Risk factors for postgastric endoscopic submucosal dissection bleeding in direct oral anticoagulant users.

OBJECTIVES: Bleeding after endoscopic submucosal dissection (ESD) for gastric tumors in patients taking antithrombotic drugs, in particular direct oral anticoagulants (DOACs), remains unresolved; therefore, we evaluated the risk factors for post-ESD bleeding and drug differences in patients taking DOACs. METHODS: We included 278 patients taking antithrombotic drugs who underwent gastric ESD between January 2017 and March 2022. Antithrombotic drugs were withdrawn following the 2017 guidelines (Appendix on anticoagulants including DOACs). To further clarify differences in antithrombotic agents' effects, the peri-cancerous mucosa in the resected specimen was pathologically evaluated according to the Updated Sydney System. Multivariate analysis was performed to assess the risk of post-ESD bleeding. RESULTS: The incidence of post-ESD bleeding in patients taking DOACs was 19.6% (10/51). Among patients taking antithrombotic drugs, DOACs were identified as a possible factor involved in post-ESD bleeding (odds ratio [OR] 4.92). Among patients taking DOACs, possible factors included resection length diameter ≥30 mm (OR 3.72), presence of neutrophil infiltration (OR 2.71), lesions occurring in the lower third of stomach (OR 2.34), and preoperative antiplatelet use (OR 2.22). Post-ESD bleeding by DOAC type was 25.0% of patients (4/16) receiving apixaban, in 20.0% (3/15) receiving edoxaban, in 21.4% (3/14) receiving rivaroxaban, and in none of those receiving dabigatran. CONCLUSIONS: The administration of DOACs was shown to be a possible factor involved in post-ESD bleeding, and risk factors for patients taking DOACs included neutrophil infiltration. The pharmacological differences in the effects of DOACs contributing to bleeding in gastric ulcers suggest comparatively less bleeding with dabigatran after ESD.

Generation of JC Polyoma Pseudovirus for High-Throughput Measurement of Neutralizing Antibodies.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS) caused by reactivation of dormant JC polyomavirus (JCPyV). PML was mainly observed in immunocompromised individuals, such as HIV-positive patients, autoimmune disease patients, and cancer patients. Given that the presence of anti-JCPyV antibodies in serum is a risk indicator for PML development, it is essential to monitor anti-JCPyV antibody levels. In the present study, we established reporter-based single-infection neutralization assays for JCPyV and the genetically similar BK polyoma virus (BKPyV). We then confirmed the lack of cross-reactivity between the two viruses using test sera obtained from mice immunized with plasmids encoding the JCPyV or BKPyV capsid. Next, we compared neutralization antibody titers in sera from healthy donors, patients with multiple sclerosis (MS), and HIV-positive patients using an in-house enzyme-linked immunosorbent assay (ELISA) with JCPyV-like particles (virus-like particles; VLPs). A positive correlation was demonstrated between the neutralization titer (75% infectious concentration; IC75) against JCPyV and the antibody titer obtained by VLP-based JCPyV ELISA. This assay system may be applied to detect antibodies against other PyVs by generation of pseudoviruses using the respective capsid expression plasmids, and is expected to contribute to the surveillance of PyV as well as basic research on these viruses.

RNaseH2A downregulation drives inflammatory gene expression via genomic DNA fragmentation in senescent and cancer cells.

Cellular senescence caused by oncogenic stimuli is associated with the development of various age-related pathologies through the senescence-associated secretory phenotype (SASP). SASP is mediated by the activation of cytoplasmic nucleic acid sensors. However, the molecular mechanism underlying the accumulation of nucleotide ligands in senescent cells is unclear. In this study, we revealed that the expression of RNaseH2A, which removes ribonucleoside monophosphates (rNMPs) from the genome, is regulated by E2F transcription factors, and it decreases during cellular senescence. Residual rNMPs cause genomic DNA fragmentation and aberrant activation of cytoplasmic nucleic acid sensors, thereby provoking subsequent SASP factor gene expression in senescent cells. In addition, RNaseH2A expression was significantly decreased in aged mouse tissues and cells from individuals with Werner syndrome. Furthermore, RNaseH2A degradation using the auxin-inducible degron system induced the accumulation of nucleotide ligands and induction of certain tumourigenic SASP-like factors, promoting the metastatic properties of colorectal cancer cells. Our results indicate that RNaseH2A downregulation provokes SASP through nucleotide ligand accumulation, which likely contributes to the pathological features of senescent, progeroid, and cancer cells.

