A chromosome-level genome assembly for the amphibious plant Rorippa aquatica reveals its allotetraploid origin and mechanisms of heterophylly upon submergence.

The ability to respond to varying environments is crucial for sessile organisms such as plants. The amphibious plant Rorippa aquatica exhibits a striking type of phenotypic plasticity known as heterophylly, a phenomenon in which leaf form is altered in response to environmental factors. However, the underlying molecular mechanisms of heterophylly are yet to be fully understood. To uncover the genetic basis and analyze the evolutionary processes driving heterophylly in R. aquatica, we assembled the chromosome-level genome of the species. Comparative chromosome painting and chromosomal genomics revealed that allopolyploidization and subsequent post-polyploid descending dysploidy occurred during the speciation of R. aquatica. Based on the obtained genomic data, the transcriptome analyses revealed that ethylene signaling plays a central role in regulating heterophylly under submerged conditions, with blue light signaling acting as an attenuator of ethylene signal. The assembled R. aquatica reference genome provides insights into the molecular mechanisms and evolution of heterophylly.

Nuclear pore complex proteins are involved in centromere distribution.

The subnuclear distribution of centromeres is cooperatively regulated by condensin II and the linker of nucleoskeleton and cytoskeleton (LINC) complex. However, other nuclear membrane structures and nuclear proteins are probably involved in centromere dynamics and distribution. Here, we focused on the nuclear pore complex (NPC), which is known to regulate gene expression, transcription memory, and chromatin structure in addition to transport between the cytoplasm and nucleoplasm. We report here that some nucleoporins (Nups), including Nup85, Nup133, CG1, Nup93b, and NUA, are involved in centromere scattering in Arabidopsis thaliana. In addition, the centromere dynamics after metaphase in nup mutants were found to be similar to that of the condensin II mutant. Furthermore, both biochemical and genetic approaches showed that the Nups interact with the LINC complex. These results suggest that Nups regulate centromere scattering cooperatively with condensin II and the LINC complex.

Septic Arthritis of Cervical Spine Facet Joints: A Case Report and Review of Imaging.

BACKGROUND Septic arthritis of the facet joint (SAFJ) has been considered a rare type of spinal infection. However, because of the aging of the population, the increase in compromised hosts, and the increase in MRI use in recent years, the number of reports has been increasing. We report the clinical progress of a rare case of septic arthritis of the cervical facet joint (SACFJ) with some imaging considerations, and we compare our findings with existing reports of SACFJ. CASE REPORT A 73-year-old Japanese woman presented with fever, paralytic symptoms, and paresthesia of the upper limbs. Here, we report a case of SACFJ in which MRI findings allowed early diagnosis, and a favorable course was obtained by conservative treatment with antibiotics. Although MRI performed 93 days after the initiation of treatment showed a slight residual signal change in the facet joints, no symptoms had recurred by the sixth month after hospital discharge. CONCLUSIONS If a patient develops neurological symptoms such as paralysis with fever and increased inflammatory response, the physician must consider the possibility of pyogenic spondylitis, including SACFJ, and order an MRI. Epidural abscess is almost inevitable in SACFJ, and surgical treatment, including abscess drainage, is required if spinal cord or paralytic symptoms progress. For patients with SACFJ, as well as pyogenic spondylitis, MRI may not be useful in determining treatment efficacy.

Analysis of Chromatin Accessibility, Histone Modifications, and Transcriptional States in Specific Cell Types Using Flow Cytometry.

The past two decades in biomedical research have experienced an explosion of cell type-specific and single-cell studies, especially concerning the concomitant dissection of regulatory and transcriptional landscapes of those under investigation. Additionally, leveraging next-generation sequencing (NGS) platforms efforts have been undertaken to evaluate the effects of chromatin accessibility, histone modifications, or even transcription factor binding sites. We have shown that Fluorescence-Activated Nuclear Sorting (FANS) is an effective means to characterize the transcriptomes of nuclei from different tissues. In light of our own technical and experimental developments, we extend this effort to combine FACS/FANS with Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), Chromatin Immunoprecipitation sequencing (ChIP-seq), and RNA sequencing (RNA-seq) for profiling individual cell types according to their chromatin and transcriptional states.

Validity of the Center-Center Method for the Syndesmotic Fixation Axis Compared to the Transsyndesmotic Axis.

