PIF1 helicase promotes break-induced replication in mammalian cells.

Break-induced replication (BIR) is a specialized homologous-recombination pathway for DNA double-strand break (DSB) repair, which often induces genome instability. In this study, we establish EGFP-based recombination reporters to systematically study BIR in mammalian cells and demonstrate an important role of human PIF1 helicase in promoting BIR. We show that at endonuclease cleavage sites, PIF1-dependent BIR is used for homology-initiated recombination requiring long track DNA synthesis, but not short track gene conversion (STGC). We also show that structure formation-prone AT-rich DNA sequences derived from common fragile sites (CFS-ATs) induce BIR upon replication stress and oncogenic stress, and PCNA-dependent loading of PIF1 onto collapsed/broken forks is critical for BIR activation. At broken replication forks, even STGC-mediated repair of double-ended DSBs depends on POLD3 and PIF1, revealing an unexpected mechanism of BIR activation upon replication stress that differs from the conventional BIR activation model requiring DSB end sensing at endonuclease-generated breaks. Furthermore, loss of PIF1 is synthetically lethal with loss of FANCM, which is involved in protecting CFS-ATs. The breast cancer-associated PIF1 mutant L319P is defective in BIR, suggesting a direct link of BIR to oncogenic processes.

Super-resolution deep learning reconstruction at coronary computed tomography angiography to evaluate the coronary arteries and in-stent lumen: an initial experience.

A super-resolution deep learning reconstruction (SR-DLR) algorithm trained using data acquired on the ultrahigh spatial resolution computed tomography (UHRCT) has the potential to provide better image quality of coronary arteries on the whole-heart, single-rotation cardiac coverage on a 320-detector row CT scanner. However, the advantages of SR-DLR at coronary computed tomography angiography (CCTA) have not been fully investigated. The present study aimed to compare the image quality of the coronary arteries and in-stent lumen between SR-DLR and model-based iterative reconstruction (MBIR). We prospectively enrolled 70 patients (median age, 69 years; interquartile range [IQR], 59-75 years; 50 men) who underwent CCTA using a 320-detector row CT scanner between January and August 2022. The image noise in the ascending aorta, left atrium, and septal wall of the ventricle was measured, and the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) in the proximal coronary arteries were calculated. Of the twenty stents, stent strut thickness and luminal diameter were quantitatively evaluated. The image noise on SR-DLR was significantly lower than that on MBIR (median 22.1 HU; IQR, 19.3-24.9 HU vs. 27.4 HU; IQR, 24.2-31.2 HU, p < 0.01), whereas the SNR (median 16.3; IQR, 11.8-21.8 vs. 13.7; IQR, 9.9-18.4, p = 0.01) and CNR (median 24.4; IQR, 15.5-30.2 vs. 19.2; IQR, 14.1-23.2, p < 0.01) on SR-DLR were significantly higher than that on MBIR. Stent struts were significantly thinner (median, 0.68 mm; IQR, 0.61-0.78 mm vs. 0.81 mm; IQR, 0.72-0.96 mm, p < 0.01) and in-stent lumens were significantly larger (median, 1.84 mm; IQR, 1.65-2.26 mm vs. 1.52 mm; IQR, 1.28-2.25 mm, p < 0.01) on SR-DLR than on MBIR. Although further large-scale studies using invasive coronary angiography as the reference standard, comparative studies with UHRCT, and studies in more challenging population for CCTA are needed, this study's initial experience with SR-DLR would improve the utility of CCTA in daily clinical practice due to the better image quality of the coronary arteries and in-stent lumen at CCTA compared with conventional MBIR.

A Comparison of Retrospective ECG-Gated CT and Surgical or Angiographical Findings in Acute Aortic Syndrome.

The best cardiac phases in retrospective ECG-gated CT for detecting an intimal tear (IT) in aortic dissection (AD) and an ulcer-like projection (ULP) in an intramural hematoma (IMH) have not been established. This study aimed to compare the detection accuracy of diastolic-phase and systolic-phase ECG-gated CT for IT in AD and ULP in IMH, with subsequent surgical or angiographical confirmation as the reference standard.In total, 81 patients (67.6 ± 11.8 years; 41 men) who underwent emergency ECG-gated CT and subsequent open surgery or thoracic endovascular aortic repair for AD (n = 52) or IMH (n = 29) were included. The accuracies of detecting IT and ULP were compared among only diastolic-phase, only systolic-phase, and both diastolic-phase and systolic-phase methods of retrospective ECG-gated CT; surgical or angiographical findings were used as the reference standard. The detection accuracy for IT and ULP using only diastolic-phase, only systolic-phase, and both diastolic-phase and systolic-phase methods of ECG-gated CT was 93% [95% CI: 87-97], 94% [95% CI: 88-97], and 95% [95% CI: 90-97], respectively. There were no significant differences in detection accuracy among the 3 acquisition methods (P = 0.55). Similarly, there were no significant differences in the accuracy of detecting IT in AD (P = 0.55) and ULP in IMH (P > 0.99) among only diastolic-phase, only systolic-phase, and both diastolic- and systolic-phase ECG-gated CT.Retrospective ECG-gated CT for detecting IT in AD and ULP in IMH yields highly accurate findings. There were no significant differences seen among only diastolic-phase, only systolic-phase, and both diastolic-phase and systolic-phase ECG-gated CT.

