Endometrial Cell-Type Specific Regulation of the Endocannabinoids System and the Impact of Menstrual Cycle and Endometriosis.

Introduction: Anandamide (AEA) and 2-arachidonoylglycerol are endogenous agonists of the cannabinoid receptors and regulate and control many cellular functions. Their activities are governed by enzymes and proteins that regulate their synthesis, receptor binding, transport, and degradation, which are known as the endocannabinoid system (ECS). The aim of this study was to investigate the regulation of endocannabinoid activity in the endometrium by studying the RNA and protein expression of the ECS within endometrial cell types and during different menstrual cycle stages and the impact of endometriosis. Materials and Methods: The RNA expression of 70 ECS genes was assessed using RNA sequencing of isolated endometrial epithelial and stromal cells. Subsequent immunofluorescence-stained endometrial samples on ECS components of interest were objectively analyzed via an agnostic and automated image analysis pipeline to extract quantitative information. Differential gene and protein expression was investigated between the two cell types, menstrual cycle phases, and endometriosis cases and controls. Results: Sufficient RNA expression was detected for 45 genes, and 17 (38%) genes were significantly different between epithelial and stromal cells. FAAH RNA was significantly higher in epithelial cells compared with stromal cells. Protein expression analysis of the main synthesizing (NAPE-PLD) and catabolizing (FAAH and NAAA) enzymes of AEA revealed a significantly stronger epithelial expression compared to stromal cells. The RNA and protein expression of CB1 receptors was very low with no significant difference between epithelial and stromal cells. Eleven ECS genes were regulated across the menstrual cycle, and there was no gene with significant difference between endometriosis cases and controls in epithelial cells. Discussion: Differential expression of ECS genes supports a cell type-specific endocannabinoid activity in the endometrium. As endocannabinoids are short-lived signaling molecules, higher RNA and protein expression of FAAH in the epithelial cells suggests an active regulation of endocannabinoid activity in epithelial cells within the endometrium.

Risk factors for proximal and distal aortic events after type A acute aortic dissection.

OBJECTIVES: Type A acute aortic dissection (TAAAD) is a life-threatening condition often requiring emergency surgery, with approximately 30% of patients needing reoperation. This study aimed to identify predictors of long-term aortic events from early postoperative computed tomography (CT) examinations. METHODS: A total of 336 cases underwent TAAAD surgery at two institutions between 2002 and 2018. Of these, 302 patients received CT examinations immediately after initial TAAAD surgery. Predictors of aortic events were evaluated from these early postoperative CT exams. Aortic events were defined as any events involving aortic-related death, open surgery, reoperation, endovascular stenting, or thoracic aorta diameter enlargement to ≥ 55 mm. RESULTS: Excluding 34 in-hospital deaths (10.1%; 34/336), the 1-, 5-, and 10-year actuarial survival rates after primary TAAAD surgery were 98.2%, 88.6%, and 81.7%, respectively. Over a mean follow-up period of 7.4 ± 5.1 years, 67 aortic events (proximal: 19, distal: 45, both: 3) were observed. Freedom from proximal aortic events was 98.6%, 93.9%, and 85.2% at 1, 5, and 10 years, respectively. Proximal anastomosis new entry was identified as a significant risk factor for aortic events, with a 92% vs. 42% incidence at 10 years (p < 0.001). Freedom from distal aortic events was 99.6%, 84.5%, and 67.2% at 1, 5, and 10 years, respectively. A false/true area ratio greater than 1 and distal anastomosis new entry were significant risk factors for aortic events (low-risk group: 83.3% vs. high-risk group: 42.3% at 10 years, p < 0.001). CONCLUSIONS: Detailed analysis of early postoperative CT scans following primary TAAAD surgery may help identify predictors of subsequent aortic events, potentially improving long-term patient management and outcomes.

Short Fatigue-Crack Growth from Crack-like Defects under Completely Reversed Loading Predicted Based on Cyclic R-Curve.

Understanding short fatigue-crack propagation behavior is inevitable in the defect-tolerant design of structures. Short cracks propagate differently from long cracks, and the amount of crack closure plays a key role in the propagation behavior of short cracks. In the present paper, the buildup of fatigue-crack closure due to plasticity with crack extension from crack-like defects is simulated with a modified strip yield model, which leaves plastic stretch in the wake of the advancing crack. Crack-like defects are assumed to be closure-free and do not close even under compression. The effect of the size of crack-like defects on the growth and arrest of short cracks was systematically investigated and the cyclic R-curve derived. The cyclic R-curve determined under constant amplitude loading of multiple specimens is confirmed to be independent of the initial defect length. Load-shedding and ΔK-constant loading tests are employed to extend the cyclic R-curve beyond the fatigue limit determined under constant amplitude loading. The initiation stage of cracks is taken into account in R-curves when applied to smooth specimens.