The BRCA2 missense mutation K2497R suppressed self-degradation and increased ATP production and cell proliferation.

Breast cancer susceptibility gene 2 (BRCA2) mediates genome maintenance during the S phase of the cell cycle, with important roles in replication stress, centrosome replication, and cytokinesis. In this study, we showed that a small heat shock protein, HSP27, interacted with and participated in the degradation of BRCA2 in estrogen-treated MCF-7 cells. BRCA2 degradation reportedly requires ubiquitination of the C-terminal region; thus, fragments of amino acid (aa) residues 2241-2940 were produced and assayed for their degradation following cycloheximide (CHX) treatment. The results showed that aa 2491-2580 affected the degradation of BRCA2, especially lysine (Lys) 2497. Furthermore, the K2497 A/R mutation increased ATP production and the proliferation of DLD-1 (BRCA2 knockout) cells compared to the cells expressing wild-type BRCA2-FLAG. Notably, a single residue, Lys2497, affected BRCA2 degradation, and K2497R is reportedly a missense mutation in hereditary breast cancer.

BRCA2 represses the transcriptional activity of pS2 by E2-ERα.

Germline mutations to the breast cancer 2 (BRCA2) gene have been associated with hereditary breast cancer. In addition to estrogen uptake, BRCA2 expression increases in the S phase of the cell cycle and largely contributes to DNA damage repair associated with DNA replication. However, the role of BRCA2 in estrogen induction remains unclear. An expression plasmid was created to induce BRCA2 activation upon the addition of estradiol by introducing mutations to the binding sequences for the transcription factors USF1, E2F1, and NF-κB within the promoter region of BRCA2. Then, the estrogen receptor (ER) sites of the proteins that interact with BRCA2 upon the addition of estradiol were identified. Both proteins were bound by the helical domain of BRCA2 and activation function-2 of the ER, suggesting that this binding may regulate the transcriptional activity of pS2, a target gene of the estradiol-ER, by suppressing the binding of SRC-1, a coactivator required for activation of the transcription factor.

Improved indocyanine green retention after short-term lenvatinib withdrawal in three patients with hepatocellular carcinoma.

Use of lenvatinib, which has a high response rate in advanced hepatocellular carcinoma, sometimes results in tumor shrinkage and resectability of previously unresectable liver cancers. In Asia, including Japan, liver reserve, one of the determinants of resectability, is mainly determined by the indocyanine green (ICG) retention rate. Three patients with advanced liver cancer treated at our institution had very poor ICG retention rates during treatment with lenvatinib. Lenvatinib may reduce blood flow in both cancerous and non-cancerous regions by inhibiting vascular endothelial growth factor. Therefore, accurate determination of liver function likely requires withdrawal of this treatment several days before ICG retention testing.

Acetylation-dependent regulation of PD-L1 nuclear translocation dictates the efficacy of anti-PD-1 immunotherapy.

Immunotherapies that target programmed cell death protein 1 (PD-1) and its ligand PD-L1 as well as cytotoxic T-lymphocyte-associated protein 4 (CTLA4) have shown impressive clinical outcomes for multiple tumours. However, only a subset of patients achieves durable responses, suggesting that the mechanisms of the immune checkpoint pathways are not completely understood. Here, we report that PD-L1 translocates from the plasma membrane into the nucleus through interactions with components of the endocytosis and nucleocytoplasmic transport pathways, regulated by p300-mediated acetylation and HDAC2-dependent deacetylation of PD-L1. Moreover, PD-L1 deficiency leads to compromised expression of multiple immune-response-related genes. Genetically or pharmacologically modulating PD-L1 acetylation blocks its nuclear translocation, reprograms the expression of immune-response-related genes and, as a consequence, enhances the anti-tumour response to PD-1 blockade. Thus, our results reveal an acetylation-dependent regulation of PD-L1 nuclear localization that governs immune-response gene expression, and thereby advocate targeting PD-L1 translocation to enhance the efficacy of PD-1/PD-L1 blockade.