Aortic Elongation in Bicuspid Aortic Valve with Aortic Stenosis Assessed by Thin-Slice Electrocardiogram-Gated Computed Tomography.

Bicuspid aortic valve (BAV) patients with aortic stenosis (AS) are known to develop dilatation of the ascending aorta at a younger age, but the morphology of the aorta in these patients is yet to be investigated. Thus, in this study, we aim to evaluate the aortic morphology of BAV patients with severe AS using thin-slice electrocardiogram (ECG) -gated computed tomography (CT) and identify the possible contributing effect of age.In this retrospective study, 122 BAV and 154 tricuspid aortic valve (TAV) patients who received aortic valve replacement for severe AS were assessed by thin-slice ECG-gated CT and three-dimensional reconstruction. The morphology of the ascending aorta was also evaluated among BAV patients aged < 70 (n = 72) and ≥ 70 (n = 50) years old. As per our findings, BAV patients with severe AS had significantly greater diameter (P < 0.01), elongation (P < 0.01), and tortuosity (P = 0.03) of the ascending aorta; minimum aortic arch angle (P < 0.01); and significantly lower calcified plaque (P < 0.01) compared with those of TAV patients even after adjusting for background. Multiple regression analysis showed that standardized partial regression coefficients (ß) of dilatation (0.5) and elongation (0.35) were higher among other measurements of aortic morphology for BAV patients. BAV patients with severe AS aged ≥ 70 years had significantly greater diameter (42.0 [37.2-46.1] mm versus 40.4 [35.2-44.2] mm, P = 0.049) and elongation (133.8 [123.5-147.3] mm versus 127.0 [111.0-140.0] mm, P = 0.01) of the ascending aorta than those aged < 70 years.BAV patients with severe AS were determined to have greater dilatation and elongation of the ascending aorta. Moreover, BAV patients older than 70 years had greater diameter and elongation of the ascending aorta.

DNA polymerase θ (POLQ) is important for repair of DNA double-strand breaks caused by fork collapse.

DNA polymerase θ (POLQ) plays an important role in alternative nonhomologous end joining or microhomology-mediated end joining (alt-NHEJ/MMEJ). Here, we show that POLQ is not only required for MMEJ to repair DNA double-strand breaks (DSBs) generated by endonucleases such as I-SceI or Cas9, but is also needed for repair of DSBs derived from DNA nicks generated by Cas9 nickase. Consistently, we found that POLQ deficiency leads to sensitivity to topoisomerase inhibitors that cause DNA single-strand break (SSB) accumulation at replication forks and to ATR inhibitors that induce replication fork collapse. These studies support the function of POLQ in coping with replication stress and repairing DSBs upon fork collapse. POLQ overexpression is present in many cancer types and is associated with poor prognosis, including breast cancer regardless of BRCA1 status. We provide proof-of-concept evidence to support a novel cancer treatment strategy that combines POLQ inhibition with administration of topoisomerase or ATR inhibitors, which induces replication stress and fork collapse. Given the prevalence of POLQ overexpression in tumors, such strategy may have a significant impact on developing targeted cancer treatment.

Ultra-high-resolution CT angiography of the artery of Adamkiewicz: a feasibility study.