The intron binding protein EMB-4 is an opposite regulator of cold and high temperature tolerance in Caenorhabditis elegans.

Adaptation and tolerance to changes in heat and cold temperature are essential for survival and proliferation in plants and animals. However, there is no clear information regarding the common molecules between animals and plants. In this study, we found that heat, and cold tolerance of the nematode Caenorhabditis elegans is oppositely regulated by the RNA-binding protein EMB-4, whose plant homolog contains polymorphism causing heat tolerance diversity. Caenorhabditis elegans alters its cold and heat tolerance depending on the previous cultivation temperature, wherein EMB-4 respectively acts as a positive and negative controller of heat and cold tolerance by altering gene expression. Among the genes whose expression is regulated by EMB-4, a phospholipid scramblase, and an acid sphingomyelinase, which are involved in membrane lipid metabolism, were found to play essential roles in the negative regulation of heat tolerance.

Intestinal epithelium dysfunctions cause IgA deposition in the kidney glomeruli of intestine-specific Ap1m2-deficient mice.

BACKGROUND: Intestinal epithelial cells (IECs) serve as robust barriers against potentially hostile luminal antigens and commensal microbiota. Epithelial barrier dysfunction enhances intestinal permeability, leading to leaky gut syndrome (LGS) associated with autoimmune and chronic inflammatory disorders. However, a causal relationship between LGS and systemic disorders remains unclear. Ap1m2 encodes clathrin adaptor protein complex 1 subunit mu 2, which facilitates polarized protein trafficking toward the basolateral membrane and contributes to the establishment of epithelial barrier functions. METHODS: We generated IEC-specific Ap1m2-deficient (Ap1m2ΔIEC) mice with low intestinal barrier integrity as an LSG model and examined the systemic impact. FINDINGS: Ap1m2ΔIEC mice spontaneously developed IgA nephropathy (IgAN)-like features characterized by the deposition of IgA-IgG immune complexes and complement factors in the kidney glomeruli. Ap1m2 deficiency markedly enhanced aberrantly glycosylated IgA in the serum owing to downregulation and mis-sorting of polymeric immunoglobulin receptors in IECs. Furthermore, Ap1m2 deficiency caused intestinal dysbiosis by attenuating IL-22-STAT3 signaling. Intestinal dysbiosis contributed to the pathogenesis of IgAN because antibiotic treatment reduced aberrantly glycosylated IgA production and renal IgA deposition in Ap1m2ΔIEC mice. INTERPRETATION: IEC barrier dysfunction and subsequent dysbiosis by AP-1B deficiency provoke IgA deposition in the mouse kidney. Our findings provide experimental evidence of a pathological link between LGS and IgAN. FUNDING: AMED, AMED-CREST, JSPS Grants-in-Aid for Scientific Research, JST CREST, Fuji Foundation for Protein Research, and Keio University Program for the Advancement of Next Generation Research Projects.

Composite Lymphoma after Treatment with Infliximab for Sarcoidosis and Ankylosing Spondylitis.

Lymphoid proliferations and lymphomas associated with immune deficiency and dysregulation (LP/L-IDD) are rare entities associated with the use of immunosuppressive drugs (ISD) for autoimmune conditions. Composite lymphomas, featuring both B-cell and T-cell lymphomas, are infrequent, and their occurrence as LP/L-IDD is rare. We herein report the case of a 70-year-old man with right pleural effusion and lymphadenopathy, who was treated with infliximab for sarcoidosis and ankylosing spondylitis. A biopsy revealed a composite lymphoma of DLBCL and PTCL-NOS. CHOP chemotherapy led to significant remission. This case report emphasizes the need to consider lymphoma in patients with autoimmune diseases such as sarcoidosis and ankylosing spondylitis, especially those treated with ISDs.

Allogeneic Hematopoietic cell Transplantation Using Alemtuzumab in Asian Patients with Inborn Errors of Immunity.