Estradiol/GPER affects the integrity of mammary duct-like structures in vitro.

High estrogen concentration leads to an inflammatory reaction in the mammary gland tissue in vivo; however, the detailed mechanism underlying its specific effects on the breast duct has not been fully clarified. We used 3D-cultured MCF-10A acini as a breast duct model and demonstrated various deleterious effects of 17-ß estradiol (E2), including the destruction of the basement membrane surrounding the acini, abnormal adhesion between cells, and cell death via apoptosis and pyroptosis. Moreover, we clarified the mechanism underlying these phenomena: E2 binds to GPER in MCF-10A cells and stimulates matrix metalloproteinase 3 (MMP-3) and interleukin-1ß (IL-1ß) secretion via JNK and p38 MAPK signaling pathways. IL-1ß activates the IL-1R1 signaling pathway and induces continuous MMP-3 and IL-1ß secretion. Collectively, our novel findings reveal an important molecular mechanism underlying the effects of E2 on the integrity of duct-like structures in vitro. Thus, E2 may act as a trigger for ductal carcinoma transition in situ.

Upregulation of CHOP participates in caspase activation and virus release in human astrovirus-infected cells.

Human astroviruses (HAstVs), non-enveloped RNA viruses with positive-sense RNA genomes, are an important cause of acute gastroenteritis in young children, although the processes that produce infectious virions are not clearly defined. To track the viral replication complex (RC) upon HAstV1 infection, the subcellular distribution of double-stranded (ds) RNA and of ORF1b, a viral RNA polymerase, was examined by immunocytochemistry. Foci that were positive for dsRNA and for ORF1b were co-localized, and both foci were also co-localized with resident proteins of the endoplasmic reticulum (ER). Focusing on the association between the HAstV RC and ER, we examined the expression of unfolded protein response (UPR) markers and found that targets of eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4), including CCAAT/enhancer-binding protein homologous protein (CHOP), a proapoptotic transcription factor, were upregulated at the late phase in HAstV-infected cells. Consistently, eIF2α was phosphorylated at the late phase of HAstV infection. The formation of foci resembling stress granules, another known downstream response to eIF2α phosphorylation, was also observed at the same period. Phosphorylation of eIF2α was attenuated in protein kinase R (PKR)-knockdown cells, suggesting that, unlike the canonical ER stress response, PKR was involved in eIF2α phosphorylation in response to HAstV infection. Studies have indicated that immature HAstV capsid protein is processed by caspases, and caspase cleavage is integral to particle release. Inhibition of CHOP upregulation reduced caspase activation and the release of HAstV RNA from cells during HAstV infection. Our results suggest that the eIF2α-ATF4-CHOP pathway participates in HAstV propagation.

The influence of pre-operative antiplatelet and anticoagulant agents on the outcomes in elderly patients undergoing early surgery for hip fracture.

BACKGROUND: Early surgery improves the prognosis of elderly patients with hip fractures. However, many patients take antiplatelet and anticoagulant therapies for comorbidities. This study compared perioperative outcomes and 1-year mortality rates with early surgery in elderly patients with hip fractures taking or not taking these agents preoperatively. METHODS: Among 418 patients undergoing surgery for hip fractures at our institution from 2014 to 2016, 266 patients over 65 years who had surgery within 48 hours of admission were enrolled. We excluded patients with high-energy injuries, multiple or pathological fractures, and patients undergoing osteosynthesis for femoral neck fractures. The study population was divided into those who underwent hemiarthroplasty for neck fractures and those who underwent osteosynthesis for trochanteric fractures. We also divided the population into patients receiving chronic anticoagulation therapy (medicated group: 19 hemiarthroplasty, 70 osteosynthesis) and patients not receiving anticoagulation therapy (non-medicated group: 47 hemiarthroplasty, 130 osteosynthesis). Comorbidities, intraoperative blood loss, estimated blood loss from admission to the first and seventh day after surgery, transfusions, length of stay, complications, and 1-year mortality rates were evaluated. RESULTS: Diabetes mellitus and cerebrovascular disorders were significantly more common in the medicated group for both surgery types. In the osteosynthesis group, estimated blood loss on the first day was 710 ml in the medicated group and 572 ml in the non-medicated group (P = 0.015). In the hemiarthroplasty group, corresponding values were 668 and 480 ml, respectively (P = 0.016). Estimated blood loss on the seventh day, complications, length of stay and 1-year mortality rate were not increased significantly. CONCLUSIONS: The medicated group had an increase in estimated blood loss on the first day. However, there was no significant increase in transfusions, complications and 1-year mortality rates. Early surgery for elderly patients with hip fractures is recommended, even for those taking antiplatelet and anticoagulant agents.

