Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid.

Recent work has shown that meningeal lymphatic vessels (mLVs), mainly in the dorsal part of the skull, are involved in the clearance of cerebrospinal fluid (CSF), but the precise route of CSF drainage is still unknown. Here we reveal the importance of mLVs in the basal part of the skull for this process by visualizing their distinct anatomical location and characterizing their specialized morphological features, which facilitate the uptake and drainage of CSF. Unlike dorsal mLVs, basal mLVs have lymphatic valves and capillaries located adjacent to the subarachnoid space in mice. We also show that basal mLVs are hotspots for the clearance of CSF macromolecules and that both mLV integrity and CSF drainage are impaired with ageing. Our findings should increase the understanding of how mLVs contribute to the neuropathophysiological processes that are associated with ageing.

Clinical outcomes of etoposide and cytarabine as consolidation in elderly patients with primary CNS lymphoma.

BACKGROUND: A consolidation strategy has not been established for transplant-ineligible elderly patients with primary central nervous system lymphoma (PCNSL). In this study, we aimed to retrospectively evaluate the clinical outcomes of etoposide and cytarabine (EA) as consolidation chemotherapy for transplant-ineligible patients with PCNSL following high-dose methotrexate (MTX)-based induction chemotherapy. MATERIALS AND METHODS: Between 2015 and 2021, newly diagnosed transplant-ineligible patients with PCNSL with diffuse large B-cell lymphoma were consecutively enrolled. All enrolled patients were over 60 years old and received EA consolidation after achieving a complete or partial response following induction chemotherapy. RESULTS: Of the 85 patients who achieved a complete or partial response to MTX-based induction chemotherapy, 51 received EA consolidation chemotherapy. Among the 25 (49.0%, 25/51) patients in partial remission before EA consolidation, 56% (n = 14) achieved complete remission after EA consolidation. The median overall survival and progression-free survival were 43 and 13 months, respectively. Hematological toxicities were most common, and all patients experienced grade 4 neutropenia and thrombocytopenia. Forty-eight patients experienced febrile neutropenia during consolidation chemotherapy, and 4 patients died owing to treatment-related complications. CONCLUSION: EA consolidation chemotherapy for transplant-ineligible, elderly patients with PCNSL improved response rates but showed a high relapse rate and short progression-free survival. The incidences of treatment-related mortality caused by hematologic toxicities and severe infections were very high, even after dose modification. Therefore, the use of EA consolidation should be reconsidered in elderly patients with PCNSL.

Comprehensive insights into AML relapse: genetic mutations, clonal evolution, and clinical outcomes.

INTRODUCTION: Acute myeloid leukemia (AML) is a complex hematologic malignancy characterized by uncontrolled proliferation of myeloid precursor cells within bone marrow. Despite advances in understanding of its molecular underpinnings, AML remains a therapeutic challenge due to its high relapse rate and clonal evolution. METHODS: In this retrospective study, we analyzed data from 24 AML patients diagnosed at a single institution between January 2017 and August 2023. Comprehensive genetic analyses, including chromosomal karyotyping, next-generation sequencing, and gene fusion assays, were performed on bone marrow samples obtained at initial diagnosis and relapse. Clinical data, treatment regimens, and patient outcomes were also documented. RESULTS: Mutations in core genes of FLT3, NPM1, DNMT3A, and IDH2 were frequently discovered in diagnostic sample and remained in relapse sample. FLT3-ITD, TP53, KIT, RUNX1, and WT1 mutation were acquired at relapse in one patient each. Gene fusion assays revealed stable patterns, while chromosomal karyotype analyses indicated a greater diversity of mutations in relapsed patients. Clonal evolution patterns varied, with some cases showing linear or branching evolution and others exhibiting no substantial change in core mutations between diagnosis and relapse. CONCLUSIONS: Our study integrates karyotype, gene rearrangements, and gene mutation results to provide a further understanding of AML heterogeneity and evolution. We demonstrate the clinical relevance of specific mutations and clonal evolution patterns, emphasizing the need for personalized therapies and measurable residual disease monitoring in AML management. By bridging the gap between genetics and clinical outcome, we move closer to tailored AML therapies and improved patient prognoses.

