary UKA at our hospital between 2013 and 2017. Each tibial cutting surface was measured in the transverse plane at 2 mm below the respective joint line. We used anteroposterior and mediolateral length to calculate the mediolateral length/anteroposterior ratio of both medial and lateral compartments. We then compared measurements across six current UKA systems: Oxford fixed tibia and fixed lateral tibia, Triathlon, TRIBRID, JOURNEY UNI, and HLS Uni Evolution. Results: We found no significant differences in morphometric data between the medial and the lateral OA. The cutting surface of lateral plateau, however, had smaller anteroposterior dimensions, greater mediolateral length, and higher mediolateral length/anteroposterior ratio than those of medial plateau. Therefore, in this Japanese population-based study, Oxford lateral tibia had good compatibility with the measurement data of lateral compartments. Conclusions: Lateral compartments had lower anteroposterior length, greater mediolateral length, and higher mediolateral length/anteroposterior ratio than those of medial compartments. We, therefore, strongly recommend using Oxford fixed lateral tibia for lateral OA over other current tibial prostheses because of superior coverage.

Critical heat flux (CHF) plays an important role as the upper limit of heat dissipation process during the in-vessel reactor external reactor vessel cooling (IVR-ERVC). IVR-ERVC is regarded as a very effective way to release the decay heat after the core melt. This severe accident mitigation countermeasure has already been applied in some advanced PWRs and is considered to be used in some advanced BWRs as well. In this paper, experiments of the macro-fin structure positioned on a slope with 5° and 10° downward inclination angle were carried out. The results of the copper bar experiments were then analyzed and compared with the results obtained from a copper bare block experiments which were conducted previously at the same test facility. It was found that the CHF of the finned structure decreased compared with that of the bare surface. This shows that the current CHF data of the bare surface cannot be used directly to the design of new advanced BWRs. The hot/dry spot theory designated for the copper bar was brought up with an attempt to explain the difference of copper bare block and copper bar. Results show that using this theory can explain the decrease of CHF of the copper bar.

A calcium silicate insulator material was investigated for its interaction with non-radioactive cesium at room temperature by batch-type adsorption experiments. An industrial-grade calcium silicate was weighted and ground into powder with an average size of 15.4 ± 0.1 μm, which was then placed into the polypropylene (PP) tubes containing cesium chloride aqueous solution. Cesium concentrations were range between 1.3×10-6–3.5×10-3 M. Adsorption kinetics of cesium was evaluated based on pseudo-first order and pseudo-second order models, while the adsorption isotherm was modeled using the Langmuir model and modified BET model. Physicochemical properties of calcium silicate before and after the adsorption was investigated using XRD, SEM/EDS, and FT-IR. Chemisorption was found as the underlying sorption process between calcium silicate insulator material and cesium.

In recent years, the diversity of cancer cells in tumor tissues as a result of intratumor heterogeneity has attracted attention. In particular, the development of single-cell analysis technology has made a significant contribution to the field; technologies that are centered on single-cell RNA sequencing (scRNA-seq) have been reported to analyze cancer constituent cells, identify cell groups responsible for therapeutic resistance, and analyze gene signatures of resistant cell groups. However, although single-cell analysis is a powerful tool, various issues have been reported, including batch effects and transcriptional noise due to gene expression variation and mRNA degradation. To overcome these issues, machine learning techniques are currently being introduced for single-cell analysis, and promising results are being reported. In addition, machine learning has also been used in various ways for single-cell analysis, such as single-cell assay of transposase accessible chromatin sequencing (ATAC-seq), chromatin immunoprecipitation sequencing (ChIP-seq) analysis, and multi-omics analysis; thus, it contributes to a deeper understanding of the characteristics of human diseases, especially cancer, and supports clinical applications. In this review, we present a comprehensive introduction to the implementation of machine learning techniques in medical research for single-cell analysis, and discuss their usefulness and future potential.

Summary: The generation of tumor-initiating cells during colon carcinogenesis is associated with the dysregulation of Wnt signaling, which is known to act on Lgr5-positive intestinal stem cells. Here, using single-cell qPCR analysis, we identified a subset of Lgr5-positive stem cells that emerged during tumorigenesis in a mouse model of colon cancer. These tumor-specific Lgr5-positive cells expressed low levels of Ceacam1 and increased levels of a specific subset of Wnt targets and showed enhanced tumorigenicity. Among the Wnt targets that were specifically expressed, the long isoform of Tcf1 was required for the proliferation of tumor organoids and drove a unique Wnt target gene expression profile. Tcf1 expression increased at an early stage of colon carcinogenesis and was associated with the nuclear accumulation of β-catenin, underscoring the importance of the induction of Tcf1 expression in generating tumorigenic colon stem cells. : Shiokawa et al. show that a tumorigenic subpopulation of Lgr5-positve cells emerges during colon tumorigenesis. The tumorigenic Lgr5-positive cells express a distinct subset of Wnt target genes. The long isoform of Tcf1 dictates the unique expression profile of the Wnt targets and mediates generation of colon tumorigenic stem cells.

