Disordered region of nuclear membrane protein Bqt4 recruits phosphatidic acid to the nuclear envelope to maintain its structural integrity.

The nuclear envelope (NE) is a permeable barrier that maintains nuclear-cytoplasmic compartmentalization and ensures nuclear function; however, it ruptures in various situations such as mechanical stress and mitosis. Although the protein components for sealing a ruptured NE have been identified, the mechanism by which lipid components are involved in this process remains to be elucidated. Here, we found that an inner nuclear membrane (INM) protein Bqt4 directly interacts with phosphatidic acid (PA) and serves as a platform for NE maintenance in the fission yeast Schizosaccharomyces pombe. The intrinsically disordered region (IDR) of Bqt4, proximal to the transmembrane domain, binds to PA and forms a solid aggregate in vitro. Excessive accumulation of Bqt4 IDR in INM results in membrane overproliferation and lipid droplet formation in the nucleus, leading to centromere dissociation from the NE and chromosome missegregation. Our findings suggest that Bqt4 IDR controls nuclear membrane homeostasis by recruiting PA to the INM, thereby maintaining the structural integrity of the NE.

Noonan syndrome-like phenotype associated with an ERF frameshift variant.

Noonan syndrome is a so-called "RASopathy," that is characterized by short stature, distinctive facial features, congenital heart defects, and developmental delay. Of individuals with a clinical diagnosis of Noonan syndrome, 80%-90% have pathogenic variants in the known genes implicated in the disorder, but the molecular mechanism is unknown in the remaining cases. Heterozygous pathogenic variants of ETS2 repressor factor (ERF), which functions as a repressor in the RAS/MAPK signaling pathway, cause syndromic craniosynostosis. Here, we report an ERF frameshift variant cosegregating with a Noonan syndrome-like phenotype in a family. The proband was a 3-year-old female who presented with dysmorphic facial features, including proptosis, hypertelorism, slightly down slanted palpebral fissures, low-set posteriorly rotated ears, depressed nasal bridge, short stature, and developmental delay. Exome sequencing of the proband identified a heterozygous ERF variant [NM_006494.4: c.185del p.(Glu62Glyfs*15)]. Her mother and sister showed a similar phenotype and had the same heterozygous ERF variant. A large proportion of the previously reported patients with syndromic craniosynostosis and pathogenic ERF variants also showed characteristic features that overlap with those of Noonan syndrome. The present finding supports an association between heterozygous ERF variants and a Noonan syndrome-like phenotype.

CENP-A and CENP-B collaborate to create an open centromeric chromatin state.

Centromeres are epigenetically defined via the presence of the histone H3 variant CENP-A. Contacting CENP-A nucleosomes, the constitutive centromere associated network (CCAN) and the kinetochore assemble, connecting the centromere to spindle microtubules during cell division. The DNA-binding centromeric protein CENP-B is involved in maintaining centromere stability and, together with CENP-A, shapes the centromeric chromatin state. The nanoscale organization of centromeric chromatin is not well understood. Here, we use single-molecule fluorescence and cryoelectron microscopy (cryoEM) to show that CENP-A incorporation establishes a dynamic and open chromatin state. The increased dynamics of CENP-A chromatin create an opening for CENP-B DNA access. In turn, bound CENP-B further opens the chromatin fiber structure and induces nucleosomal DNA unwrapping. Finally, removal of CENP-A increases CENP-B mobility in cells. Together, our studies show that the two centromere-specific proteins collaborate to reshape chromatin structure, enabling the binding of centromeric factors and establishing a centromeric chromatin state.

A ubiquitin-proteasome pathway degrades the inner nuclear membrane protein Bqt4 to maintain nuclear membrane homeostasis.

Aberrant accumulation of inner nuclear membrane (INM) proteins is associated with deformed nuclear morphology and mammalian diseases. However, the mechanisms underlying the maintenance of INM homeostasis remain poorly understood. In this study, we explored the degradation mechanisms of the INM protein Bqt4 in the fission yeast Schizosaccharomyces pombe. We have previously shown that Bqt4 interacts with the transmembrane protein Bqt3 at the INM and is degraded in the absence of Bqt3. Here, we reveal that excess Bqt4, unassociated with Bqt3, is targeted for degradation by the ubiquitin-proteasome system localized in the nucleus and Bqt3 antagonizes this process. The degradation process involves the Doa10 E3 ligase complex at the INM. Bqt4 is a tail-anchored protein and the Cdc48 complex is required for its degradation. The C-terminal transmembrane domain of Bqt4 was necessary and sufficient for proteasome-dependent protein degradation. Accumulation of Bqt4 at the INM impaired cell viability with nuclear envelope deformation, suggesting that quantity control of Bqt4 plays an important role in nuclear membrane homeostasis.