Assessment of unloading effects after open wedge high tibial osteotomy using quantitative bone scintigraphy.

INTRODUCTION: We evaluated changes in bone tracer uptake (BTU) in open wedge high tibial osteotomy (OWHTO) and determined if BTU correlates with clinical symptoms, postoperative alignment, or cartilage regeneration after OWHTO. MATERIALS AND METHODS: Seventy-five knees in 64 patients who underwent OWHTO for medial compartment osteoarthritis were enrolled in this retrospective study. All cases were assessed preoperatively and at plate removal using bone scintigraphy. Visual analog scale (VAS), Japanese Orthopedic Association (JOA) score, Oxford Knee Score (OKS), Knee Injury and Osteoarthritis Outcome Score (KOOS), and the weight-bearing line ratio (WBLR) were assessed preoperatively and at plate removal. In addition, cartilage regeneration was evaluated at plate removal. We assessed changes in BTU for the medial and lateral compartment after OWHTO and the correlations between BTU of the medial compartment and all other parameters were analyzed. RESULTS: Postoperatively, all outcome measures significantly improved: mean VAS 61.4 ± 18.3 to 9.5 ± 8.2, mean JOA score 65.1 ± 11.5 to 94.7 ± 6.0, mean OKS 29.4 ± 8.1 to 42.3 ± 4.1, mean KOOS 57.0 ± 14.3 to 83.7 ± 9.6, mean WBLR 22.8 ± 10.9 to 70.0 ± 9.4. Cartilage regeneration was observed in 53 knees (70.7%). BTU of the medial compartment significantly decreased after OWTHO, whereas no increased postoperative BTU was found in the lateral compartment. Postoperative BTU of the medial compartment significantly correlated with VAS, KOOS, and WBLR. No statistically significant associations were found between BTU and cartilage regeneration. CONCLUSIONS: OWHTO significantly decreased BTU of the medial compartment, which correlated with knee pain and postoperative mechanical alignment. Unloading effects of OWHTO led to pain relief after surgery, regardless of cartilage regeneration.

The effect of a multidisciplinary approach on geriatric hip fractures in Japan.

BACKGROUND: This study aimed to report results of the multidisciplinary treatment approach for geriatric hip fractures and evaluate its effectiveness compared with conventional treatment. Patients aged 65 years and older who presented with a hip fracture at our hospital on or after 2014 were treated according to a multidisciplinary approach. METHOD: Two groups of patients with hip fracture were compared. Group I (n = 364) was treated according to the new multidisciplinary approach in 2014-2016, and Group II (n = 105) which received conventional treatment in 2012. Time to surgery, length of hospital stay, postoperative complications, osteoporosis treatment, functional recovery, in-hospital mortality, 90-day mortality, and 1-year mortality were evaluated. The medical costs of multidisciplinary treatment were compared with those in other hospitals every year. RESULTS: There were no significant differences in the time to surgery between Group I and Group II, but each was considerably shorter than the average time in other Japanese hospitals. The length of hospital stay was longer in Group I. The overall postoperative complication rate was lower in Group I, but there was no significant difference for each individual complication. The rate of anti-osteoporosis pharmacotherapy administration at the time of discharge was significantly higher in Group I. Moreover, the proportion of patients who recovered to their pre-injury functional level was significantly higher in Group I. The mortality rates did not significantly differ year on year. The total hospitalization medical cost per patient for the multidisciplinary treatment was lower than other hospital costs every year. CONCLUSIONS: Multidisciplinary treatment produced no significant improvement in time to surgery, length of hospital stay, or postoperative complications. However, the use of the multidisciplinary treatment approach led to a significant increase in osteoporosis treatment rate and better functional recovery. Furthermore, the total medical costs for multidisciplinary treatment were lower than the acute care hospital costs.