Summary: Cancer stem cells (CSCs) are associated with the refractory nature of cancer, and elucidating the targetable pathways for CSCs is crucial for devising innovative antitumor therapies. We find that the proliferation of CSC-enriched colon spheroids from clinical specimen is dependent on mTORC1 kinase, which is activated by reactive oxygen species (ROS) produced by NOX1, an NADPH oxidase. In the spheroid-derived xenograft tumors, NOX1 is preferentially expressed in LGR5-positive cells. Dependence on NOX1 expression or mTOR kinase activity is corroborated in the xenograft tumors and mouse colon cancer-derived organoids. NOX1 co-localizes with mTORC1 in VPS41-/VPS39-positive lysosomes, where mTORC1 binds to S100A9, a member of S100 calcium binding proteins, in a NOX1-produced ROS-dependent manner. S100A9 is oxidized by NOX1-produced ROS, which facilitates binding to mTORC1 and its activation. We propose that NOX1-dependent mTORC1 activation via S100A9 oxidation in VPS41-/VPS39-positive lysosomes is crucial for colon CSC proliferation and colon cancer progression. : Using patient-derived colon cancer spheroids to investigate biological pathways for cancer stem cell (CSC) proliferation, Ohata et al. demonstrate that induction of NOX1, an NAPDH oxidase, induces activation of mTORC1 via S100A9 oxidation in endolysosomes. The resulting activation of mTORC1 is essential for proliferation of the spheroids and colon cancer. Keywords: colon cancer, NOX1, mTORC1, cancer stem cells, ROS, S100A9

Summary: Degradation of mitochondria by selective autophagy, termed mitophagy, contributes to the control of mitochondrial quality. Bcl2-L-13 is a mammalian homolog of Atg32, which is an essential mitophagy receptor in yeast. However, the molecular machinery involved in Bcl2-L-13-mediated mitophagy remains to be elucidated. Here, we show that the ULK1 (unc-51-like kinase) complex is required for Bcl2-L-13 to process mitophagy. Screening of a series of yeast Atg mutants revealed that a different set of ATG genes is used for Bcl2-L-13- and Atg32-mediated mitophagy in yeast. The components of the Atg1 complex essential for starvation-induced autophagy were indispensable in Bcl2-L-13-, but not Atg32-mediated, mitophagy. The ULK1 complex, a counterpart of the Atg1 complex, is necessary for Bcl2-L-13-mediated mitophagy in mammalian cells. We propose a model where, upon mitophagy induction, Bcl2-L-13 recruits the ULK1 complex to process mitophagy and the interaction of LC3B with ULK1, as well as Bcl2-L-13, is important for the mitophagy. : Upon starvation, autophagy degrades cellular components to obtain nutrition for survival. Damaged mitochondria are removed by mitophagy, which is a specific form of autophagy. Bcl2-L-13 protein is involved in the process. Murakawa et al. find that the ULK1 complex, essential for autophagy, is necessary for Bcl2-L-13-mediated mitophagy. Keywords: Atg32, Bcl2-L-13, mitochondria, mitophagy

TRAF2 and NCK-interacting protein kinase (TNIK) is a key regulatory component of the TCF4 and β-catenin transcriptional complex. In this study, the authors identify a TNIK inhibitor that blocks Wnt signalling and Wnt-driven colorectal tumorigenesis in mice.

To develop Pu-burner high temperature gas-cooled reactor (HTGR) attaining very high burn-up around 500 GWd/t, the security and safety fuel (3S-TRISO fuel) is proposed. The 3S-TRISO fuel employs the coated fuel particle with a fuel kernel made of plutonium dioxide (PuO2) and yttria stabilized zirconia (YSZ) as an inert matrix. Especially, a zirconium carbide (ZrC) coating is one of key technologies of the 3S-TRISO, which performs as an oxygen getter to reduce the fuel failure due to internal pressure during the irradiation. R&Ds on ZrC coating directly on the dummy kernel made of YSZ which surrogates PuO2-YSZ have been started in the Japanese fiscal year 2015. As results of ZrC coating tests on YSZ particle by the bromide process developed in Japan Atomic Energy Agency, stoichiometric ZrC coatings with 18 - 21 μm of thicknesses were obtained with 0.1 kg of particle loading weight. As indicated by a thermochemical analysis on the chemical stability of YSZ against the ZrC bromide process, no deterioration of YSZ exposed by source gases of ZrC bromide process was observed through the characterization by Scanning Transmission Electron Microscope (STEM) observation. Finally, reproducibility of stoichiometric ZrC coating on YSZ particle was demonstrated by the bromide chemical vapor deposition process.

Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) is considered a representative marker of intestinal stem cells from both non-tumor and tumor tissues. However, it remains unclear whether all or only a fraction of Lgr5-positive cells behave as stem cells. Recently, we reported that Lgr5-positive cells from non-tumor and tumor tissues can be classified into overlapping yet distinct groups and that the tumor-specific groups are associated with tumorigenic capability, suggesting that these cells could represent targets for therapeutic intervention.

Spondylolysis is a major cause of back pain in children and adolescents. The prevalence of spondylolysis depends on the sex, race, and congenital abnormality. These biases suggest hereditary predisposition as an etiology of spondylolysis. However, no conclusive evidence still exists regarding the inheritance for spondylolysis. Herein, we report rare cases with familial occurrence of lumbar spondylolysis. In two generations of a Japanese family, three brothers including identical twins complained of back pain related to sports activities. Clinical, radiographic, and computed tomographic examinations identified spondylolysis at L4 and L5 in all these boys. The father experienced long-term back pain and had spondylolisthesis at L4–L5 and healed spondylolysis at L5. The daughter and mother did not show any lumbar spondylolysis. The frequent development of spondylolysis at L4, a rarely affected segment, in four of six family members (66.7%) support that lumbar spondylolysis has an underlying genetic etiology, primarily autosomal dominant inheritance.

The creep-buckling behaviors of cylindrical stainless-steel tubes subjected to radial external pressure load at elevated temperatures—800, 900, and 1000 °C—were experimentally investigated. Prior to the creep-buckling tests, the buckling pressure was measured under each temperature condition. Then, in creep-buckling experiments, the creep-buckling failure time was measured by reducing the external pressure load for two different tube specimens—representing the first and second buckling modes—to examine the relationship between the external pressure and the creep-buckling failure time. The measured failure time ranged from

Non-muscle invasive (superficial) bladder cancer is a low-grade malignancy with good prognosis, while muscle invasive (invasive) bladder cancer is a high-grade malignancy with poor prognosis. N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induces superficial bladder cancers with papillary morphology in rats and invasive bladder cancers with infiltrating phenotype in mice. In this study, we analyzed genomic landscapes of rodent BBN-induced bladder cancers using array-based comparative genomic hybridization (array CGH). While no significant copy number alterations were detected in superficial bladder tumors in rats, copy number gains in chromosomal regions 2D-E1, 7qA3, 9F2, and 11C-D were detected in invasive bladder tumors in mice. Amplification of representative genes located on 2D-E1 and 7qA3 chromosomal regions was confirmed by quantitative PCR. Cyp2a22 and Cyp2a5 genes but not Cyp2g1, Cyp2a12, and Rab4b genes on mouse chromosome 7qA3 were amplified in invasive bladder cancers. Although the human ortholog gene of Cyp2a22 has not been confirmed, the mouse Cyp2a5 gene is the ortholog of the human CYP2A6 gene located in chromosomal region 19q13.2, and CYP2A6 was identified by database search as one of the closest human homolog to mouse Cyp2a22. Considering a possibility that this region may be related to mouse 7qA3, we analyzed CYP2A6 copy number and expression in human bladder cancer using cell lines and resected tumor specimens. Although only one of eight cell lines showed more than one copy increase of the CYP2A6 gene, CYP2A6 amplification was detected in six out of 18 primary bladder tumors where it was associated with the invasive phenotype. Immunohistochemical analyses of 118 primary bladder tumors revealed that CYP2A6 protein expression was also higher in invasive tumors, especially in those of the scattered type. Together, these findings indicate that the amplification and overexpression of the CYP2A6 gene are characteristic of human bladder cancers with increased malignancy and that CYP2A6 can be a candidate prognostic biomarker in this type of cancer.

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.

This paper introduces our own hierarchical data visualization technique, HeiankyoView, and discusses its potential for fluid science. HeiankyoView represents hierarchy as nested rectangles, and displays thousands of data elements in one display space. The paper briefly introduces the algorithm and various applications of HeiankyoView, and discusses our attempts for visualization of computational fluid dynamics data. Finally, the paper introduces our work on visualization of atomic plant data using HeiankyoView.