Alemtuzumab is used with reduced-toxicity conditioning (RTC) in allogeneic hematopoietic cell transplantation (HCT), demonstrating efficacy and feasibility for patients with inborn errors of immunity (IEI) in Western countries; however, the clinical experience in Asian patients with IEI is limited. We retrospectively analyzed patients with IEI who underwent the first allogeneic HCT with alemtuzumab combined with RTC regimens in Japan. A total of 19 patients were included and followed up for a median of 18 months. The donors were haploidentical parents (n = 10), matched siblings (n = 2), and unrelated bone marrow donors (n = 7). Most patients received RTC regimens containing fludarabine and busulfan and were treated with 0.8 mg/kg alemtuzumab with intermediate timing. Eighteen patients survived and achieved stable engraftment, and no grade 3-4 acute graft-versus-host disease was observed. Viral infections were observed in 11 patients (58%) and 6 of them presented symptomatic. The median CD4+ T cell count was low at 6 months (241/µL) but improved at 1 year (577/µL) after HCT. Whole blood cells continued to exhibit > 80% donor type in most cases; however, 3/10 patients exhibited poor donor chimerism only among T cells and also showed undetectable levels of T-cell receptor recombination excision circles (TRECs) at 1 year post-HCT. This study demonstrated the efficacy and safety of alemtuzumab; however, patients frequently developed viral infections and slow reconstitution or low donor chimerism in T cells, emphasizing the importance of monitoring viral status and T-cell-specific chimerism. (238 < 250 words).

[Successful bridging therapy with alectinib prior to allogeneic stem cell transplantation for refractory ALK-positive anaplastic large cell lymphoma].

Although alectinib is effective for relapsed or refractory ALK-positive anaplastic large cell lymphoma (ALCL) and has a favorable safety profile, its role as a bridging therapy for allogeneic hematopoietic stem cell transplantation (allo-HSCT) and the role of allo-HSCT itself in this setting are unknown. A 35-year-old man with ALK-positive ALCL experienced relapse after first-line therapy with CHOP. Brentuximab vedotin led to partial response and high-dose chemotherapy combined with autologous HSCT was performed. However, disease progressed 15 months after transplantation, and alectinib was initiated. Complete response (CR) was achieved after three months of treatment, and alectinib was continued for 5 months. After cessation of alectinib, allogeneic bone marrow transplantation from an HLA 1-locus mismatched unrelated donor was performed after conditioning with fludarabine, busulfan, and total body irradiation. GVHD prophylaxis consisted of tacrolimus and short-term methotrexate. The post-transplant course was unremarkable except for grade I acute GVHD. The lymphoma has not recurred for 2 years after allo-HSCT without resuming alectinib. The clinical course of our case suggests that alectinib bridging therapy and allo-HSCT are effective in relapsed/refractory ALK-positive ALCL.

Impact of BK polyomavirus viremia on the outcomes of allogeneic hematopoietic stem cell transplantation.

Although it is known that BK polyomavirus (BKPyV) causes hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation (HSCT), the clinical significance of BKPyV viremia has not been fully evaluated. We retrospectively analyzed the results of quantitative polymerase chain reaction (PCR) evaluations for detecting BKPyV in the whole blood samples of patients undergoing allogeneic HSCT during the period from January 2010 to June 2020 at a single institute, Tokyo Medical and Dental University. BKPyV was detected in the blood of 28 of the 107 evaluated patients, and the cumulative incidence of was 27.9% (95%CI: 20.2-37.9%). HC due to BKPyV developed in four of the 28 patients with BKPyV viremia (14.3%) and in two of the 79 patients without it (2.5%; P < 0.05). BKPyV viremia itself did not affect the patients' post-transplant estimated glomerular filtration rate (eGFR), but BKPyV viremia with a high viral load was significantly associated with decreased eGFR values (P < 0.05). BKPyV viremia was also associated with significantly lower progression-free survival at 3 years (35.1% [95%CI: 17.8-53.1%] vs. 60.4% [95%CI: 48.4-70.5], P < 0.05). Our findings demonstrated that BKPyV viremia was associated with onset of HC, an early decline of renal function, and poorer survival after allogeneic HSCT. Further studies are needed to test these results and elucidate the mechanisms of renal dysfunction associated with BKPyV viremia.

QTL analysis of femaleness in monoecious spinach and fine mapping of a major QTL using an updated version of chromosome-scale pseudomolecules.