Prognostic impact of EGFR/ALK alterations in leptomeningeal metastasis from lung adenocarcinoma treated with whole-brain radiotherapy.

The prognosis and prognostic factors of patients receiving whole-brain radiotherapy (WBRT) for leptomeningeal metastasis (LM) from lung adenocarcinoma have not been established. Particularly, the impact of EGFR mutations and ALK rearrangements on survival remains unclear. This retrospective study evaluated the prognosis and prognostic factors of patients receiving WBRT for LM. We evaluated overall survival (OS) from WBRT initiation and clinical variables in 80 consecutive patients receiving WBRT for LM from lung adenocarcinoma at our institution between June 2013 and June 2021. After a median follow-up of 5.2 (range 0.5-56.5) months, the median OS was 6.2 months (95% CI 4.4-12.4). Of the 80 patients, 51 were classified as EGFR/ALK mutant (EGFR: 44; ALK: 6; both: 1) and 29 as wild-type. The median OS was 10.4 (95% CI 5.9-20.9) versus 3.8 (95% CI 2.5-7.7) months in the EGFR/ALK-mutant versus wild-type patients (HR = 0.49, P = 0.0063). Multivariate analysis indicated that EGFR/ALK alterations (HR = 0.54, P = 0.021) and Eastern Cooperative Oncology Group performance status (ECOG PS) of 0-1 (HR = 0.25, P < 0.001) were independent factors associated with favorable OS. Among the patients who underwent brain MRI before and after WBRT, intracranial progression-free survival was longer in the 26 EGFR/ALK-mutant than 13 wild-type patients (HR = 0.31, P = 0.0039). Although the prognosis of patients receiving WBRT for LM remains poor, EGFR/ALK alterations and good ECOG PS may positively impact OS in those eligible for WBRT.

Who should receive single-fraction palliative radiotherapy for gastric cancer bleeding?: An exploratory analysis of a multicenter prospective observational study (JROSG 17-3).

Purpose: Although the Palliative Prognostic Index (PPI) has been used to predict survival in various cancers, to our knowledge, no study has examined its applicability in gastric cancer. This study aimed to determine the baseline PPI cutoff value for recommending single-fraction radiotherapy in patients with bleeding gastric cancer. Materials and methods: This was a secondary analysis of the Japanese Radiation Oncology Study Group (JROSG) 17-3, a multicenter prospective study of palliative radiotherapy for bleeding gastric cancer. Discrimination was evaluated using a time-dependent receiver operating characteristic curve, and the optimal cutoff value was determined using the Youden index. A calibration plot was used to assess the agreement between predicted and observed survival. Results: We enrolled 55 patients in JROSG 17-3. The respective median survival times were 6.7, 2.8, and 1.0 months (p = 0.021) for patients with baseline PPI scores of ≤ 2, 2 < PPI ≤ 4, and PPI > 4. The areas under the curve for predicting death within 2, 3, 4, and 5 months were 0.813, 0.787, 0.775, and 0.721, respectively. The negative predictive value was highest when survival < 2 months was predicted and the Youden index was highest when the cutoff PPI value was 2. The calibration curve showed a reasonable agreement between the predicted and observed survival. Conclusion: Baseline PPI is useful for estimating short-term prognosis in patients treated with palliative radiotherapy for gastric cancer bleeding. A cutoff PPI value of 2 for estimating survival ≤ 2 months should be used to recommend single-fraction radiotherapy.

Ceramide synthase homolog Tlc4 maintains nuclear envelope integrity via its Golgi translocation.

Maintaining the integrity of the nuclear envelope (NE) is essential for preventing genomic DNA damage. Recent studies have shown that enzymes that catalyze lipid synthesis are involved in NE maintenance, but the underlying mechanism remains unclear. Here, we found that the ceramide synthase (CerS) homolog in the fission yeast Schizosaccharomyces pombe Tlc4 (SPAC17A2.02c) suppressed NE defects in cells lacking the NE proteins Lem2 and Bqt4. Tlc4 possesses a TRAM/LAG1/CLN8 domain that is conserved in CerS proteins and functions through its non-catalytic activity. Tlc4 was localized at the NE and endoplasmic reticulum, similar to CerS proteins, and also showed unique additional localization at the cis- and medial-Golgi cisternae. Growth and mutation analyses revealed that Golgi localization of Tlc4 was tightly linked to its activity of suppressing the defects in the double-deletion mutant of Lem2 and Bqt4. Our results suggest that Lem2 and Bqt4 control the translocation of Tlc4 from the NE to the Golgi, which is necessary for maintaining NE integrity.