Utility of Plasma Microbial Cell-Free DNA Whole-Genome Sequencing for Diagnosis of Invasive Aspergillosis in Patients With Hematologic Malignancy or COVID-19.

BACKGROUND: We evaluated the clinical accuracy and utility of whole-genome sequencing (WGS) of plasma microbial cell-free DNA (cfDNA) as a novel noninvasive method in diagnosing invasive aspergillosis (IA) in patients with hematologic malignancy (HM) or coronavirus disease 2019 (COVID-19). METHODS: Adults with HM or COVID-19 and suspected IA were recruited. IA cases were retrospectively diagnosed according to EORTC/MSG definitions and ECMM/ISHAM criteria for HM and COVID-19 patients, respectively. The results of cfDNA WGS were compared with the conventional diagnosis. RESULTS: Microbial cfDNA WGS was performed 53 times from 41 participants (19 from HM, 16 from COVID-19, and 7 from the control group). In participants with HM, Aspergillus cfDNA was detected in 100% of proven IA and 91.7% of probable IA cases. In participants with COVID-19, 50.0% of probable IA were positive for Aspergillus in cfDNA WGS. Concordance between Aspergillus cfDNA detection and proven/probable IA conventional diagnosis was significantly higher in participants with HM than in those with COVID-19. IA diagnosed using EORTC/MGS definitions showed significantly high concordance between Aspergillus cfDNA detection and proven/probable IA. CONCLUSIONS: Aspergillus cfDNA detection strongly correlated with proven/probable IA diagnosed using EORTC/MSG definitions and could be used as an additional diagnostic tool for IA.

Cyclin-dependent kinase 1 depolymerizes nuclear lamin filaments by disrupting the head-to-tail interaction of the lamin central rod domain.

Nuclear lamins maintain the nuclear envelope structure by forming long linear filaments via two alternating molecular arrangements of coiled-coil dimers, known as A11 and A22 binding modes. The A11 binding mode is characterized by the antiparallel interactions between coil 1b domains, whereas the A22 binding mode is facilitated by interactions between the coil 2 domains of lamin. The junction between A11- and A22-interacting dimers in the lamin tetramer produces another parallel head-tail interaction between coil 1a and the C-terminal region of coil 2, called the ACN interaction. During mitosis, phosphorylation in the lamin N-terminal head region by the cyclin-dependent kinase (CDK) complex triggers depolymerization of lamin filaments, but the associated mechanisms remain unknown at the molecular level. In this study, we revealed using the purified proteins that phosphorylation by the CDK1 complex promotes disassembly of lamin filaments by directly abolishing the ACN interaction between coil 1a and the C-terminal portion of coil 2. We further observed that this interaction was disrupted as a result of alteration of the ionic interactions between coil 1a and coil 2. Combined with molecular modeling, we propose a mechanism for CDK1-dependent disassembly of the lamin filaments. Our results will help to elucidate the cell cycle-dependent regulation of nuclear morphology at the molecular level.

Epigenetic regulation of DHRS2 by SUV420H2 inhibits cell apoptosis in renal cell carcinoma.

