Structural snapshots of actively translating human ribosomes.

Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.

Pulmonary MRI with ultra-short TE using single- and dual-echo methods: comparison of capability for quantitative differentiation of non- or minimally invasive adenocarcinomas from other lung cancers with that of standard-dose thin-section CT.

OBJECTIVE: The purpose of this study was thus to compare capabilities for quantitative differentiation of non- and minimally invasive adenocarcinomas from other of pulmonary MRIs with ultra-short TE (UTE) obtained with single- and dual-echo techniques (UTE-MRISingle and UTE-MRIDual) and thin-section CT for stage IA lung cancer patients. METHODS: Ninety pathologically diagnosed stage IA lung cancer patients who underwent thin-section standard-dose CT, UTE-MRISingle, and UTE-MRIDual, surgical treatment and pathological examinations were included in this retrospective study. The largest dimension (Dlong), solid portion (solid Dlong), and consolidation/tumor (C/T) ratio of each nodule were assessed. Two-tailed Student's t-tests were performed to compare all indexes obtained with each method between non- and minimally invasive adenocarcinomas and other lung cancers. Receiver operating characteristic (ROC)-based positive tests were performed to determine all feasible threshold values for distinguishing non- or minimally invasive adenocarcinoma (MIA) from other lung cancers. Sensitivity, specificity, and accuracy were then compared by means of McNemar's test. RESULTS: Each index showed significant differences between the two groups (p < 0.0001). Specificities and accuracies of solid Dlong for UTE-MRIDual2nd echo and CTMediastinal were significantly higher than those of solid Dlong for UTE-MRISingle and UTE-MRIDual1st echo and all C/T ratios except CTMediastinal (p < 0.05). Moreover, the specificities and accuracies of solid Dlong and C/T ratio were significantly higher than those of Dlong for each method (p < 0.05). CONCLUSION: Pulmonary MRI with UTE is considered at least as valuable as thin-section CT for quantitative differentiation of non- and minimally invasive adenocarcinomas from other stage IA lung cancers. CLINICAL RELEVANCE STATEMENT: Pulmonary MRI with UTE's capability for quantitative differentiation of non- and minimally invasive adenocarcinomas from other lung cancers in stage IA lung cancer patients is equal or superior to that of thin-section CT. KEY POINTS: • Correlations were excellent for pathologically examined nodules with the largest dimensions (Dlong) and a solid component (solid Dlong) for all indexes (0.95 ≤ r ≤ 0.99, p < 0.0001). • Pathologically examined Dlong and solid Dlong obtained with all methods showed significant differences between non- and minimally invasive adenocarcinomas and other lung cancers (p < 0.0001). • Solid tumor components are most accurately measured by UTE-MRIDual2nd echo and CTMediastinal, whereas the ground-glass component is imaged by UTE-MRIDual1st echo and CTlung with high accuracy. UTE-MRIDual predicts tumor invasiveness with 100% sensitivity and 87.5% specificity at a C/T threshold of 0.5.

Chemical Exchange Saturation Transfer MRI: Capability for Predicting Therapeutic Effect of Chemoradiotherapy on Non-Small Cell Lung Cancer Patients.