Large opening gaps, unstable hinge fractures, and osteotomy line below the safe zone cause delayed bone healing after open-wedge high tibial osteotomy.

PURPOSE: To evaluate bone formation in the osteotomy gap after open-wedge high tibial osteotomy (OWHTO), including after plate removal, and to investigate risk factors for delayed bone healing. METHODS: Ninety-three patients (102 knees) who underwent OWHTO without bone grafting were enrolled. The osteotomy gap was divided into the lateral hinge and the four zones on anteroposterior radiographs, and we defined the zone in which trabecular bone continuity could be observed as gap filling. Bone formation in the osteotomy gap was evaluated according to this definition at 3, 6, and 12 months postoperatively; at plate removal; and at the final follow-up (mean, 62.3 ± 30.2 months). We also investigated the risk factors for delayed bone healing. RESULTS: The lateral hinge united at 3 months postoperatively in 92 knees (90.2%). At 1 year postoperatively, 98 knees (96.1%) reached zone 1 and 92 knees (90.2%) reached zone 2. At plate removal, gap filling reached zone 2 in all cases and progressed further without loss of correction after plate removal. Opening width over 13.0 mm [odds ratio (OR): 1.61, P = 0.02], Takeuchi's classification type II lateral hinge fracture (OR: 20.4, P < 0.01), and osteotomy line below the safe zone (OR: 8.98, P < 0.01) significantly delayed bone formation after OWHTO. CONCLUSIONS: Gap filling progressed from lateral to medial after OWHTO without bone grafting and progressed further after plate removal. Large opening gaps, unstable hinge fractures, and osteotomy line below the safe zone cause delayed bone healing after OWHTO.

Crosstalk of DNA double-strand break repair pathways in poly(ADP-ribose) polymerase inhibitor treatment of breast cancer susceptibility gene 1/2-mutated cancer.

Germline mutations in breast cancer susceptibility gene 1 or 2 (BRCA1 or BRCA2) significantly increase cancer risk in hereditary breast and ovarian cancer syndrome (HBOC). Both genes function in the homologous recombination (HR) pathway of the DNA double-strand break (DSB) repair process. Therefore, the DNA-repair defect characteristic of cancer cells brings about a therapeutic advantage for poly(ADP-ribose) polymerase (PARP) inhibitor-induced synthetic lethality. PARP inhibitor-based therapeutics initially cause cancer lethality but acquired resistance mechanisms have been found and need to be elucidated. In particular, it is essential to understand in detail the mechanism of DNA damage and repair to PARP inhibitor treatment. Further investigations have shown the roles of BRCA1/2 and its associations to other molecules in the DSB repair system. Notably, the repair pathway chosen in BRCA1-deficient cells could be entirely different from that in BRCA2-deficient cells after PARP inhibitor treatment. The present review describes synthetic lethality and acquired resistance mechanisms to PARP inhibitor through the DSB repair pathway and subsequent repair process. In addition, recent knowledge of resistance mechanisms is discussed. Our model should contribute to the development of novel therapeutic strategies.

Effect of Low-Intensity Pulsed Ultrasound on Bone Healing at Osteotomy Sites After Open Wedge High Tibial Osteotomy.