Renal cell carcinoma (RCC), also known as kidney cancer, is a common malignant tumor of the urinary system. While surgical treatment is essential, novel therapeutic targets and corresponding drugs for RCC are still needed due to the high relapse rate and low five-year survival rate. In this study, we found that SUV420H2 is overexpressed in renal cancers and that high SUV420H2 expression is associated with a poor prognosis, as evidenced by RCC RNA-seq results derived from the TCGA. SUV420H2 knockdown using siRNA led to growth suppression and cell apoptosis in the A498 cell line. Furthermore, we identified DHRS2 as a direct target of SUV420H2 in the apoptosis process through a ChIP assay with a histone 4 lysine 20 (H4K20) trimethylation antibody. Rescue experiments showed that cotreatment with siSUV420H2 and siDHRS2 attenuated cell growth suppression induced by SUV420H2 knockdown only. Additionally, treatment with the SUV420H2 inhibitor A-196 induced cell apoptosis via upregulation of DHRS2. Taken together, our findings suggest that SUV420H2 may be a potential therapeutic target for the treatment of renal cancer.

A natural variation in the RNA polymerase of severe fever with thrombocytopenia syndrome virus enhances viral replication and in vivo virulence.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease with high mortality in Eastern Asia. The disease is caused by the SFTS virus (SFTSV), also known as Dabie bandavirus, which has a segmented RNA genome consisting of L, M, and S segments. Previous studies have suggested differential viral virulence depending on the genotypes of SFTSV; however, the critical viral factor involved in the differential viral virulence is unknown. Here, we found a significant difference in viral replication in vitro and virulence in vivo between two Korean isolates belonging to the F and B genotypes, respectively. By generating viral reassortants using the two viral strains, we demonstrated that the L segment, which encodes viral RNA-dependent RNA polymerase (RdRp), is responsible for the enhanced viral replication and virulence. Comparison of amino acid sequences and viral replication rates revealed a point variation, E251K, on the surface of RdRp to be the most significant determinant for the enhanced viral replication rate and in vivo virulence. The effect of the variation was further confirmed using recombinant SFTSV generated by reverse genetic engineering. Therefore, our results indicate that natural variations affecting the viral replicase activity could significantly contribute to the viral virulence of SFTSV.

Real-world data on prognostic value of measurable residual disease assessment by fragment analysis or next-generation sequencing in multiple myeloma.

Measurable residual disease (MRD) negativity is a strong prognostic indicator in multiple myeloma (MM). However, the optimal use of MRD in daily clinical practice has been hampered by the limited feasibility of MRD testing. Therefore, we examined the clinical relevance of commercially available MRD modalities based on clonality assays by fragment analysis with IdentiClone® (n = 73 patients) and next-generation sequencing (NGS) with LymphoTrack® (n = 116 patients) in newly diagnosed patients with MM who received autologous stem cell transplantation (ASCT). MRD was assessed at the end of induction (pre-ASCT) and/or at 100 days after ASCT (post-ASCT). MRD could not predict survival when assessed by fragment analysis. However, NGS-based MRD negativity at pre- or post-ASCT was beneficial in terms of progression-free and overall survival. Moreover, NGS-based MRD negativity was independently associated with improved progression-free and overall survival, and MRD-positive patients both pre- and post-ASCT had worst outcome. Indeed, initial adverse prognostic features by high-risk cytogenetics could be mitigated upon achieving MRD negativity by NGS. We demonstrate the feasibility and clinical benefit of achieving MRD negativity by commercially available clonality-based MRD assays in MM and support incorporating NGS, but not fragment analysis, to tailor therapeutic strategies in real-world practice.