Although spinach is predominantly dioecious, monoecious plants with varying proportions of female and male flowers are also present. Recently, monoecious inbred lines with highly female and male conditions have been preferentially used as parents for F1-hybrids, rather than dioecious lines. Accordingly, identifying the loci for monoecism is an important issue for spinach breeding. We here used long-read sequencing and Hi-C technology to construct SOL_r2.0_pseudomolecule, a set of six pseudomolecules of spinach chromosomes (total length: 879.2 Mb; BUSCO complete 97.0%) that are longer and more genetically complete than our previous version of pseudomolecules (688.0 Mb; 81.5%). Three QTLs, qFem2.1, qFem3.1, and qFem6.1, responsible for monoecism were mapped to SOL_r2.0_pseudomolecule. qFem3.1 had the highest LOD score and corresponded to the M locus, which was previously identified as a determinant of monoecious expression, by genetic analysis of progeny from female and monoecious plants. The other QTLs were shown to modulate the ratio of female to male flowers in monoecious plants harboring a dominant allele of the M gene. Our findings will enable breeders to efficiently produce highly female- and male-monoecious parental lines for F1-hybrids by pyramiding the three QTLs. Through fine-mapping, we narrowed the candidate region for the M locus to a 19.5 kb interval containing three protein-coding genes and one long non-coding RNA gene. Among them, only RADIALIS-like-2a showed a higher expression in the reproductive organs, suggesting that it might play a role in reproductive organogenesis. However, there is no evidence that it is involved in the regulation of stamen and pistil initiation, which are directly related to the floral sex differentiation system in spinach. Given that auxin is involved in reproductive organ formation in many plant species, genes related to auxin transport/response, in addition to floral organ formation, were identified as candidates for regulators of floral sex-differentiation from qFem2.1 and qFem6.1.

Mutations in nuclear pore complex promote osmotolerance in Arabidopsis by suppressing the nuclear translocation of ACQOS and its osmotically induced immunity.

We have previously reported a wide variation in salt tolerance among Arabidopsis thaliana accessions and identified ACQOS, encoding a nucleotide-binding leucine-rich repeat (NLR) protein, as the causal gene responsible for the disturbance of acquired osmotolerance induced after mild salt stress. ACQOS is conserved among Arabidopsis osmosensitive accessions, including Col-0. In response to osmotic stress, it induces detrimental autoimmunity, resulting in suppression of osmotolerance, but how ACQOS triggers autoimmunity remains unclear. Here, we screened acquired osmotolerance (aot) mutants from EMS-mutagenized Col-0 seeds and isolated the aot19 mutant. In comparison with the wild type (WT), this mutant had acquired osmotolerance and decreased expression levels of pathogenesis-related genes. It had a mutation in a splicing acceptor site in NUCLEOPORIN 85 (NUP85), which encodes a component of the nuclear pore complex. A mutant with a T-DNA insertion in NUP85 acquired osmotolerance similar to aot19. The WT gene complemented the osmotolerant phenotype of aot19. We evaluated the acquired osmotolerance of five nup mutants of outer-ring NUPs and found that nup96, nup107, and aot19/nup85, but not nup43 or nup133, showed acquired osmotolerance. We examined the subcellular localization of the GFP-ACQOS protein and found that its nuclear translocation in response to osmotic stress was suppressed in aot19. We suggest that NUP85 is essential for the nuclear translocation of ACQOS, and the loss-of-function mutation of NUP85 results in acquired osmotolerance by suppressing ACQOS-induced autoimmunity in response to osmotic stress.

Golgi apparatus-localized CATION CALCIUM EXCHANGER4 promotes osmotolerance of Arabidopsis.