BACKGROUND: Placement of clamp forceps along the transsyndesmotic (TS) axis reduces the risk of iatrogenic syndesmotic malreduction during ankle fracture surgery with tibiofibular diastasis. This study aimed to measure the difference between the TS axis and the axis determined by an intraoperative fluoroscopic technique using the center-center (CC) method. We also compared the values obtained when the CC method was performed at different heights from the tibial plafond. METHODS: We evaluated the computed tomography scans of 150 patients with normal syndesmosis. The CC method was simulated using digitally reconstructed radiographs. The TS and CC axes were projected onto an axial computed tomographic image linked to digitally reconstructed radiography. The angle between the two axes (interaxis angle) and the distance between the intersection of these axes and the medial tibial cortex (interaxis distance) were measured. The procedures were performed 0, 10, and 20 mm proximal to the tibial plafond, and the measurements were compared among the different heights using a 1-way repeated-measures analysis of variance. RESULTS: The TS axis was found to be externally rotated to the CC axis, with an interaxis angle of 8.5 degrees (SD, 6.8 degrees). The interaxis angle increased from 1.9 degrees at a height of 0 mm to a greater angle at higher heights (P < .001). The overall interaxis distance was 7.7 (SD, 6.3) mm, increasing from 2.0 mm at a height of 0 mm to a greater distance at higher heights (P < .001). CONCLUSION: The TS axis was externally rotated relative to the CC axis, and the difference between the 2 axes was greater when the CC method was performed on the higher heights from the tibial plafond. To clamp the syndesmosis along the TS axis, the CC method should be applied close to the ankle joint. LEVEL OF EVIDENCE: Level IV, case series.

Readability and quality of online patient resources regarding knee osteoarthritis and lumbar spinal stenosis in Japan.

BACKGROUND: This study aimed to quantify the readability and quality of online patient resources on knee osteoarthritis and lumbar spinal stenosis in Japan. METHODS: Three search engines (Google, Yahoo, and Bing) were searched for the terms knee osteoarthritis and lumbar spinal stenosis. The first 30 websites of each search were screened. Duplicate websites and those unrelated to the searched diseases were excluded. The remaining 125 websites (62 on knee osteoarthritis, 63 on lumbar spinal stenosis) were analyzed. The text readability was assessed using two web-based programs (Obi-3 and Readability Research Lab) and lexical density. Website quality was evaluated using the DISCERN score, Clear Communication Index, and Journal of American Medical Association benchmark criteria. RESULTS: Readability scores were high, indicating that the texts were difficult to understand. Only 24 (19%) and six (5%) websites were classified as average difficulty readability according to Obi-3 and Readability Research Lab, respectively. The overall quality of information was low, with only four (3%) being rated as having sufficient quality based on the Clear Communication Index and Journal of American Medical Association benchmark criteria. None of the websites satisfied the DISCERN quality criteria. CONCLUSIONS: Patient information on Japanese websites regarding knee osteoarthritis and lumbar spinal stenosis were difficult to understand. Moreover, the quality of the websites was insufficient. Orthopaedic surgeons should contribute to the creation of high-quality easy-to-read websites to facilitate patient-physician communication.

Chromatin dynamics and subnuclear gene positioning for transcriptional regulation.

Plants have been found to exhibit diverse characteristics and functions of chromatin organization, showing both similarities and differences to animals. It is becoming clear how chromatin organization is linked to transcriptional regulation in response to environmental stresses. Regulation of specific chromatin positions in the nuclear space is important for transcription, and the mechanisms that enable such chromatin dynamics are gradually being unveiled. Genes move between subdomains responsible for transcriptional activation or suppression in the subnuclear space in a gene repositioning cycle. We propose a model of localized chromatin interaction in nuclear subdomains, in which the dynamics of local chromatin interactions have a more important impact on the regulation of gene expression than large-scale chromatin organization. In this mini-review, we highlight recent findings on chromatin dynamics, particularly involving transcriptional regulation, and discuss future directions in the study of chromatin organization in plants.

The plant nuclear lamina disassembles to regulate genome folding in stress conditions.