PURPOSE: Preoperative identification of the artery of Adamkiewicz can help prevent postoperative spinal cord injury following thoracic and thoracoabdominal aortic repair. Several studies have demonstrated the feasibility of evaluating the artery of Adamkiewicz using multi-detector row computed tomography (CT), but precise visualization remains a challenge. The present study was conducted to evaluate the usefulness of ultra-high-resolution CT for visualizing the artery of Adamkiewicz with a slice thickness of 0.25 versus 0.5 mm in patients with aortic aneurysms. METHODS: Our institutional review board approved this study. Twenty-four patients with thoracic and thoracoabdominal aneurysms were scanned with beam collimation of 0.25 mm × 128. Images were reconstructed with slice thicknesses of 0.25 and 0.5 mm. The signal-to-noise ratio (SNR) of the aorta and contrast-to-noise ratio (CNR) between the anterior spinal artery and spinal cord were measured. Two independent observers evaluated visualization of the artery of Adamkiewicz and its continuity between the anterior spinal artery and the aorta using a four-point scale. RESULTS: No significant differences in the SNR of the aorta or CNR of the anterior spinal artery were observed between 0.25- and 0.5-mm slices. The average visualization score was significantly higher for 0.25-mm slices (3.58 ± 0.78) than for 0.5-mm slices (3.13 ± 0.99) (p = 0.01). The percentage of patients with nondiagnostic image quality was significantly lower for 0.25-mm slices (8.3%) than for 0.5-mm slices (33.3%) (p = 0.03). CONCLUSION: In patients with aortic aneurysms, ultra-high-resolution CT with 0.25-mm slices significantly improves visualization of the artery of Adamkiewicz compared to 0.5-mm slices.

Hand grip strength as a predictor of postoperative complications in esophageal cancer patients undergoing esophagectomy.

BACKGROUND: Radical esophagectomy remains the primary treatment option for resectable esophageal cancer. However, it sometimes induces postoperative complications due to its invasive nature. Recently, the impact of loss of muscle mass on postoperative complications and survival among cancer patients has been highlighted. This study aimed to identify the impact of low hand grip strength (HGS) on postoperative complications after esophagectomy. METHODS: A total of 188 patients (male: 166, female: 22) who underwent radical esophagectomy with gastric tube reconstruction between 2008 and 2014 were included. The correlation between HGS and age was analyzed using Pearson's correlation coefficient. Due to the small patient numbers, only male patients were stratified into two groups according to age (<70 years: non-elderly group, ≥70 years: elderly group). Receiver operating characteristic curve analysis was performed for each group using postoperative complication occurrence as the endpoint to determine an optimal HGS cutoff value. RESULTS: Postoperative complications occurred in 60.9% of the elderly group and 47.4% of the non-elderly group. When the cutoff values were set at 30.5 and 37 kg for the elderly and non-elderly group, respectively, low HGS was an independent predictive factor of postoperative complications on multivariate analysis only in the elderly group (p = 0.008). In the elderly group, the incidence of postoperative pneumonia was 39.5% among patients with low HGS vs. 3.8% among patients with high HGS. CONCLUSION: Preoperative HGS is an independent predictive factor of postoperative complications, especially postoperative pneumonia, for elderly male patients with esophageal cancer treated with radical esophagectomy.

Herquline A, produced by Penicillium herquei FKI-7215, exhibits anti-influenza virus properties.

In the course of screening for new anti-influenza virus antibiotics, we isolated herquline A from a culture broth of the fungus, Penicillium herquei FKI-7215. Herquline A inhibited replication of influenza virus A/PR/8/34 strain in a dose-dependent manner without exhibiting cytotoxicity against several human cell lines. It did not inhibit the viral neuraminidase.

Design, Synthesis and Biological Evaluation of a Structurally Simplified Syringolin A Analogues.

In this study, we designed and synthesized a structurally simplified syringolin A analogue 4, which could have a switched hydrogen bonding interaction with the ß5 subunit of 20S proteasome. This analogue exhibits potent ß5 proteasome inhibitory activity with an IC50 value of 107 nM. It also shows cytotoxicity against a range of human cancer cells at submicromolar level (109-254 nM). This analogue is expected to be a lead compound as a next generation proteasome inhibitor because of its simple structure.

NPY antagonism reduces adiposity and attenuates age-related imbalance of adipose tissue metabolism.

An orexigenic hormone, neuropeptide Y (NPY), plays a role not only in the hypothalamic regulation of appetite, but also in the peripheral regulation of lipid metabolism. However, the intracellular mechanisms triggered by NPY to regulate lipid metabolism are poorly understood. Here we report that NPY deficiency reduces white adipose tissue (WAT) mass and ameliorates the age-related imbalance of adipose tissue metabolism in mice. Gene expression involved in adipogenesis/lipogenesis was found to decrease, whereas proteins involved in lipolysis increased in gonadal WAT (gWAT) of NPY-knockout mice. These changes were associated with an activated SIRT1- and PPARγ-mediated pathway. Moreover, the age-related decrease of de novo lipogenesis in gWAT and thermogenesis in inguinal WAT was inhibited by NPY deficiency. Further analysis using 3T3-L1 cells showed that NPY inhibited lipolysis through the Y1 receptor and enhanced lipogenesis following a reduction in cAMP response element-binding protein (CREB) and SIRT1 protein expression. Therefore, NPY appears to act as a key regulator of adipose tissue metabolism via the CREB-SIRT1 signaling pathway. Taken together, NPY deficiency reduces adiposity and ameliorates the age-related imbalance of adipose tissue metabolism, suggesting that antagonism of NPY may be a promising target for drug development to prevent age-related metabolic diseases.