Calcium (Ca2+) is a major ion in living organisms, where it acts as a second messenger for various biological phenomena. The Golgi apparatus retains a higher Ca2+ concentration than the cytosol and returns cytosolic Ca2+ to basal levels after transient elevation in response to environmental stimuli such as osmotic stress. However, the Ca2+ transporters localized in the Golgi apparatus of plants have not been clarified. We previously found that a wild-type (WT) salt-tolerant Arabidopsis (Arabidopsis thaliana) accession, Bu-5, showed osmotic tolerance after salt acclimatization, whereas the Col-0 WT did not. Here, we isolated a Bu-5 background mutant gene, acquired osmotolerance-defective 6 (aod6), which reduces tolerance to osmotic, salt, and oxidative stresses, with a smaller plant size than the WT. The causal gene of the aod6 mutant encodes CATION CALCIUM EXCHANGER4 (CCX4). The aod6 mutant was more sensitive than the WT to both deficient and excessive Ca2+. In addition, aod6 accumulated higher Ca2+ than the WT in the shoots, suggesting that Ca2+ homeostasis is disturbed in aod6. CCX4 expression suppressed the Ca2+ hypersensitivity of the csg2 (calcium sensitive growth 2) yeast (Saccharomyces cerevisiae) mutant under excess CaCl2 conditions. We also found that aod6 enhanced MAP kinase 3/6 (MPK3/6)-mediated immune responses under osmotic stress. Subcellular localization analysis of mGFP-CCX4 showed GFP signals adjacent to the trans-Golgi apparatus network and co-localization with Golgi apparatus-localized markers, suggesting that CCX4 localizes in the Golgi apparatus. These results suggest that CCX4 is a Golgi apparatus-localized transporter involved in the Ca2+ response and plays important roles in osmotic tolerance, shoot Ca2+ content, and normal growth of Arabidopsis.

Clinical factors for central nervous system progression and survival in primary vitreoretinal lymphoma.

Primary vitreoretinal lymphoma (PVRL) is a rare subtype of malignant lymphoma with a poor prognosis because of high frequency of central nervous system (CNS) progression. Identification of factors associated with CNS progression is essential to improve the prognosis of patients with PVRL. We conducted a retrospective study of 54 patients diagnosed with PVRL and treated at our hospital to identify factors associated with CNS progression and prognosis. All patients were treated with intravitreal methotrexate (MTX) injections in the affected eyes until lesion resolution. Twenty-four patients were treated with systemic administration of high-dose MTX (systemic HD-MTX) every other week for a total of five cycles following intravitreal MTX injection. Of 24 patients, 20 completed five cycles of systemic HD-MTX. The 5-year cumulative incidence of CNS progression and overall survival (OS) rate were 78.0% and 69.0% respectively. By univariate and multivariate analyses, bilateral disease and the detection of B-cell clonality confirmed by flow cytometric analysis were risk factors associated with CNS progression. Moreover, systemic HD-MTX completion reduced the risk of CNS progression and was identified as a factor affecting OS. In this study, factors for CNS progression identified may potentially contribute to the optimized therapeutic stratification to improve the survival of patients with PVRL.

Untreated atrial septal defect after intracardiac repair for tetralogy of Fallot.

Tetralogy of Fallot was repaired previously in a preschooler through right ventriculotomy, providing excellent exposure for ventricular septal defect closure and right ventricular outflow tract obstruction relief. Herein, we describe the preschooler as a 50-year-old man with untreated atrial septal defect found at pulmonary valve replacement in the remote period after tetralogy of Fallot repair. It was inferred intraoperatively that the previous tetralogy of Fallot was repaired only through right ventriculotomy, and atrial septal defect closure was performed together with pulmonary valve replacement.

[Acute promyelocytic leukemia with asymptomatic cerebral hemorrhage].

A 43-year-old man presenting with oral bleeding was diagnosed with acute promyelocytic leukemia (APL). Induction chemotherapy consisting of all-trans retinoic acid and idarubicin was initiated, and disseminated intravascular coagulation (DIC) was treated with fresh frozen plasma and recombinant thrombomodulin infusions. The patient was free from neurological symptoms throughout the clinical course. However, cerebral hemorrhagic lesions were detected incidentally on magnetic resonance imaging performed to screen for leukemic central nervous system invasion at 2 weeks after treatment initiation. Imaging findings suggested subacute or later-phase cerebral hemorrhage. Platelet transfusions and other supportive care was provided. Serial imaging evaluations confirmed reduction of the hemorrhagic lesions. Hematological remission was achieved after induction chemotherapy, and no symptoms due to cerebral hemorrhage developed during the subsequent consolidation therapy. As patients with APL characteristically experience hemorrhagic events due to bleeding tendency caused by DIC, physicians should be aware of the possibility of asymptomatic cerebral hemorrhage in these patients.

Effects of Counterion on the Formation and Hydration Behavior of α-Form Hydrated Crystals (α-Gels).