Flow-induced acoustic resonance in a piping system containing closed coaxial side-branches was investigated experimentally. Resonance characteristics of the piping system were examined by a microphone. The results revealed that the resonance frequencies of the shear layer instability were locked in corresponding to the natural frequencies of the side-branches. Phase-averaged velocity fields were obtained two-dimensionally in the junction of coaxial side-branches by dynamic particle image velocimetry (PIV), while the acoustic resonance was induced at the first and second hydrodynamic modes. Patterns of jet correspond to two hydrodynamic modes were derived from the phase-averaged velocity fields. The dynamic PIV can acquire time-series velocity fluctuations, then, two-dimensional phase delay maps under resonance and off-resonance conditions in the junction of coaxial side-branches were obtained. Experimental results show that the proposed phase delay map method costs less experiment and computation time and achieves a better accuracy and repetition than the phase-locking technique. In addition, the phase delay map method can obtain phase difference under the different frequency components. This is important when two different acoustic modes were induced in one experimental condition.

Androgen receptor is a primary transcription factor involved in the proliferation of prostate cancer cells. Thus, hormone therapy using antiandrogens, such as bicalutamide, is a first-line treatment for the disease. Although hormone therapy initially reduces the tumor burden, many patients eventually relapse, developing tumors with acquired endocrine resistance. Elucidation of the molecular mechanisms underlying endocrine resistance is therefore a fundamental issue for the understanding and development of alternative therapeutics for advanced prostate cancer. In the present study, we performed short hairpin RNA (shRNA)-mediated functional screening to identify genes involved in bicalutamide-mediated effects on LNCaP prostate cancer cells. Among such candidate genes selected by screening using volcano plot analysis, ribosomal protein L31 (RPL31) was found to be essential for cell proliferation and cell-cycle progression in bicalutamide-resistant LNCaP (BicR) cells, based on small interfering RNA (siRNA)-mediated knockdown experiments. Of note, RPL31 mRNA is more abundantly expressed in BicR cells than in parental LNCaP cells, and clinical data from ONCOMINE and The Cancer Genome Altas showed that RPL31 is overexpressed in prostate carcinomas compared with benign prostate tissues. Intriguingly, protein levels of the tumor suppressor p53 and its targets, p21 and MDM2, were increased in LNCaP and BicR cells treated with RPL31 siRNA. We observed decreased degradation of p53 protein after RPL31 knockdown. Moreover, the suppression of growth and cell cycle upon RPL31 knockdown was partially recovered with p53 siRNA treatment. These results suggest that RPL31 is involved in bicalutamide-resistant growth of prostate cancer cells. The shRNA-mediated functional screen in this study provides new insight into the molecular mechanisms and therapeutic targets of advanced prostate cancer.

Haploid embryonic stem cells (ESCs) are useful for studying mammalian genes because disruption of only one allele can cause loss-of-function phenotypes. Here, we report the use of haploid ESCs and the CRISPR RNA-guided Cas9 nuclease gene-targeting system to manipulate mammalian genes. Co-transfection of haploid ESCs with vectors expressing Cas9 nuclease and single-guide RNAs (sgRNAs) targeting Tet1, Tet2, and Tet3 resulted in the complete disruption of all three genes and caused a loss-of-function phenotype with high efficiency (50%). Co-transfection of cells with vectors expressing Cas9 and sgRNAs targeting two loci on the same chromosome resulted in the creation of a large chromosomal deletion and a large inversion. Thus, the use of the CRISPR system in combination with haploid ESCs provides a powerful platform to manipulate the mammalian genome.

Accomplishing the retrieval of fuel debris from Fukushima Daiichi Nuclear Power Plant (1F) Unit 3 (1F3) requires an understanding of its distribution. In this study, we performed real-scale corium spreading and sedimentation behavior analyses using Lagrangian moving particle hydrodynamics and large eddy simulation methods. These methods allowed us to calculate the spreading of corium with various shear viscosities under water conditions and to propose the best estimation for the fuel debris distribution in 1F3. To minimize uncertainties arising from unknown boundary conditions, we investigated relevant parameters through literature review. Our analyses showed that highly viscous corium tends to pile up within the pedestal region under strong convective vapor and boiling heat transfer, while low-viscosity corium spreads to the outside of the pedestal regions regardless of cooling efficiency. We identified three cooling modes based on initial shear viscosity and cooling efficiency and predicted the fuel debris distribution in 1F3 by comparing our results to those of the Tokyo Electric Power Company (TEPCO) and Organisation for Economic Co-operation and Development/Nuclear Energy Agency (OECD/NEA) Benchmark Study of the Accident at the Fukushima Daiichi Nuclear Power Station (BSAF) project. The distribution estimation of highly viscous corium derived from oxidic corium is consistent with the three-dimensional reconstructed image by TEPCO and the calculated results by the OECD/NEA BSAF project. [ABSTRACT FROM AUTHOR]