Structure-activity relationship study of syringolin A as a potential anticancer agent.

A detailed structure-activity relationship of syringolin A (1), which is a promising antitumor natural product, was described. We previously developed syringolin A analog 2 as a potent proteasome inhibitor by the structure-based drug design of syringolin A. In this Letter, we synthesized a range of analogs of 2, having a different length of the lipophilic chain and substituted aryl group, and their cytotoxicity against human cancer cells was evaluated. It turned out that these modifications greatly affected the cytotoxicity. Further optimization would lead to develop a novel proteasome inhibitor.

Total synthesis of syringolin A and improvement of its biological activity.

The development process for syringolin A analogues having improved proteasome inhibitory and antitumor activity is described. The strategy was to first establish a convergent synthesis of syringolin A using a rare intramolecular Ugi three-component reaction in the last stage of the synthesis, so as to gain access toa set of structure-based analogues. The inhibitory activity of chymotrypsin-like activity of 20S proteasome was largely improved by targeting the S3 subsite of the ß5 subunit. Cytotoxic activity was also improved by installing the membrane-permeable substituent. These biological properties are comparable to those of bortezomib, a clinically used first-line proteasome inhibitor.

Dbf4 is direct downstream target of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) protein to regulate intra-S-phase checkpoint.

Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells.

Caenorhabditis elegans transfers across a gap under an electric field as dispersal behavior.

Interactions between different animal species are a critical determinant of each species' evolution and range expansion. Chemical, visual, and mechanical interactions have been abundantly reported, but the importance of electric interactions is not well understood. Here, we report the discovery that the nematode Caenorhabditis elegans transfers across electric fields to achieve phoretic attachment to insects. First, we found that dauer larvae of C. elegans nictating on a substrate in a Petri dish moved directly to the lid through the air due to the electrostatic force from the lid. To more systematically investigate the transfer behavior, we constructed an assay system with well-controlled electric fields: the worms flew up regardless of whether a positive or negative electric field was applied, suggesting that an induced charge within the worm is related to this transfer. The mean take-off speed is 0.86 m/s, and the worm flies up under an electric field exceeding 200 kV/m. This worm transfer occurs even when the worms form a nictation column composed of up to 100 worms; we term this behavior "multiworm transfer." These observations led us to conclude that C. elegans can transfer and attach to the bumblebee Bombus terrestris, which was charged by rubbing with flower pollen in the lab. The charge on the bumblebee was measured with a coulomb-meter to be 806 pC, which was within the range of bumblebee charges and of the same order of flying insect charges observed in nature, suggesting that electrical interactions occur among different species.

Moderate protein intake percentage in mice for maintaining metabolic health during approach to old age.

Nutritional requirements for maintaining metabolic health may vary with each life stage, such as young, middle, and old age. To investigate the appropriate ratio of nutrients, particularly proteins, for maintaining metabolic health while approaching old age, young (6-month-old) and middle-aged (16-month-old) mice were fed isocaloric diets with varying protein percentages (5%, 15%, 25%, 35%, and 45% by calorie ratio) for two months. The low-protein diet developed mild fatty liver, with middle-aged mice showing more lipids than young mice, whereas the moderate-protein diet suppressed lipid contents and lowered the levels of blood glucose and lipids. Self-organizing map (SOM) analysis revealed that plasma amino acid profiles differed depending on age and difference in protein diet and were associated with hepatic triglyceride and cholesterol levels. Results indicate that the moderate protein intake percentages (25% and 35%) are required for maintaining metabolic health in middle-aged mice, which is similar to that in young mice.

Release of TNF-α from macrophages is mediated by small GTPase Rab37.