The nuclear lamina is a complex network of nuclear lamins and lamin-associated nuclear membrane proteins, which scaffold the nucleus to maintain structural integrity. In Arabidopsis thaliana, nuclear matrix constituent proteins (NMCPs) are essential components of the nuclear lamina and are required to maintain the structural integrity of the nucleus and specific perinuclear chromatin anchoring. At the nuclear periphery, suppressed chromatin overlapping with repetitive sequences and inactive protein-coding genes are enriched. At a chromosomal level, plant chromatin organization in interphase nuclei is flexible and responds to various developmental cues and environmental stimuli. On the basis of these observations in Arabidopsis, and given the role of NMCP genes (CRWN1 and CRWN4) in organizing chromatin positioning at the nuclear periphery, one can expect considerable changes in chromatin-nuclear lamina interactions when the global chromatin organization patterns are being altered in plants. Here we report the highly flexible nature of the plant nuclear lamina, which disassembles substantially under various stress conditions. Focusing on heat stress, we reveal that chromatin domains, initially tethered to the nuclear envelope, remain largely associated with CRWN1 and become scattered in the inner nuclear space. By investigating the three-dimensional chromatin contact network, we further reveal that CRWN1 proteins play a structural role in shaping the changes in genome folding under heat stress. Also, CRWN1 acts as a negative transcriptional coregulator to modulate the shift of the plant transcriptome profile in response to heat stress.

NAC103 mutation alleviates DNA damage in an Arabidopsis thaliana mutant sensitive to excess boron.

Excess boron (B) is toxic to plants and thereby causes DNA damage and cell death in root meristems. However, the underlying mechanisms which link boron and DNA damage remain unclear. It has been reported that the rpt5a-6 mutant of the 26S proteasome is sensitive to excess boron, resulting in more frequent cell death in root meristem and reduced root elongation. In this study, we showed that a reduction in root growth in the rpt5a mutant in the presence of high boron levels is repressed by a mutation in NAC domain containing transcription factor NAC103, a substrate of the proteasome, which functions in the unfolded protein response pathway. The mutation in NAC103 alleviated excess-B-induced DNA damage and cell death in root meristems of the rpt5a mutant. Superoxide ( O 2 - ) staining with nitroblue tetrazolium revealed that boron stress causes O 2 - accumulation in root tips, which was higher in the rpt5a-6 mutant, whereas the accumulation was lower in the rpt5a-6 nac103-3 double mutant. Our work demonstrates the overall involvement of NAC103 in maintaining healthy root meristem under excess boron conditions in the absence of RPT5A proteasome subunit.

[The Six Cases of Recurrence of Lung Metastasis after Radical Resection of Pancreatic Cancer Performed at Our Hospital].

Prognosis of pancreatic cancer is extremely poor due to recurrence in the early postoperative period. However, there are some reports that the prognosis for lung recurrence alone is relatively better than that of others. In this study, we report the cases of lung recurrence alone after radical resection of pancreatic cancer performed at our hospital. Among 255 cases of radical resection of pancreatic cancer performed at our hospital between July 2010 and August 2021, 6 cases in which the initial recurrence site of recurrence was lung alone were included in the study. The median age of the patients was 72 years (62-82), and there were 5 males and 1 female. Four patients had undergone pancreaticoduodenectomy and 2 patients underwent distal pancreatectomy. Four patients received adjuvant chemotherapy, 3 with S-1 and 1 with GEM. No patients underwent surgical resection for recurrence of lung metastasis, and all patients were treated with chemotherapy. The median time to recurrence was 351 days. As initial therapy after recurrence, 3 patients received GEM plus nab-PTX combination therapy and 3 patients received S-1. The median overall survival after recurrence was 1,979 days, and the 1-year and 3-year overall survival rates after recurrence were 100% and 100%, respectively. The prognosis of patients with recurrence of lung metastasis after surgery for pancreatic cancer at our hospital was similarly good as the previous reports.

Antecedents and Outcomes of Work Engagement among Psychiatric Nurses in Japan.