Characterization of terminal-ileal and colonic Crohn's disease in treatment-naïve paediatric patients based on transcriptomic profile using logistic regression.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic and idiopathic inflammatory disorder of the gastrointestinal tract and comprises ulcerative colitis (UC) and Crohn's disease (CD). Crohn's disease can affect any part of the gastrointestinal tract, but mainly the terminal ileum and colon. In the present study, we aimed to characterize terminal-ileal CD (ICD) and colonic CD (CCD) at the molecular level, which might enable a more optimized approach for the clinical care and scientific research of CD. METHODS: We analyzed differentially expressed genes in samples from 23 treatment-naïve paediatric patients with CD and 25 non-IBD controls, and compared the data with previously published RNA-Seq data using multi-statistical tests and confidence intervals. We implemented functional profiling and proposed statistical methods for feature selection using a logistic regression model to identify genes that are highly associated in ICD or CCD. We also validated our final candidate genes in independent paediatric and adult cohorts. RESULTS: We identified 550 genes specifically expressed in patients with CD compared with those in healthy controls (p < 0.05). Among these DEGs, 240 from patients with CCD were mainly involved in mitochondrial dysfunction, whereas 310 from patients with ICD were enriched in the ileum functions such as digestion, absorption, and metabolism. To choose the most effective gene set, we selected the most powerful genes (p-value ≤ 0.05, accuracy ≥ 0.8, and AUC ≥ 0.8) using logistic regression. Consequently, 33 genes were identified as useful for discriminating CD location; the accuracy and AUC were 0.86 and 0.83, respectively. We then validated the 33 genes with data from another independent paediatric cohort (accuracy = 0.93, AUC = 0.92) and adult cohort (accuracy = 0.88, AUC = 0.72). CONCLUSIONS: In summary, we identified DEGs that are specifically expressed in CCD and ICD compared with those in healthy controls and patients with UC. Based on the feature selection analysis, 33 genes were identified as useful for discriminating CCD and ICD with high accuracy and AUC, for not only paediatric patients but also independent cohorts. We propose that our approach and the final gene set are useful for the molecular classification of patients with CD, and it could be beneficial in treatments based on disease location.

Maturation of human intestinal organoids in vitro facilitates colonization by commensal lactobacilli by reinforcing the mucus layer.

Lactobacilli, which are probiotic commensal bacteria that mainly reside in the human small intestine, have attracted attention for their ability to exert health-promoting effects and beneficially modulate host immunity. However, host epithelial-commensal bacterial interactions are still largely unexplored because of limited access to human small intestinal tissues. Recently, we described an in vitro maturation technique for generating adult-like, mature human intestinal organoids (hIOs) from human pluripotent stem cells (hPSCs) that closely resemble the in vivo tissue structure and cellular diversity. Here, we established an in vitro human model to study the response to colonization by commensal bacteria using luminal microinjection into mature hIOs, allowing for the direct examination of epithelial-bacterial interactions. Lactobacillus reuteri and Lactobacillus plantarum were more likely to survive and colonize when microinjected into the lumen of mature hIOs than when injected into immature hIOs, as determined by scanning electron microscopy, colony formation assay, immunofluorescence, and real-time imaging with L plantarum expressing red fluorescent protein. The improved mature hIO-based host epithelium system resulted from enhanced intestinal epithelial integrity via upregulation of mucus secretion and tight junction proteins. Our study indicates that mature hIOs are a physiologically relevant in vitro model system for studying commensal microorganisms.

YAP and TAZ Negatively Regulate Prox1 During Developmental and Pathologic Lymphangiogenesis.

RATIONALE: The Hippo pathway governs cellular differentiation, morphogenesis, and homeostasis, but how it regulates these processes in lymphatic vessels is unknown. OBJECTIVE: We aimed to reveal the role of the final effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), in lymphatic endothelial cell (LEC) differentiation, morphogenesis, and homeostasis. METHODS AND RESULTS: During mouse embryonic development, LEC-specific depletion of Yap/Taz disturbed both plexus patterning and valve initiation with upregulated Prox1 (prospero homeobox 1). Conversely, LEC-specific YAP/TAZ hyperactivation impaired lymphatic specification and restricted lymphatic sprouting with profoundly downregulated Prox1. Notably, lymphatic YAP/TAZ depletion or hyperactivation aggravated or attenuated pathological lymphangiogenesis in mouse cornea. Mechanistically, VEGF (vascular endothelial growth factor)-C activated canonical Hippo signaling pathway in LECs. Indeed, repression of PROX1 transcription by YAP/TAZ hyperactivation was mediated by recruitment of NuRD (nucleosome remodeling and histone deacetylase) complex and endogenous binding activity of TEAD (TEA domain family members) to the PROX1 promoter. Furthermore, YAP/TAZ hyperactivation enhanced MYC signaling and inhibited CDKN1C, leading to cell cycle dysregulation and aberrant proliferation. CONCLUSIONS: We find that YAP and TAZ play promoting roles in remodeling lymphatic plexus patterning and postnatal lymphatic valve maintenance by negatively regulating Prox1 expression. We further show that YAP and TAZ act as plastic regulators of lymphatic identity and define the Hippo signaling-mediated PROX1 transcriptional programing as a novel dynamic checkpoint underlying LEC plasticity and pathophysiology.