BACKGROUND: Amide proton transfer (APT) weighted chemical exchange saturation transfer CEST (APTw/CEST) magnetic resonance imaging (MRI) has been suggested as having the potential for assessing the therapeutic effect of brain tumors or rectal cancer. Moreover, diffusion-weighted imaging (DWI) and positron emission tomography fused with computed tomography by means of 2-[fluorine-18]-fluoro-2-deoxy-D-glucose (FDG-PET/CT) have been suggested as useful in same setting. PURPOSE: To compare the capability of APTw/CEST imaging, DWI, and FDG-PET/CT for predicting therapeutic effect of chemoradiotherapy (CRT) on stage III non-small cell lung cancer (NSCLC) patients. STUDY TYPE: Prospective. POPULATION: Eighty-four consecutive patients with Stage III NSCLC, 45 men (age range, 62-75 years; mean age, 71 years) and 39 women (age range, 57-75 years; mean age, 70 years). All patients were then divided into two groups (Response Evaluation Criteria in Solid Tumors [RECIST] responders, consisting of the complete response and partial response groups, and RECIST non-responders, consisting of the stable disease and progressive disease groups). FIELD STRENGTH/SEQUENCE: 3 T, echo planar imaging or fast advanced spin-echo (FASE) sequences for DWI and 2D half Fourier FASE sequences with magnetization transfer pulses for CEST imaging. ASSESSMENT: Magnetization transfer ratio asymmetry (MTRasym ) at 3.5 ppm, apparent diffusion coefficient (ADC), and maximum standard uptake value (SUVmax, ) on PET/CT were assessed by means of region of interest (ROI) measurements at primary tumor. STATISTICAL TESTS: Kaplan-Meier method followed by log-rank test and Cox proportional hazards regression analysis with multivariate analysis. A P value <0.05 was considered statistically significant. RESULTS: Progression-free survival (PFS) and overall survival (OS) had significant difference between two groups. MTRasym at 3.5 ppm (hazard ratio [HR] = 0.70) and SUVmax (HR = 1.41) were identified as significant predictors for PFS. Tumor staging (HR = 0.57) was also significant predictors for OS. DATA CONCLUSION: APTw/CEST imaging showed potential performance as DWI and FDG-PET/CT for predicting the therapeutic effect of CRT on stage III NSCLC patients. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 1.

Computed DWI MRI Results in Superior Capability for N-Stage Assessment of Non-Small Cell Lung Cancer Than That of Actual DWI, STIR Imaging, and FDG-PET/CT.

BACKGROUND: Computed diffusion-weighted imaging (cDWI) is a mathematical computation technique that generates DWIs for any b-value by using actual DWI (aDWI) data with at least two different b-values and may improve differentiation of metastatic from nonmetastatic lymph nodes. PURPOSE: To determine the appropriate b-value for cDWI to achieve a better diagnostic capability for lymph node staging (N-staging) in non-small cell lung cancer (NSCLC) patients compared to aDWI, short inversion time (TI) inversion recovery (STIR) imaging, or positron emission tomography with 2-[fluorine-18] fluoro-2-deoxy-d-glucose combined with computed tomography (FDG-PET/CT). STUDY TYPE: Prospective. SUBJECTS: A total of 245 (127 males and 118 females; mean age 72 years) consecutive histopathologically confirmed NSCLC patients. FIELD STRENGTH/SEQUENCE: A 3 T, half-Fourier single-shot turbo spin-echo sequence, electrocardiogram (ECG)-triggered STIR fast advanced spin-echo (FASE) sequence with black blood and STIR acquisition and DWI obtained by FASE with b-values of 0 and 1000 sec/mm2 . ASSESSMENT: From aDWIs with b-values of 0 and 1000 (aDWI1000 ) sec/mm2 , cDWI using 400 (cDWI400 ), 600 (cDWI600 ), 800 (cDWI800 ), and 2000 (cDWI2000 ) sec/mm2 were generated. Then, 114 metastatic and 114 nonmetastatic nodes (mediastinal and hilar lymph nodes) were selected and evaluated with a contrast ratio (CR) for each cDWI and aDWI, apparent diffusion coefficient (ADC), lymph node-to-muscle ratio (LMR) on STIR, and maximum standard uptake value (SUVmax ). STATISTICAL TESTS: Receiver operating characteristic curve (ROC) analysis, Youden index, and McNemar's test. RESULTS: Area under the curve (AUC) of CR600 was significantly larger than the CR400 , CR800 , CR2000 , aCR1000 , and SUVmax . Comparison of N-staging accuracy showed that CR600 was significantly higher than CR400 , CR2000 , ADC, aCR1000 , and SUVmax , although there were no significant differences with CR800 (P = 0.99) and LMR (P = 0.99). DATA CONCLUSION: cDWI with b-value at 600 sec/mm2 may have potential to improve N-staging accuracy as compared with aDWI, STIR, and PET/CT. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

Three-Dimensional Gradient-Echo-Based Amide Proton Transfer-Weighted Imaging of Brain Tumors: Comparison With Two-Dimensional Spin-Echo-Based Amide Proton Transfer-Weighted Imaging.