Inner nuclear membrane proteins Lem2 and Bqt4 interact with different lipid synthesis enzymes in fission yeast.

The nuclear envelope (NE) is a double-membrane structure consisting of inner and outer membranes that spatially separate the nucleus from the cytoplasm, and its function is critical for cellular functions such as genome maintenance. In the fission yeast, Schizosaccharomyces pombe, the inner nuclear membrane proteins, Lem2 and Bqt4, play pivotal roles in maintaining the NE structure. We previously found that the double deletion of lem2+ and bqt4+ causes a synthetic lethal defect associated with severe NE rupture, and overexpression of Elo2, a solo very-long-chain fatty acid elongase, suppresses this defect by restoring the NE. However, the molecular basis of this restoration remains elusive. To address this, we identified Lem2- and Bqt4-binding proteins via immunoprecipitation and mass spectrometry in this study. Forty-five and 23 proteins were identified as Lem2- and Bqt4-binding proteins, respectively. Although these binding proteins partially overlapped, Lem2 and Bqt4 interacted with different types of lipid metabolic enzymes: Cho2, Ole1 and Erg11 for Lem2 and Cwh43 for Bqt4. These enzymes are known to be involved in various lipid synthesis processes, suggesting that Lem2 and Bqt4 may contribute to the regulation of lipid synthesis by binding to these enzymes.

Incidence of and risk factors for non-hematologic toxicity with combined radiotherapy and CDK4/6 inhibitors in metastatic breast cancer using dose-volume parameters analysis: a multicenter cohort study.

BACKGROUND: There is a lack of data on combined radiotherapy (RT) and cyclin-dependent kinase 4 and 6 inhibitor (CDK4/6i) risk factors and toxicity. This study aimed to assess the incidence of and risk factors for non-hematologic toxicities in patients treated with combined RT and CDK4/6i using dose-volume parameter analysis. METHODS: We conducted a retrospective multicenter cohort study of patients with metastatic breast cancer receiving RT within 14 days of CDK4/6i use. The endpoint was non-hematologic toxicities. Patient characteristics and RT treatment planning data were compared between the moderate or higher toxicities (≥ grade 2) group and the non-moderate toxicities group. RESULTS: Sixty patients were included in the study. CDK4/6i was provided at a median daily dose of 125 mg and 200 mg for palbociclib and abemaciclib, respectively. In patients who received concurrent RT and CDK4/6i (N = 29), the median concurrent prescribed duration of CDK4/6i was 14 days. The median delivered RT dose was 30 Gy and 10 fractions. The rate of grade 2 and 3 non-hematologic toxicities was 30% and 2%, respectively. There was no difference in toxicity between concurrent and sequential use of CDK4/6i. The moderate pneumonitis group had a larger lung V20 equivalent dose of 2 Gy per fraction and planning target volume than the non-moderate pneumonitis group. CONCLUSIONS: Moderate toxicities are frequent with combined RT and CDK4/6i. Caution is necessary concerning the combined RT and CDK4/6i. Particularly, reducing the dose to normal organs is necessary for combined RT and CDK4/6i.

Contrasting patterns of nitrogen release from fine roots and leaves driven by microbial communities during decomposition.

Leachate from decaying root and leaf litter plays crucial roles in soil biogeochemical processes in forest ecosystems. Unlike for leaf litter, however, the chemical composition and microbial community of root litter leachate are poorly understood. We hypothesized that both leachate nitrogen (N) composition and microbial communities differ between plant organs and decomposition stages and that leachate composition affects microbial community composition. We conducted a 2.5-year laboratory incubation using root and leaf substrate from Cryptomeria japonica and Chamaecyparis obtusa. We monitored the N forms released and used metabarcoding to characterize the microbial communities. Leachate N accounted for 40 % and 30 % of net N losses from C. japonica and C. obtusa roots, respectively; the remainder was probably lost in gaseous forms. In contrast, leaves absorbed N during the incubation regardless of tree species. The predominant N form in root leachate was nitrate (NO3-); cumulative NO3- quantity was 22.6 and 25.5 times greater in root than in leaf leachate for C. japonica and C. obtusa, respectively. A nitrifying bacterium was selected as the indicator taxon in root substrates, whereas many families of N-fixing bacteria were selected in leaf substrates. At the end of the incubation period, bacterial taxonomic diversity was high in both organs from both tree species, ranging from 177 to 339 taxa and increasing with time. However, fungal diversity was low for both organs (72 to 155 taxa). Shifts in bacterial community structure were related to NO3- concentration and leachate pH, whereas shifts in fungal community structure were related to leachate pH. These results suggest that the contrasting N dynamics of root and leaf substrates are strongly affected by the characteristics of and the microbes recruited by their leachates. Understanding organ-specific litter N dynamics is indispensable for predicting N cycling for optimal management of forest ecosystems in a changing world.