Activated macrophages at wound sites release many cytokines which positively affect skin wound healing. However, the molecular mechanisms controlling cytokine secretion from macrophages have not been elucidated. In the present study, we performed an RT-PCR analysis and found that 19 small GTPase Rab isoforms were expressed at skin wound sites, with six of them (i.e. Rab3B, Rab27B, Rab30, Rab33A, Rab37, and Rab40C) being upregulated during the inflammation and proliferation/migration phase of skin repair. We also found that gene expression of Rab37 in murine primary and RAW264.7 macrophages was significantly induced after stimulation with LPS. Overexpression of wild type and constitutively active Rab37 in RAW264.7 cells significantly increased TNF-α secretion, whereas knockdown of Rab37 by siRNA significantly decreased it. We also identified 29 putative Rab37-interacting proteins, including the membrane fusion regulating Munc13-1, using liquid chromatography/linear ion trap mass spectrometry (LC-MS/MS). Immunocytochemical analysis further revealed that TNF-α-containing vesicles were colocalized with both Rab37 and Munc13-1 in activated macrophages. Knockdown of Munc13-1 by siRNA significantly decreased TNF-α secretion. Taken together, these findings demonstrate that Rab37 interacts with Munc13-1 to control TNF-α secretion from activated macrophages.

Protein reporter bioassay systems for the phenotypic screening of candidate drugs: a mouse platform for anti-aging drug screening.

Recent drug discovery efforts have utilized high throughput screening (HTS) of large chemical libraries to identify compounds that modify the activity of discrete molecular targets. The molecular target approach to drug screening is widely used in the pharmaceutical and biotechnology industries, because of the amount of knowledge now available regarding protein structure that has been obtained by computer simulation. The molecular target approach requires that the structure of target molecules, and an understanding of their physiological functions, is known. This approach to drug discovery may, however, limit the identification of novel drugs. As an alternative, the phenotypic- or pathway-screening approach to drug discovery is gaining popularity, particularly in the academic sector. This approach not only provides the opportunity to identify promising drug candidates, but also enables novel information regarding biological pathways to be unveiled. Reporter assays are a powerful tool for the phenotypic screening of compound libraries. Of the various reporter genes that can be used in such assays, those encoding secreted proteins enable the screening of hit molecules in both living cells and animals. Cell- and animal-based screens enable simultaneous evaluation of drug metabolism or toxicity with biological activity. Therefore, drug candidates identified in these screens may have increased biological efficacy and a lower risk of side effects in humans. In this article, we review the reporter bioassay systems available for phenotypic drug discovery.

[Investigation of a Compensation Filter for Reduction of Dark Band Artifact in the Head and Neck CT].

PURPOSE: Dark band (DB) artifact in head and neck computed tomography (CT) is caused by beam hardening (BH), and decreased CT values in the X-ray target become a problem. Therefore, we investigated whether it is possible to reduce DB artifact in the head and neck with a compensation filter. METHODS: We made 2 types of filters with alcohol and water. We set each of these filters in front of the chest phantom's clavicle and evaluated DB artifact. The evaluation method measured CT values in the DB artifact area and background (BG) area by changing each compensation filter thickness and the distance between the chest phantom's surface and each compensation filter. In addition, we measured average standard deviation (SD) in the BG area by the presence of each compensation filter. RESULTS: CT values in the DB artifact area were approximate to those in the BG area by setting the thickness of each compensation filter to more than 30 mm. Furthermore, these CT values were decreased by separating the distance between the chest phantom's surface and each compensation filter. Average SD in the BG area showed no significant difference between no filter and each compensation filter. CONCLUSION: It was possible to reduce DB artifact by a compensation filter for DB.

A Comparison of Gene Expression Profiles of Rat Tissues after Mild and Short-Term Calorie Restrictions.

Many studies have shown the beneficial effects of calorie restriction (CR) on rodents' aging; however, the molecular mechanism explaining these beneficial effects is still not fully understood. Previously, we conducted transcriptomic analysis on rat liver with short-term and mild-to-moderate CR to elucidate its early response to such diet. Here, we expanded transcriptome analysis to muscle, adipose tissue, intestine, and brain and compared the gene expression profiles of these multiple organs and of our previous dataset. Several altered gene expressions were found, some of which known to be related to CR. Notably, the commonly regulated genes by CR include nicotinamide phosphoribosyltransferase and heat shock protein 90, which are involved in declining the aging process and thus potential therapeutic targets for aging-related diseases. The data obtained here provide information on early response markers and key mediators of the CR-induced delay in aging as well as on age-associated pathological changes in mammals.

Diatretol, an α, α'-dioxo-diketopiperazine, is a potent in vitro and in vivo antimalarial.

A fungal metabolite, diatretol, has shown to be a promising antimalarial agent. Diatretol displayed potent in vitro antiparasitic activity against the Plasmodium falciparum K1 strain, with an IC50 value of 378 ng ml-1, as well as in vivo efficacy in a Plasmodium berghei-infected mice model, with ca. 50% inhibition at 30 mg/kg (p.o.).