OBJECTIVE: Although amide proton transfer-weighted (APTw) imaging is reported by 2-dimensional (2D) spin-echo-based sequencing, 3-dimensional (3D) APTw imaging can be obtained by gradient-echo-based sequencing. The purpose of this study was to compare the efficacy of APTw imaging between 2D and 3D imaging in patients with various brain tumors. METHODS: A total of 49 patients who had undergone 53 examinations [5 low-grade gliomas (LGG), 16 high-grade gliomas (HGG), 6 malignant lymphomas, 4 metastases, and 22 meningiomas] underwent APTw imaging using 2D and 3D sequences. The magnetization transfer ratio asymmetry (MTR asym ) was assessed by means of region of interest measurements. Pearson correlation was performed to determine the relationship between MTR asym for the 2 methods, and Student's t test to compare MTR asym for LGG and HGG. The diagnostic accuracy to differentiate HGG from LGG of the 2 methods was compared by means of the McNemar test. RESULTS: Three-dimensional APTw imaging showed a significant correlation with 2D APTw imaging ( r = 0.79, P < 0.0001). The limits of agreement between the 2 methods were -0.021 ± 1.42%. The MTR asym of HGG (2D: 1.97 ± 0.96, 3D: 2.11 ± 0.95) was significantly higher than those of LGG (2D: 0.46 ± 0.89%, P < 0.01; 3D: 0.15 ± 1.09%, P < 0.001). The diagnostic performance of the 2 methods to differentiate HGG from LGG was not significantly different ( P = 1). CONCLUSIONS: The potential capability of 3D APTw imaging is equal to or greater than that of 2D APTw imaging and is considered at least as valuable in patients with brain tumors.

Effect of fibrinogen replacement therapy on bleeding outcomes and 1-year mortality in patients undergoing thoracic aortic surgery: a retrospective cohort study.

PURPOSE: This study aimed to examine the effect of fibrinogen replacement therapy with cryoprecipitate or fibrinogen concentrate on bleeding outcomes and 1-year mortality in patients undergoing thoracic aortic surgery. METHODS: We retrospectively studied 439 consecutive patients who underwent thoracic aortic surgery with cardiopulmonary bypass between January 1st, 2010 and December 31st, 2019 and identified patients who received cryoprecipitate or fibrinogen concentrate (the fibrinogen replacement group) and those who did not (the control group). Multivariate analyses were performed to examine the associations of fibrinogen replacement therapy with perioperative major bleeding (i.e., excessive hemorrhage or blood transfusion), re-exploration for bleeding, and 1-year mortality. RESULTS: There were 285 patients in the fibrinogen replacement group who received 2.2 ± 1.0 g of concentrated fibrinogen amount and 154 patients in the control group. The incidence of major bleeding in the fibrinogen replacement group was less than that in the control group in patients with fibrinogen level < 150 mg/dL during cardiopulmonary bypass (49.7% versus 74.6%, p = 0.0007, multivariate odds ratio; 0.33, 95% confidence intervals; 0.12-0.91, p = 0.03), but not in patients with fibrinogen level ≥ 150 mg/dL (25.0% versus 29.6%, p = 0.51). No significant difference was found in re-exploration for bleeding (1.0% versus 1.3%, p = 1.00) or 1-year mortality (10.4% versus 5.3%, multivariate Cox proportional-hazard ratio; 1.03, 95% confidence intervals; 0.82-1.31, p = 0.74) between the fibrinogen replacement group and the control group. CONCLUSIONS: The results of this study indicate that 2-3 g of fibrinogen replacement reduces the incidence of major bleeding in patients with hypofibrinogenemia during cardiopulmonary bypass in thoracic aortic surgery.