Crosstalk between WNT and STAT3 is mediated by galectin-3 in tumor progression.

BACKGROUND: Aberrant activation of the WNT/ß-catenin and STAT3 signaling pathways plays a critical role in cancer progression. However, direct targeting of these pathways as an anti-cancer therapeutic approach needs to be reconsidered due to its serious side effects. Here, we demonstrate that overexpression of WNT induces STAT3 activation in a galectin-3-dependent manner. METHODS: We investigated how galectin-3 mediates the crosstalk between WNT/ß-catenin and STAT3 signaling and whether inhibition of galectin-3 can reduce gastric cancer. The molecular mechanisms were analyzed by biochemical assays using cultured gastric cancer cells, patient tissues, and genetically engineered mice. Moreover, we confirm of therapeutic effects of GB1107, a cell-penetrating galectin-3 specific inhibitor, using orthotopic gastric cancer-bearing mice RESULTS: Increased levels of galectin-3 and STAT3 phosphorylation were detected in the stomach tissues of WNT1-overexpressing mouse models. Also, high expression levels and co-localization of ß-catenin, pSTAT3, and galectin-3 in patients with advanced gastric cancer were correlated with a poorer prognosis. Galectin-3 depletion significantly decreased STAT3 Tyr705 phosphorylation, which regulates its nuclear localization and transcriptional activation. A peptide of galectin-3 (Y45-Q48) directly bound to the STAT3 SH2 domain and enhanced its phosphorylation. GB1107, a specific membrane-penetrating inhibitor of galectin-3, significantly reduced the activation of both STAT3 and ß-catenin and inhibited tumor growth in orthotopic gastric cancer-bearing mice. CONCLUSIONS: We propose that galectin-3 mediates the crosstalk between the WNT and STAT3 signaling pathways. Therefore GB1107, a galectin-3-specific inhibitor, maybe a potent agent with anti-gastric cancer activity. Further studies are needed for its clinical application in gastric cancer therapy.

Deferoxamine Reduces Inflammation and Osteoclastogenesis in Avulsed Teeth.

Replacement and inflammatory resorption are serious complications associated with the delayed replantation of avulsed teeth. In this study, we aimed to assess whether deferoxamine (DFO) can suppress inflammation and osteoclastogenesis in vitro and attenuate inflammation and bone resorption in a replanted rat tooth model. Cell viability and inflammation were evaluated in RAW264.7 cells. Osteoclastogenesis was confirmed by tartrate-resistant acid phosphatase staining, reactive oxygen species (ROS) measurement, and quantitative reverse transcriptase-polymerase chain reaction in teeth exposed to different concentrations of DFO. In vivo, molars of 31 six-week-old male Sprague-Dawley rats were extracted and stored in saline (n = 10) or DFO solution (n = 21) before replantation. Micro-computed tomography (micro-CT) imaging and histological analysis were performed to evaluate inflammation and root and alveolar bone resorption. DFO downregulated the genes related to inflammation and osteoclastogenesis. DFO also reduced ROS production and regulated specific pathways. Furthermore, the results of the micro-CT and histological analyses provided evidence of the decrease in inflammation and hard tissue resorption in the DFO group. Overall, these results suggest that DFO reduces inflammation and osteoclastogenesis in a tooth replantation model, and thus, it has to be further investigated as a root surface treatment option for an avulsed tooth.