OBJECTIVE: The purpose of this study was to evaluate the effects of low-intensity pulsed ultrasound (LIPUS) on bone healing at osteotomy sites after open wedge high tibial osteotomy (OWHTO). MATERIALS AND METHODS: Twenty-six patients underwent OWHTO without bone grafting. Thirty cases treated with LIPUS (group L) after surgery were compared with 13 cases without LIPUS treatment (group C). We divided the osteotomy gap into the lateral hinge and 4 zones on anteroposterior radiography, and the progression of gap filling was evaluated at 1, 3, and 6 months post-operatively in both groups. RESULTS: In group L, the lateral hinge formed a union at 3 months postoperatively in 11 knees (84.6%). At 6 months, gap filling in 10 knees (76.9%) reached to zone 2. In group C, while the lateral hinge formed a union at 3 months postoperatively in all cases. At 6 months, gap filling in 6 knees (46.1%) reached to zone 2. The progression of gap filling were 8.5%, 18.9%, and 32.9% (at 1, 3, and 6 months after surgery, respectively) in group L, while the progress levels at the same time points were 7.7%, 16.6%, and 24.4% in group C. There were significant differences at 6 months after surgery between the 2 groups. DISCUSSION: LIPUS accelerated bone healing at 6 months after OWHTO. It is important that the accuracy of LIPUS methods be further studied and improved.

Patellofemoral Osteoarthritis Progression and Alignment Changes after Open-Wedge High Tibial Osteotomy Do Not Affect Clinical Outcomes at Mid-term Follow-up.

PURPOSE: To evaluate the clinical and radiological outcomes of open-wedge high tibial osteotomy (OWHTO) with respect to the patellofemoral joint and to assess whether patellofemoral osteoarthritis (OA) progression and alignment changes after OWHTO affect clinical outcomes. METHODS: Inclusion criteria were consecutive patients who underwent OWHTO from March 2005 to September 2013. Exclusion criteria were loss to follow-up within 2 years and absence of second-look arthroscopy findings at the time of plate removal. The clinical parameters, including anterior knee pain while climbing stairs, Japanese Orthopedic Association score, and Oxford Knee Score, were evaluated. Radiological outcomes, including weight-bearing line ratio, modified Blackburne-Peel ratio, posterior tibial slope, tilting angle, lateral shift ratio, and patellofemoral OA (Kellgren-Lawrence grade), were evaluated preoperatively and at the final follow-up. Cartilage status (International Cartilage Repair Society grade) was evaluated at the initial HTO and at plate removal. RESULTS: Fifty-three patients (60 knees) were included in this study. The mean follow-up was 58.2 ± 22.4 months. Two knees (3%) presented with mild anterior knee pain after OWHTO. The mean Japanese Orthopedic Association score (66.9 ± 11.2 to 91.2 ± 9.7) significantly improved (P < .001), and the mean Oxford Knee Score at the final follow-up was 42.0 ± 5.3. The mean modified Blackburne-Peel ratio (0.9 ± 0.1 to 0.7 ± 0.1, P < .001) and tilting angle (6.8 ± 3.7 to 5.6 ± 3.4, P = .033) significantly decreased after OWHTO, whereas no significant changes in posterior tibial slope (P = .511) and lateral shift ratio (P = .522) were observed. Radiologically, patellofemoral OA had progressed in 15 knees (27%), and arthroscopically patellofemoral cartilage degeneration had progressed in 27 knees (45%). However, there was no significant correlation between changes in patellofemoral alignment and clinical outcomes. CONCLUSIONS: Changes in patellofemoral alignment and patellofemoral OA progression did not affect the clinical outcomes of OWHTO at mid-term follow-up. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

FKBP51 regulates cell motility and invasion via RhoA signaling.

FK506 binding protein 51 (FKBP51), a member of the immunophilin family, is involved in multiple signaling pathways, tumorigenesis, and chemoresistance. FKBP51 expression correlates with metastatic potential in melanoma and prostate cancer. However, the functions of FKBP51, particularly involving the regulation of cell motility and invasion, are not fully understood. We discovered two novel interacting partner proteins of FKBP51, i.e., deleted in liver cancer 1 (DLC1) and deleted in liver cancer 2 (DLC2), using immunoprecipitation and mass spectrometry. DLC1 and DLC2 are Rho GTPase-activating proteins that are frequently downregulated in various cancers. Next, we demonstrated that overexpression of FKBP51 enhances cell motility and invasion of U2OS cells via upregulation of RhoA activity and enhanced Rho-ROCK signaling. Moreover, FKBP51-depleted cells displayed a cortical distribution of actin filaments and decreased cell motility and invasion. Consistent with this phenotype, FKBP51 depletion caused a downregulation of RhoA activity. Considered together, our results demonstrate that FKBP51 positively controls cell motility by promoting RhoA and ROCK activation; thus, we have revealed a novel role for FKBP51 in cytoskeletal rearrangement and cell migration and invasion.