Deep Learning Reconstruction of Diffusion-weighted MRI Improves Image Quality for Prostatic Imaging.

Background Deep learning reconstruction (DLR) may improve image quality. However, its impact on diffusion-weighted imaging (DWI) of the prostate has yet to be assessed. Purpose To determine whether DLR can improve image quality of diffusion-weighted MRI at b values ranging from 1000 sec/mm2 to 5000 sec/mm2 in patients with prostate cancer. Materials and Methods In this retrospective study, images of the prostate obtained at DWI with a b value of 0 sec/mm2, DWI with a b value of 1000 sec/mm2 (DWI1000), DWI with a b value of 3000 sec/mm2 (DWI3000), and DWI with a b value of 5000 sec/mm2 (DWI5000) from consecutive patients with biopsy-proven cancer from January to June 2020 were reconstructed with and without DLR. Image quality was assessed using signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) from region-of-interest analysis and qualitatively assessed using a five-point visual scoring system (1 [very poor] to 5 [excellent]) for each high-b-value DWI sequence with and without DLR. The SNR, CNR, and visual score for DWI with and without DLR were compared with the paired t test and the Wilcoxon signed rank test with Bonferroni correction, respectively. Apparent diffusion coefficients (ADCs) from DWI with and without DLR were also compared with the paired t test with Bonferroni correction. Results A total of 60 patients (mean age, 67 years; age range, 49-79 years) were analyzed. DWI with DLR showed significantly higher SNRs and CNRs than DWI without DLR (P < .001); for example, with DWI1000 the mean SNR was 38.7 ± 0.6 versus 17.8 ± 0.6, respectively (P < .001), and the mean CNR was 18.4 ± 5.6 versus 7.4 ± 5.6, respectively (P < .001). DWI with DLR also demonstrated higher qualitative image quality than DWI without DLR (mean score: 4.8 ± 0.4 vs 4.0 ± 0.7, respectively, with DWI1000 [P = .001], 3.8 ± 0.7 vs 3.0 ± 0.8 with DWI3000 [P = .002], and 3.1 ± 0.8 vs 2.0 ± 0.9 with DWI5000 [P < .001]). ADCs derived with and without DLR did not differ substantially (P > .99). Conclusion Deep learning reconstruction improves the image quality of diffusion-weighted MRI scans of prostate cancer with no impact on apparent diffusion coefficient quantitation with a 3.0-T MRI system. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Turkbey in this issue.

Efficacy of Ultrashort Echo Time Pulmonary MRI for Lung Nodule Detection and Lung-RADS Classification.

Background Pulmonary MRI with ultrashort echo time (UTE) has been compared with chest CT for nodule detection and classification. However, direct comparisons of these methods' capabilities for Lung CT Screening Reporting and Data System (Lung-RADS) evaluation remain lacking. Purpose To compare the capabilities of pulmonary MRI with UTE with those of standard- or low-dose thin-section CT for Lung-RADS classification. Materials and Methods In this prospective study, standard- and low-dose chest CT (270 mA and 60 mA, respectively) and MRI with UTE were used to examine consecutive participants enrolled between January 2017 and December 2020 who met American College of Radiology Appropriateness Criteria for lung cancer screening with low-dose CT. Probability of nodule presence was assessed for all methods with a five-point visual scoring system by two board-certified radiologists. All nodules were then evaluated in terms of their Lung-RADS classification using each method. To compare nodule detection capability of the three methods, consensus for performances was rated by using jackknife free-response receiver operating characteristic analysis, and sensitivity was compared by means of the McNemar test. In addition, weighted κ statistics were used to determine the agreement between Lung-RADS classification obtained with each method and the reference standard generated from standard-dose CT evaluated by two radiologists who were not included in the image analysis session. Results A total of 205 participants (mean age: 64 years ± 7 [standard deviation], 106 men) with 1073 nodules were enrolled. Figure of merit (FOM) (P < .001) had significant differences among three modalities (standard-dose CT: FOM = 0.91, low-dose CT: FOM = 0.89, pulmonary MRI with UTE: FOM = 0.94), with no evidence of false-positive findings in participants with all modalities (P > .05). Agreements for Lung-RADS classification between all modalities and the reference standard were almost perfect (standard-dose CT: κ = 0.82, P < .001; low-dose CT: κ = 0.82, P < .001; pulmonary MRI with UTE: κ = 0.82, P < .001). Conclusion In a lung cancer screening population, ultrashort echo time pulmonary MRI was comparable to standard- or low-dose CT for Lung CT Screening Reporting and Data System classification. © RSNA, 2021 Online supplemental material is available for this article. See also the editorial by Wielpütz in this issue.