Structural and Functional Characterizations of Cancer Targeting Nanoparticles Based on Hepatitis B Virus Capsid.

Cancer targeting nanoparticles have been extensively studied, but stable and applicable agents have yet to be developed. Here, we report stable nanoparticles based on hepatitis B core antigen (HBcAg) for cancer therapy. HBcAg monomers assemble into spherical capsids of 180 or 240 subunits. HBcAg was engineered to present an affibody for binding to human epidermal growth factor receptor 1 (EGFR) and to present histidine and tyrosine tags for binding to gold ions. The HBcAg engineered to present affibody and tags (HAF) bound specifically to EGFR and exterminated the EGFR-overexpressing adenocarcinomas under alternating magnetic field (AMF) after binding with gold ions. Using cryogenic electron microscopy (cryo-EM), we obtained the molecular structures of recombinant HAF and found that the overall structure of HAF was the same as that of HBcAg, except with the affibody on the spike. Therefore, HAF is viable for cancer therapy with the advantage of maintaining a stable capsid form. If the affibody in HAF is replaced with a specific sequence to bind to another targetable disease protein, the nanoparticles can be used for drug development over a wide spectrum.

Non-cryogenic structure of a chloride pump provides crucial clues to temperature-dependent channel transport efficiency.

Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.

Colorectal adenocarcinoma-derived EGFR mutants are oncogenic and sensitive to EGFR-targeted monoclonal antibodies, cetuximab and panitumumab.

Somatic mutations of epidermal growth factor receptor (EGFR) occur in ~3% of colorectal cancer (CRC) patients. Here, through systematic functional screening of 21 recurrent EGFR mutations selected from public data sets, we show that 11 colon cancer-derived EGFR mutants (G63R, E114K, R165Q, R222C, S492R, P596L, K708R, E709K, G719S, G724S and L858R) are oncogenic and able to transform cells in a ligand-independent manner. We demonstrate that cellular transformation by these mutants requires receptor dimerization. Importantly, the EGF-induced and constitutive oncogenic potential of these EGFR mutants are inhibited by cetuximab or panitumumab in vivo and in vitro. Taken together, we propose that a subset of EGFR mutations can serve as genomic predictors for response to anti-EGFR antibodies and that metastatic CRC patients with such mutations may benefit from these drugs as part of the first-line therapy.

Suppression of HIF-1α accumulation by betulinic acid through proteasome activation in hypoxic cervical cancer.

Betulinic acid (BA) exhibits various biological activities such as anti-bacterial, anti-inflammatory, anti-human papilloma virus (HPV), and anti-cancer activities. HPV infection is associated with a high risk of cervical cancer, which is the leading cause of deaths among women worldwide. Therefore, BA is an attractive therapeutic agent for treating cervical cancer. In this study, we investigated the role of BA in regulating the hypoxia-mediated response in HeLa cells and clarified the underlying mechanism of action. We found that BA inhibited the hypoxia-induced accumulation of HIF-1α without affecting HIF-1α mRNA levels and suppressed the expression of HIF target genes, including VEGF, GLUT1, and PDK1 in HeLa cells. Additionally, BA enhanced the ß1, ß2, and ß5 activities of the proteasome, which resulted in reduced levels of ubiquitinated proteins and HIF-1α protein in HeLa cells. However, BA treatment did not affect the deubiquitinase enzyme activity in HeLa cells. These results indicate that inhibition of HIF-1α accumulation by BA is mediated by activation of the proteasome, and BA is a potential anticancer agent for the regulation of the HIF signaling pathway.

RNA-seq based transcriptome analysis of EHMT2 functions in breast cancer.