While previous studies have examined antecedents and outcomes of work engagement among general nurses, studies among psychiatric nurses remain limited. This study aimed to explore the antecedents (i.e., job crafting and nursing practice environment) and outcomes (i.e., strength-oriented care attitudes, mental health, and turnover intention) of work engagement among psychiatric nurses in Japan. This cross-sectional study included 309 nurses from three psychiatric hospitals in Japan (valid response rate: 60.4%). Data collection using the self-administered questionnaire took place from July to August 2021. We performed Structural Equation Modeling to examine the directional relationships among variables. Job crafting (ß = 0.57, p < 0.01) and nursing practice environment (ß = 0.23, p = 0.01) exhibited positive effects on work engagement. Work engagement had positive effects on strength-oriented care attitudes (ß = 0.15, p = 0.04) and mental health (ß = 0.37, p < 0.01) as well as negative effects on intention to resign from their profession as a nurse (ß = -0.17, p = 0.01). Job crafting and a healthier nursing practice environment could help enhance work engagement. Higher work engagement could contribute to improving strength-oriented care attitudes, mental health, and intention to resign from their profession as a nurse.

Histone deacetylation regulates de novo shoot regeneration.

During de novo plant organ regeneration, auxin induction mediates the formation of a pluripotent cell mass called callus, which regenerates shoots upon cytokinin induction. However, molecular mechanisms underlying transdifferentiation remain unknown. Here, we showed that the loss of HDA19, a histone deacetylase (HDAC) family gene, suppresses shoot regeneration. Treatment with an HDAC inhibitor revealed that the activity of this gene is essential for shoot regeneration. Further, we identified target genes whose expression was regulated through HDA19-mediated histone deacetylation during shoot induction and found that ENHANCER OF SHOOT REGENERATION 1 and CUP-SHAPED COTYLEDON 2 play important roles in shoot apical meristem formation. Histones at the loci of these genes were hyperacetylated and markedly upregulated in hda19. Transient ESR1 or CUC2 overexpression impaired shoot regeneration, as observed in hda19. Therefore, HDA19 mediates direct histone deacetylation of CUC2 and ESR1 loci to prevent their overexpression at the early stages of shoot regeneration.

Synovial Fluid Derived from Human Knee Osteoarthritis Increases the Viability of Human Adipose-Derived Stem Cells through Upregulation of FOSL1.

Knee osteoarthritis (Knee OA) is an irreversible condition that causes bone deformity and degeneration of the articular cartilage that comprises the joints, resulting in chronic pain and movement disorders. The administration of cultured adipose-derived stem cells (ADSCs) into the knee joint cavity improves the clinical symptoms of Knee OA; however, the effect of synovial fluid (SF) filling the joint cavity on the injected ADSCs remains unclear. In this study, we investigated the effect of adding SF from Knee OA patients to cultured ADSCs prepared for therapeutic use in an environment that mimics the joint cavity. An increase in the viability of ADSCs was observed following the addition of SF. Gene expression profiling of SF-treated ADSCs using DNA microarrays revealed changes in several genes involved in cell survival. Of these genes, we focused on FOSL1, which is involved in the therapeutic effect of ADSCs and the survival and proliferation of cancer stem cells. We confirmed the upregulation of FOSL1 mRNA and protein expression using RT-PCR and western blot analysis, respectively. Next, we knocked down FOSL1 in ADSCs using siRNA and observed a decrease in cell viability, indicating the involvement of FOSL1 in the survival of ADSCs. Interestingly, in the knockdown cells, ADSC viability was also decreased by SF exposure. These results suggest that SF enhances cell viability by upregulating FOSL1 expression in ADSCs. For therapy using cultured ADSCs, the therapeutic effect of ADSCs may be further enhanced if an environment more conducive to the upregulation of FOSL1 expression in ADSCs can be established.

Genomic analysis of an ultrasmall freshwater green alga, Medakamo hakoo.

Ultrasmall algae have attracted the attention of biologists investigating the basic mechanisms underlying living systems. Their potential as effective organisms for producing useful substances is also of interest in bioindustry. Although genomic information is indispensable for elucidating metabolism and promoting molecular breeding, many ultrasmall algae remain genetically uncharacterized. Here, we present the nuclear genome sequence of an ultrasmall green alga of freshwater habitats, Medakamo hakoo. Evolutionary analyses suggest that this species belongs to a new genus within the class Trebouxiophyceae. Sequencing analyses revealed that its genome, comprising 15.8 Mbp and 7629 genes, is among the smallest known genomes in the Viridiplantae. Its genome has relatively few genes associated with genetic information processing, basal transcription factors, and RNA transport. Comparative analyses revealed that 1263 orthogroups were shared among 15 ultrasmall algae from distinct phylogenetic lineages. The shared gene sets will enable identification of genes essential for algal metabolism and cellular functions.