Comparison of utility of deep learning reconstruction on 3D MRCPs obtained with three different k-space data acquisitions in patients with IPMN.

OBJECTIVE: To compare the utility of deep learning reconstruction (DLR) for improving acquisition time, image quality, and intraductal papillary mucinous neoplasm (IPMN) evaluation for 3D MRCP obtained with parallel imaging (PI), multiple k-space data acquisition for each repetition time (TR) technique (Fast 3D mode multiple: Fast 3Dm) and compressed sensing (CS) with PI. MATERIALS AND METHODS: A total of 32 IPMN patients who had undergone 3D MRCPs obtained with PI, Fast 3Dm, and CS with PI and reconstructed with and without DLR were retrospectively included in this study. Acquisition time, signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) obtained with all protocols were compared using Tukey's HSD test. Results of endoscopic ultrasound, ERCP, surgery, or pathological examination were determined as standard reference, and distribution classifications were compared among all 3D MRCP protocols by McNemar's test. RESULTS: Acquisition times of Fast 3Dm and CS with PI with and without DLR were significantly shorter than those of PI with and without DLR (p < 0.05). Each MRCP sequence with DLR showed significantly higher SNRs and CNRs than those without DLR (p < 0.05). IPMN distribution accuracy of PI with and without DLR and Fast 3Dm with DLR was significantly higher than that of Fast 3Dm without DLR and CS with PI without DLR (p < 0.05). CONCLUSION: DLR is useful for improving image quality and IPMN evaluation capability on 3D MRCP obtained with PI, Fast 3Dm, or CS with PI. Moreover, Fast 3Dm and CS with PI may play as substitution to PI for MRCP in patients with IPMN. KEY POINTS: • Mean examination times of multiple k-space data acquisitions for each TR and compressed sensing with parallel imaging were significantly shorter than that of parallel imaging (p < 0.0001). • When comparing image quality of 3D MRCPs with and without deep learning reconstruction, deep learning reconstruction significantly improved signal-to-noise ratio and contrast-to-noise ratio (p < 0.05). • IPMN distribution accuracies of parallel imaging with and without deep learning reconstruction (with vs. without: 88.0% vs. 88.0%) and multiple k-space data acquisitions for each TR with deep learning reconstruction (86.0%) were significantly higher than those of others (p < 0.05).

[Relationship between the rate of a decreased oral function and the nutrient intake in community-dwelling older persons: An examination using oral function-related items in a questionnaire for latter-stage elderly people].

AIM: To determine the rate of a decreased oral function using questions from the Kihon checklist corresponding to the Questionnaire for Latter-stage Elderly People and to clarify nutrient intake in older persons. METHODS: This study targeted 511 older people (217 men, 294 women, average age 73.1±5.6 years old). Their oral function was evaluated using questions on the masticatory function and swallowing function from the Kihon checklist, corresponding to questions on the oral function in the Questionnaire for Latter-stage Elderly People. Participants who had at least one symptom measured were defined as the applicable group (AG). In addition, to evaluate the nutrient intake of the participants, interviews were conducted using the Food Frequency Questionnaire Based on Food Groups. RESULTS: The rate of inclusion in the AG was 32.9% for the total sample, 28.2% for early-stage elderly people, and 40.1% for latter-stage elderly people. The AG rates did not differ significantly between men and women. For latter-stage elderly people, the protein-energy ratio and intakes of total energy, protein, pantothenic acid, folic acid, vitamin B6, niacin, vitamin K, copper, zinc, phosphorus, magnesium, potassium, and total dietary fiber were significantly lower in the AG than in the non-AG. CONCLUSION: The evaluation of placement in the AG through questions on the oral function from the Kihon checklist corresponding to the Questionnaire for Latter-stage Elderly People demonstrated that the rate of a decreased oral function was higher in latter-stage elderly people than in early-stage elderly people. In addition, the latter-stage elderly people in the AG had a lower nutrient intake.