For breast cancer treatment, hormone therapy is effective for hormone receptor-positive breast cancer but not for TNBC (triple-negative breast cancer). Thus, many researchers have attempted to identify more effective therapeutic candidates for all subtypes of breast cancer. In this study, we established an RNA-seq analytical pipeline to analyze the subtype-specific functions of EHMT2 in the MB231 and MCF7 cell lines. After EHMT2 knockdown, we identified subtype-specific DEGs (differentially expressed genes) and overlapping DEGs. Through GO (Gene Ontology) analysis, GSEA (gene set enrichment analysis), and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis using the DEGs, we identified the subtype-specific functions of EHMT2 in the MB231 and MCF7 cell lines. Therefore, herein, we suggest that EHMT2 is an attractive therapeutic target for the treatment of all types of breast cancer.

RSL Class II Transcription Factors Guide the Nuclear Localization of RHL1 to Regulate Root Hair Development.

Root hairs are important for absorption of nutrients and water from the rhizosphere. The Root Hair Defective-Six Like (RSL) Class II family of transcription factors is expressed preferentially in root hairs and has a conserved role in root hair development in land plants. We functionally characterized the seven members of the RSL Class II subfamily in the rice (Oryza sativa) genome. In root hairs, six of these genes were preferentially expressed and four were strongly expressed. Phenotypic analysis of each mutant revealed that Os07g39940 plays a major role in root hair formation, based on observations of a short root hair phenotype in those mutants. Overexpression (OX) for each of four family members in rice resulted in an increase in the density and length of root hairs. These four members contain a transcription activation domain and are targeted to the nucleus. They interact with rice Root Hairless1 (OsRHL1), a key regulator of root hair development. When heterologously expressed in epidermal cells of Nicotiana benthamiana leaves, OsRHL1 was predominantly localized to the cytoplasm. When coexpressed with each of the four RSL Class II members, however, OsRLH1 was translocated to the nucleus. Transcriptome analysis using Os07g39940-OX plants revealed that 86 genes, including Class III peroxidases, were highly up-regulated. Furthermore, reactive oxygen species levels in the root hairs were increased in Os07g39940-OX plants but were drastically reduced in the os07g39940 and rhl1 mutants. Our results demonstrate that RSL Class II members function as essential regulators of root hair development in rice.

The in vivo functions of ARPF2 and ARRS1 in ribosomal RNA processing and ribosome biogenesis in Arabidopsis.

Yeast Rpf2 plays a critical role in the incorporation of 5S rRNA into pre-ribosomes by forming a binary complex with Rrs1. The protein characteristics and overexpression phenotypes of Arabidopsis Ribosome Production Factor 2 (ARPF2) and Arabidopsis Regulator of Ribosome Synthesis 1 (ARRS1) have been previously studied. Here, we analyze loss-of-function phenotypes of ARPF2 and ARRS1 using virus-induced gene silencing to determine their functions in pre-rRNA processing and ribosome biogenesis. ARPF2 silencing in Arabidopsis led to pleiotropic developmental defects. RNA gel blot analysis and circular reverse transcription-PCR revealed that ARPF2 depletion delayed pre-rRNA processing, resulting in the accumulation of multiple processing intermediates. ARPF2 fractionated primarily with the 60S ribosomal subunit. Metabolic rRNA labeling and ribosome profiling suggested that ARPF2 deficiency mainly affected 25S rRNA synthesis and 60S ribosome biogenesis. ARPF2 and ARRS1 formed the complex that interacted with the 60S ribosomal proteins RPL5 and RPL11. ARRS1 silencing resulted in growth defects, accumulation of processing intermediates, and ribosome profiling similar to those of ARPF2-silenced plants. Moreover, depletion of ARPF2 and ARRS1 caused nucleolar stress. ARPF2-deficient plants excessively accumulated anthocyanin and reactive oxygen species. Collectively, these results suggest that the ARPF2-ARRS1 complex plays a crucial role in plant growth and development by modulating ribosome biogenesis.