Novel whole-mount FISH analysis for intact root of Arabidopsis thaliana with spatial reference to 3D visualization.

Whole-mount fluorescent in situ hybridization (WM-FISH) is an effective tool to observe chromosome behavior in tissues or organs. However, it is difficult to obtain a precise spatial profile of fluorescent signals in roots using conventional WM-FISH mainly because of the severe damage caused during the processing. To address this problem, we established a novel WM-FISH analysis for intact roots of Arabidopsis thaliana and successfully obtained a precise spatial profile of nuclear size and centromere signals. The two main improvements in the novel WM-FISH analysis are: (i) hybridization was performed directly on MAS-coated glass slides covered with silicon wells and (ii) conditions for enzyme treatment were optimized (37 °C, 45 s). After the WM-FISH using a centromere probe, we analyzed the results by 3D data processing to quantify the nuclear volume and number of centromere signals of the obtained cortical cell files and determined the position of each nucleus in intact roots. Then we plotted the nuclear volume and number of centromere signals versus distance from the quiescent center to evaluate the precise spatial profile of each parameter.

The Deterioration of Cervical Kyphosis During Neck Flexion after Laminoplasty Affects the Surgical Outcome of Cervical Spondylotic Myelopathy.

STUDY DESIGN: A retrospective single-center study. OBJECTIVE: The present study investigated whether postoperative cervical alignment changes, in addition to preoperative patient backgrounds, imaging parameters, and disease severity, affect outcomes 1 year after laminoplasty. METHODS: One hundred and three cervical spondylotic myelopathy (CSM) patients who underwent laminoplasty were enrolled. Preoperative and postoperative (1-year) Japanese Orthopedic Association (JOA) scores, cervical alignment, and balance on X-rays were assessed. Patients were classified into 2 groups for a univariate analysis according to the status of the recovery rate (RR) of the JOA score ≥50%. A multiple logistic regression analysis was performed to identify factors associated with good surgical outcomes a. RESULTS: The mean RR of the JOA score was 47.5% and the loss of cervical lordosis in the neutral position was 5.5°. The univariate analysis revealed slight differences in age, sex, and the duration of disease. Preoperative C2-7 angles were not significantly different. The C2-7 angle during flexion after surgery was significantly smaller in Group F. In the multiple logistic regression analysis, significant factors associated with the status of RR≥50% were a younger age (OR: .75, 95%CI: .59-.96), shorter duration of disease (OR: .94, 95%CI: .89-.99), and a lordotic C2-7 angle during neck flexion after surgery (OR: 1.47, 95%CI: 1.1-1.95). CONCLUSION: We retrospectively assessed the surgical outcomes of laminoplasty in 103 CSM cases. In addition to an older age and longer duration of disease, postoperative deteriorations in cervical kyphosis during neck flexion had a negative impact on outcomes.

The Detection of Immunity against WT1 and SMAD4P130L of EpCAM+ Cancer Cells in Malignant Pleural Effusion.

Malignant pleural effusion (MPE) provides a liquid tumor microenvironment model that includes cancer cells and immune cells. However, the characteristics of tumor antigen-specific CD8+ T cells have not been investigated in detail. Here, we analyzed MPE samples taken from a patient with pancreatic cancer who received a dendritic cell vaccine targeting Wilms' Tumor 1 (WT1) antigen over the disease course (two points at MPE1st and 2nd, two months after MPE1st). Epithelial cell adhesion molecule (EpCAM)+ cancer cells (PD-L1- or T cell immunoglobulin mucin-3, TIM-3-), both PD-1 or TIM-3 positive CD8+ T cells, and CD14+CD68+CD163+TIM-3+ macrophages increased from the MPE1st to MPE2nd. The ratio of WT1-specific cytotoxic lymphocytes (WT1-CTLs) to MPE CD8+ T cells and IFN-γ secretion of WT1-CTLs were reduced with disease progression. Coincidentally, the fraction of central memory T (TCM) of WT1-CTLs was decreased. On the other hand, CD8+ T cells in response to SMAD4P130L, which is homogeneously expressed in EpCAM+ cancer cells, were detected using in vitro expansion with the HLA-A*11:01 restrictive SVCVNLYH neoantigen. Furthermore, the CD8+ T cell response to SMAD4P130L was diminished following remarkably decreased numbers of CD8+ TCM in MPE samples. In conclusion, CD8+ T cells responding to WT1 or SMAD4P130L neoantigen expressed in EpCAM+ pancreatic cancer cells were detected in MPE. A tumor antigen-specific immune response would provide novel insight into the MPE microenvironment.