Molecular architecture of the ribosome-bound Hepatitis C Virus internal ribosomal entry site RNA.

Internal ribosomal entry sites (IRESs) are structured cis-acting RNAs that drive an alternative, cap-independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo-EM reconstructions of the ribosome 80S- and 40S-bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Šfor the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P-site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA-driven translation initiation.

TORC1 regulates the DNA damage checkpoint via checkpoint protein levels.

Target of rapamycin complex 1 (TORC1) protein kinase, a master controller of cell growth, is thought to be involved in genome integrity. However, the molecular mechanisms associated with this are unclear. Here, we show that TORC1 inactivation causes decreases in the levels of a wide range of proteins involved in the DNA damage checkpoint (DDC) signaling including Tel1, Mre11, Rad9, Mrc1, and Chk1 in budding yeast. Furthermore, TORC1 inactivation compromised DDC activation, DNA repair, and cell survival after DNA damage. TORC1 inactivation promoted proteasomal degradation of Rad9 and Mre11 in a manner dependent on Skp1-Cullin-F-box protein (SCF). Finally, CDK promoted the degradation of Rad9. This study revealed that TORC1 is essential for genome integrity via the maintenance of DDC signaling.

70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria.

According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1-IF3). Here, we describe a novel type of initiation termed "70S-scanning initiation," where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine-Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.

CDK phosphorylation regulates Mcm3 degradation in budding yeast.

Accurate regulation of activity and level of the MCM complex is critical for precise DNA replication and genome transmission. Cyclin-dependent kinase (CDK) negatively regulates nuclear localization of the MCM complex via phosphorylation of the Mcm3 subunit. More recently, we found that Mcm3 is degraded via the Skp1-Cullin-F-box (SCF)-proteasome axis in budding yeast. However, how Mcm3 degradation is regulated is largely unknown. Here, we show that CDK represses Mcm3 degradation. Phosphorylated Mcm3 was excluded from the nucleus, where SCF is predominantly located, although CDK-mediated phosphorylation itself generated a phosphodegron of Mcm3, stimulating the degradation of Mcm3 resident in the nucleus. Thus, CDK negatively regulated nuclear MCM levels by exclusion from the nucleus and degradation in the nucleus via Mcm3 phosphorylation. We will discuss the physiological importance of Mcm3 degradation.

TORC1 signaling regulates DNA replication via DNA replication protein levels.

Accurate DNA replication is at the heart of faithful genome transmission in dividing cells. DNA replication is strictly controlled by various factors. However, how environmental stresses such as nutrient starvation impact on these factors and DNA replication is largely unknown. Here we show that DNA replication is regulated by target of rapamycin complex 1 (TORC1) protein kinase, which is a central regulator of cell growth and proliferation in response to nutrients. TORC1 inactivation reduced the levels of various proteins critical for DNA replication initiation, such as Mcm3, Orc3, Cdt1, and Sld2, and retarded DNA replication. TORC1 inactivation promoted proteasome-mediated Mcm3 degradation. Skp1-Cullin-F-box (SCF)-Grr1 and PEST motif mediated Mcm3 degradation. TORC1-downstream factors PP2A-Cdc55 protein phosphatase and protein kinase A regulated Mcm3 degradation. This study showed that TORC1 signaling modulates DNA replication to coordinate cell growth and genome replication in response to nutrient availability.