Functional receptor molecules CD300lf and CD300ld within the CD300 family enable murine noroviruses to infect cells.

Norovirus is the leading cause of acute gastroenteritis worldwide. Since the discovery of human norovirus (HuNoV), an efficient and reproducible norovirus replication system has not been established in cultured cells. Although limited amounts of virus particles can be produced when the HuNoV genome is directly transfected into cells, the HuNoV cycle of infection has not been successfully reproduced in any currently available cell-culture system. Those results imply that the identification of a functional cell-surface receptor for norovirus might be the key to establishing a norovirus culture system. Using a genome-wide CRISPR/Cas9 guide RNA library, we identified murine CD300lf and CD300ld as functional receptors for murine norovirus (MNV). The treatment of susceptible cells with polyclonal antibody against CD300lf significantly reduced the production of viral progeny. Additionally, ectopic CD300lf expression in nonsusceptible cell lines derived from other animal species enabled MNV infection and progeny production, suggesting that CD300lf has potential for dictating MNV host tropism. Furthermore, CD300ld, which has an amino acid sequence in the N-terminal region of its extracellular domain that is highly homologous to that of CD300lf, also functions as a receptor for MNV. Our results indicate that direct interaction of MNV with two cell-surface molecules, CD300lf and CD300ld, dictates permissive noroviral infection.

Identification of PPM1D as an essential Ulk1 phosphatase for genotoxic stress-induced autophagy.

Autophagy is an evolutionary conserved process that degrades subcellular constituents. Unlike starvation-induced autophagy, the molecular mechanism of genotoxic stress-induced autophagy has not yet been fully elucidated. In this study, we analyze the molecular mechanism of genotoxic stress-induced autophagy and identify an essential role of dephosphorylation of the Unc51-like kinase 1 (Ulk1) at Ser637, which is catalyzed by the protein phosphatase 1D magnesium-dependent delta isoform (PPM1D). We show that after exposure to genotoxic stress, PPM1D interacts with and dephosphorylates Ulk1 at Ser637 in a p53-dependent manner. The PPM1D-dependent Ulk1 dephosphorylation triggers Ulk1 puncta formation and induces autophagy. This happens not only in mouse embryonic fibroblasts but also in primary thymocytes, where the genetic ablation of PPM1D reduces the dephosphorylation of Ulk1 at Ser637, inhibits autophagy, and accelerates apoptosis induced by X-ray irradiation. This acceleration of apoptosis is caused mainly by the inability of the autophagic machinery to degrade the proapoptotic molecule Noxa. These findings indicate that the PPM1D-Ulk1 axis plays a pivotal role in genotoxic stress-induced autophagy.

BRCA2 mediates centrosome cohesion via an interaction with cytoplasmic dynein.

BRCA2 is responsible for familial breast and ovarian cancer and has been linked to DNA repair and centrosome duplication. Here we analyzed the mechanism by which the centrosomal localization signal (CLS) of BRCA2 interacts with cytoplasmic dynein 1 to localize BRCA2 to the centrosome. In vitro pull-down assays demonstrated that BRCA2 directly binds to the cytoplasmic dynein 1 light intermediate chain 2. A dominant-negative HA-CLS-DsRed fusion protein, the depletion of dynein by siRNA, and the inactivation of dynein by EHNA, inhibited the localization of BRCA2 at centrosomes and caused the separation of centrosome pairs during the S-phase. The double depletion of BRCA2 and C-Nap1 caused a larger dispersion of centrosome distances than the silencing of C-Nap1. These results suggest that cytoplasmic dynein 1 binds to BRCA2 through the latter's CLS and BRCA2 mediates the cohesion between centrosomes during the S phase, potentially serving as a cell-cycle checkpoint.