Overlapping Myocarditis and Postural Orthostatic Tachycardia Syndrome After COVID-19 Messenger RNA Vaccination: A Case Report.

The worldwide spread of the coronavirus disease 2019 (COVID-19) pandemic and the significant morbidity and mortality rate associated with it led to the rapid development of several COVID-19 vaccines. While serious side effects related to the vaccines are rare, various adverse events have been reported to occur after COVID-19 messenger RNA (mRNA) vaccination, including myocarditis, Guillain-Barré syndrome, and thrombosis. Postural orthostatic tachycardia syndrome (POTS) is a chronic cardiovascular dysautonomia among young and middle-aged individuals. Although the pathophysiology of POTS is thought to be heterogeneous, vaccine-induced immune-mediated autonomic dysfunction is hypothesized to be one cause of the syndrome. In this report, we present a case of myocarditis and POTS occurring in a 13-year-old male following COVID-19 mRNA vaccination. He presented with persistent severe fatigue and headache. The patient's symptoms improved after intravenous immunoglobulin for myocarditis, non-pharmacologic interventions, and multiple medications for POTS.

ATP levels influence cell movement during the mound phase in Dictyostelium discoideum as revealed by ATP visualization and simulation.

Cell migration plays an important role in multicellular organism development. The cellular slime mold Dictyostelium discoideum is a useful model organism for the study of cell migration during development. Although cellular ATP levels are known to determine cell fate during development, the underlying mechanism remains unclear. Here, we report that ATP-rich cells efficiently move to the central tip region of the mound against rotational movement during the mound phase. A simulation analysis based on an agent-based model reproduces the movement of ATP-rich cells observed in the experiments. These findings indicate that ATP-rich cells have the ability to move against the bulk flow of cells, suggesting a mechanism by which high ATP levels determine the cell fate of differentiation.

The inner nuclear membrane protein Lem2 coordinates RNA degradation at the nuclear periphery.

Transcriptionally silent chromatin often localizes to the nuclear periphery. However, whether the nuclear envelope (NE) is a site for post-transcriptional gene repression is not well understood. Here we demonstrate that Schizosaccharomyces pombe Lem2, an NE protein, regulates nuclear-exosome-mediated RNA degradation. Lem2 deletion causes accumulation of RNA precursors and meiotic transcripts and de-localization of an engineered exosome substrate from the nuclear periphery. Lem2 does not directly bind RNA but instead interacts with the exosome-targeting MTREC complex and its human homolog PAXT to promote RNA recruitment. This pathway acts largely independently of nuclear bodies where exosome factors assemble. Nutrient availability modulates Lem2 regulation of meiotic transcripts, implying that this pathway is environmentally responsive. Our work reveals that multiple spatially distinct degradation pathways exist. Among these, Lem2 coordinates RNA surveillance of meiotic transcripts and non-coding RNAs by recruiting exosome co-factors to the nuclear periphery.

Fission yeast Ish1 and Les1 interact with each other in the lumen of the nuclear envelope.

The nuclear envelope (NE) provides a permeable barrier that separates the eukaryotic genome from the cytoplasm. NE is a double membrane composed of inner and outer nuclear membranes. Ish1 is a stress-responsive NE protein in the fission yeast, Schizosaccharomyces pombe. Les1 is another NE protein that shares several similar domains with Ish1, but the relationship between them remains unknown. In this study, using fluorescence and electron microscopy, we found that most regions of these proteins were localized within the NE lumen. We also found that Ish1 interacted with Les1 via its C-terminal region in the NE lumen and that the NE localization of Ish1 depended on the C-terminal region of Les1. Ish1 and Les1 were co-localized at the NE in interphase cells, but when the nucleus divided at the end of mitosis (closed mitosis), they showed distinguishable localization at the midzone membrane domain. These results suggest the regulated interaction between Ish1 and Les1 in the NE lumen, although this interaction does not appear to be essential for cell survival.

Comprehensive screening for drugs that modify radiation-induced immune responses.