Unique coenzyme binding mode of hyperthermophilic archaeal sn-glycerol-1-phosphate dehydrogenase from Pyrobaculum calidifontis.

A gene encoding an sn-glycerol-1-phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (Vmax ) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2' of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild-type enzyme with NADPH, which suggests that the biased interactions around the C2'-phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)-dependent dehydrogenases. Proteins 2016; 84:1786-1796. © 2016 Wiley Periodicals, Inc.

[Evaluation of Drug Interaction between S-1 and Warfarin].

Prolonged prothrombin time is observed in patients taking warfarin (WF) with a fluoropyrimidine, such as S-1. When WF is combined with S-1, the prothrombin time-international normalized ratio (PT-INR) and dose adjustment of WF should be closely monitored. To date, no clinical data have been reported in terms of the relation between temporal variation of PT-INR and its therapeutic range. In this study, we retrospectively collected patients' clinical data including PT-INR. We identified 21 patients receiving WF therapy before the start of S-1 treatment. Patient characteristics were male/female: 18/3, median age: 69 (range 48-81) years old, cancer of gastric/lung/pancreatic/other: 8/5/4/4, and history of deep vein thrombosis (DVT)/atrial fibrillation (AF)/cerebral infarction (CI)/other: 11/6/2/2. The PT-INR of 16 patients exceeded normal upper limits after taking S-1 with WF. The median time to exceed the PT-INR upper therapeutic range is 25 (range 3-77) days. Patients receiving WF anticoagulant therapy concomitant with S-1 should have their PT-INR closely monitored and WF doses adjusted accordingly.

S-nitrosylation of mouse galectin-2 prevents oxidative inactivation by hydrogen peroxide.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. A proteomic analysis identified Gal-2 as a protein that was S-nitrosylated when mouse gastric mucosal lysates were reacted with S-nitrosoglutathione, a physiologically relevant S-nitrosylating agent. In the present study, recombinant mouse (m)Gal-2 was S-nitrosylated using nitrosocysteine (CysNO), which had no effect on the sugar-binding specificity and dimerization capacity of the protein. On the other hand, mGal-2 oxidation by H2O2 resulted in the loss of sugar-binding ability, while S-nitrosylation prevented H2O2-inducted inactivation, presumably by protecting the Cys residue(s) in the protein. These results suggest that S-nitrosylation by nitric oxides protect Gal-2 from oxidative stress in the gastrointestinal tract.

Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast.

Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. (1)H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be the potential materials for discovering further novel lifespan genes.

RsfA (YbeB) proteins are conserved ribosomal silencing factors.

The YbeB (DUF143) family of uncharacterized proteins is encoded by almost all bacterial and eukaryotic genomes but not archaea. While they have been shown to be associated with ribosomes, their molecular function remains unclear. Here we show that YbeB is a ribosomal silencing factor (RsfA) in the stationary growth phase and during the transition from rich to poor media. A knock-out of the rsfA gene shows two strong phenotypes: (i) the viability of the mutant cells are sharply impaired during stationary phase (as shown by viability competition assays), and (ii) during transition from rich to poor media the mutant cells adapt slowly and show a growth block of more than 10 hours (as shown by growth competition assays). RsfA silences translation by binding to the L14 protein of the large ribosomal subunit and, as a consequence, impairs subunit joining (as shown by molecular modeling, reporter gene analysis, in vitro translation assays, and sucrose gradient analysis). This particular interaction is conserved in all species tested, including Escherichia coli, Treponema pallidum, Streptococcus pneumoniae, Synechocystis PCC 6803, as well as human mitochondria and maize chloroplasts (as demonstrated by yeast two-hybrid tests, pull-downs, and mutagenesis). RsfA is unrelated to the eukaryotic ribosomal anti-association/60S-assembly factor eIF6, which also binds to L14, and is the first such factor in bacteria and organelles. RsfA helps cells to adapt to slow-growth/stationary phase conditions by down-regulating protein synthesis, one of the most energy-consuming processes in both bacterial and eukaryotic cells.