Different In Vitro-Generated MUTZ-3-Derived Dendritic Cell Types Secrete Dexosomes with Distinct Phenotypes and Antigen Presentation Potencies.

Human dendritic cell (DC) dexosomes were evaluated for their function and preclinical validation for vaccines. Dexosomes are small DC-secreted vesicles that contain absorbing immune signals. Vaccine manufacturing requires a significant number of monocyte-derived DCs (Mo-DCs) from donor blood; thus, Mo-DC dexosomes are expected to serve as novel materials for cancer vaccination. In this study, we characterized a potential dexosome model using immature and mature MUTZ3-derived DCs (M-imIL-4-DC, M-imIFN-DC, M-mIL-4-DC, and M-mIFN-DC) and their dexosomes (M-imIL-4-Dex, M-imIFN-Dex, M-mIL4-Dex, and M-mIFN-Dex). Despite the lack of significant differences in viability, M-mIFN-DC showed a significantly higher level of yield and higher levels of maturation surface markers, such as CD86 and HLA-ABC, than M-mIL-4-DC. In addition, M-mIFN-Dex expressed a higher level of markers, such as HLA-ABC, than M-mIL-4-Dex. Furthermore, M-mIFN-Dex exhibited a higher level of antigen presentation potency, as evaluated using a MART-1 system, than either M-imIFN-Dex or M-mIL-4-Dex. We found that M-mIFN-Dex is one of the four types of MUTZ3-derived DCs that harbor potential immunogenicity, suggesting that DC dexosomes could be useful resources in cancer immunotherapy.

Two-step regulation of centromere distribution by condensin II and the nuclear envelope proteins.

The arrangement of centromeres within the nucleus differs among species and cell types. However, neither the mechanisms determining centromere distribution nor its biological significance are currently well understood. In this study, we demonstrate the importance of centromere distribution for the maintenance of genome integrity through the cytogenic and molecular analysis of mutants defective in centromere distribution. We propose a two-step regulatory mechanism that shapes the non-Rabl-like centromere distribution in Arabidopsis thaliana through condensin II and the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Condensin II is enriched at centromeres and, in cooperation with the LINC complex, induces the scattering of centromeres around the nuclear periphery during late anaphase/telophase. After entering interphase, the positions of the scattered centromeres are then stabilized by nuclear lamina proteins of the CROWDED NUCLEI (CRWN) family. We also found that, despite their strong impact on centromere distribution, condensin II and CRWN proteins have little effect on chromatin organization involved in the control of gene expression, indicating a robustness of chromatin organization regardless of the type of centromere distribution.

Sustained defense response via volatile signaling and its epigenetic transcriptional regulation.

Plants perceive volatiles emitted from herbivore-damaged neighboring plants to urgently adapt or prime their defense responses to prepare for forthcoming herbivores. Mechanistically, these volatiles can induce epigenetic regulation based on histone modifications that alter the transcriptional status of defense genes, but little is known about the underlying mechanisms. To understand the roles of such epigenetic regulation of plant volatile signaling, we explored the response of Arabidopsis (Arabidopsis thaliana) plants to the volatile ß-ocimene. Defense traits of Arabidopsis plants toward larvae of Spodoptera litura were induced in response to ß-ocimene, through enriched histone acetylation and elevated transcriptional levels of defense gene regulators, including ethylene response factor genes (ERF8 and ERF104) in leaves. The enhanced defense ability of the plants was maintained for 5 d but not over 10 d after exposure to ß-ocimene, and this coincided with elevated expression of those ERFs in their leaves. An array of histone acetyltransferases, including HAC1, HAC5, and HAM1, were responsible for the induction and maintenance of the anti-herbivore property. HDA6, a histone deacetylase, played a role in the reverse histone remodeling. Collectively, our findings illuminate the role of epigenetic regulation in plant volatile signaling.