BACKGROUND: Combination therapy based on radiotherapy and immune checkpoint inhibitors (ICIs) was recently reported as effective for various cancers. The radiation-induced immune response (RIIR) is an essential feature in ICI-combined radiotherapy; however, the effects of drugs used concomitantly with RIIR remain unclear. We screened for drugs that can modify RIIR to understand the mutual relationship between radiotherapy and combined drugs in ICI-combined radiotherapy. METHODS: We established a high-throughput system with reporter gene assays for evaluating RIIR, focusing on factors acting downstream of the STING-IRF pathway, which can stimulate cancer cells, T cells, and dendritic cells. We further quantified the effects of 2595 drugs, including those approved by the Food and Drug Administration, on RIIR in vitro. RESULTS: The reporter assay results correlated well with the expression of immune response proteins such as programmed death-ligand 1. This high-throughput system enabled the identification of drugs including cytotoxic agents, molecular-targeted agents, and other agents that activate or suppress RIIR. CONCLUSIONS: Our study provides an encyclopedic catalogue of clinically approved drugs based on their effect on RIIR. In ICIs combined radiotherapy, activation of STING-IFN may improve the therapeutic effect and our result could form a biological basis for further clinical trials combining radiotherapy with ICIs.

Impacts of the STING-IFNAR1-STAT1-IRF1 pathway on the cellular immune reaction induced by fractionated irradiation.

Radiotherapy (RT) combined with immune checkpoint inhibitors has recently produced outstanding results and is expected to be adaptable for various cancers. However, the precise molecular mechanism by which immune reactions are induced by fractionated RT is still controversial. We aimed to investigate the mechanism of the immune response regarding multifractionated, long-term radiation, which is most often combined with immunotherapy. Two human esophageal cancer cell lines, KYSE-450 and OE-21, were irradiated by fractionated irradiation (FIR) daily at a dose of 3 Gy in 5 d/wk for 2 weeks. Western blot analysis and RNA sequencing identified type I interferon (IFN) and the stimulator of IFN genes (STING) pathway as candidates that regulate immune response by FIR. We inhibited STING, IFNAR1, STAT1, and IFN regulatory factor 1 (IRF1) and investigated the effects on the immune response in cancer cells and the invasion of surrounding immune cells. We herein revealed type I IFN-dependent immune reactions and the positive feedback of STING, IRF1, and phosphorylated STAT1 induced by FIR. Knocking out STING, IFNAR1, STAT1, and IRF1 resulted in a poorer immunological response than that in WT cells. The STING-KO KYSE-450 cell line showed significantly less invasion of PBMCs than the WT cell line under FIR. In the analysis of STING-KO cells and migrated PBMCs, we confirmed the occurrence of STING-dependent immune activation under FIR. In conclusion, we identified that the STING-IFNAR1-STAT1-IRF1 axis regulates immune reactions in cancer cells triggered by FIR and that the STING pathway also contributes to immune cell invasion of cancer cells.

Mobility of kinetochore proteins measured by FRAP analysis in living cells.

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase.

DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.

Treatment response after palliative radiotherapy for bleeding gastric cancer: a multicenter prospective observational study (JROSG 17-3).

BACKGROUND: Palliative radiotherapy seems to be rarely performed for incurable gastric cancer. In this first multicenter study, we examined the effectiveness of palliative radiotherapy and investigated whether biologically effective dose (BED) is associated with survival, response, or re-bleeding. METHODS: Eligibility criteria included blood transfusion or hemoglobin levels < 8.0 g/dL. The primary endpoint was the intention-to-treat (ITT) bleeding response rate at 4 weeks. Response entailed all of the following criteria: (i) hemoglobin levels ≥ 8.0 g/dL; (ii) 7 consecutive days without blood transfusion anytime between enrollment and blood sampling; and (iii) no salvage treatment (surgery, endoscopic treatment, transcatheter embolization, or re-irradiation) for bleeding gastric cancer. Re-bleeding was defined as the need for blood transfusion or salvage treatment. RESULTS: We enrolled 55 patients from 15 institutions. The ITT response rates were 47%, 53%, and 49% at 2, 4, and 8 weeks, respectively. The per-protocol response rates were 56%, 78%, and 90% at 2, 4, and 8 weeks, respectively. Neither response nor BED (α/ß = 10) predicted overall survival. Multivariable Fine-Gray model showed that BED was not a significant predictor of response. Univariable Cox model showed that BED was not significantly associated with re-bleeding. Grades 1, 2, 3, and, ≥ 4 radiation-related adverse events were reported in 11, 9, 1, and 0 patients, respectively. CONCLUSIONS: The per-protocol response rate increased to 90% during the 8-week follow-up. The frequent occurrence of death starting shortly after enrollment lowered the ITT response rate. BED was not associated with survival, bleeding response, or re-bleeding.