α-Form hydrated crystals form a lamellar gel in which the alkyl chains of the amphiphilic molecules are hexagonally arranged within bilayers below the gel-liquid crystal phase transition temperature. In practice, the lamellar gel network with excess water is called an "α-gel", particularly in the cosmetics industry. In this study, the hydration or water sorption of amphiphilic materials in water vapor was assessed using a humidity-controlled quartz crystal microbalance with dissipation monitoring (QCM-D) technique. The amphiphilic materials used in this study were hexadecyl phosphate salts neutralized with L-arginine (C16P-Arg), CsOH (C16P-Cs), KOH (C16P-K), and NaOH (C16P-Na). Small- and wide-angle X-ray scattering measurements revealed that C16P-Arg and C16P-Cs yielded α-form hydrated crystals. Humidity-controlled QCM-D measurements demonstrated that C16P-Arg and C16P-Cs more readily underwent hydration or water sorption than C16P-K and C16P-Na. The key conclusion is that the significant hydration ability of C16P-Arg and C16P-Cs promotes the formation of the corresponding α-form hydrated crystals.

MOS4-associated complex contributes to proper splicing and suppression of ER stress under long-term heat stress in Arabidopsis.

Plants are often exposed not only to short-term (S-) but also to long-term (L-)heat stress over several consecutive days. A few Arabidopsis mutants defective in L-heat tolerance have been identified, but the molecular mechanisms are less understood for this tolerance than for S-heat stress tolerance. To elucidate the mechanisms of the former, we used a forward genetic screen for sensitive to long-term heat (sloh) mutants and isolated sloh3 and sloh63. The mutants were hypersensitive to L- but not to S-heat stress, and sloh63 was also hypersensitive to salt stress. We identified the causal genes, SLOH3 and SLOH63, both of which encoded splicing-related components of the MOS4-associated complex (MAC). This complex is widely conserved in eukaryotes and has been suggested to interact with spliceosomes. Both genes were induced by L-heat stress in a time-dependent manner, and some abnormal splicing events were observed in both mutants under L-heat stress. In addition, endoplasmic reticulum (ER) stress and subsequent unfolded protein response occurred in both mutants under L-heat stress and were especially prominent in sloh63, suggesting that enhanced ER stress is due to the salt hypersensitivity of sloh63. Splicing inhibitor pladienolide B led to concentration-dependent disturbance of splicing, decreased L-heat tolerance, and enhanced ER stress. These findings suggest that maintenance of precise mRNA splicing under L-heat stress by the MAC is important for L-heat tolerance and suppressing ER stress in Arabidopsis.

LHT1/MAC7 contributes to proper alternative splicing under long-term heat stress and mediates variation in the heat tolerance of Arabidopsis.

Natural genetic variation has facilitated the identification of genes underlying complex traits such as stress tolerances. We here evaluated the long-term (L-) heat tolerance (37°C for 5 days) of 174 Arabidopsis thaliana accessions and short-term (S-) heat tolerance (42°C, 50 min) of 88 accessions and found extensive variation, respectively. Interestingly, L-heat-tolerant accessions are not necessarily S-heat tolerant, suggesting that the tolerance mechanisms are different. To elucidate the mechanisms underlying the variation, we performed a chromosomal mapping using the F2 progeny of a cross between Ms-0 (a hypersensitive accession) and Col-0 (a tolerant accession) and found a single locus responsible for the difference in L-heat tolerance between them, which we named Long-term Heat Tolerance 1 (LHT1). LHT1 is identical to MAC7, which encodes a putative RNA helicase involved in mRNA splicing as a component of the MOS4 complex. We found one amino acid deletion in LHT1 of Ms-0 that causes a loss of function. Arabidopsis mutants of other core components of the MOS4 complex-mos4-2, cdc5-1, mac3a mac3b, and prl1 prl2-also showed hypersensitivity to L-heat stress, suggesting that the MOS4 complex plays an important role in L-heat stress responses. L-heat stress induced mRNA processing-related genes and compromised alternative splicing. Loss of LHT1 function caused genome-wide detrimental splicing events, which are thought to produce nonfunctional mRNAs that include retained introns under L-heat stress. These findings suggest that maintaining proper alternative splicing under L-heat stress is important in the heat tolerance of A. thaliana.

Insights into the catalytic mechanism of Grimontia hollisae collagenase through structural and mutational analyses.

Grimontia hollisae collagenase (Ghcol) exhibits high collagen-degrading activity. To explore its catalytic mechanism, its substrate (Gly-Pro-Hyp-Gly-Pro-Hyp, GPOGPO)-complexed crystal structure was determined at 2.0 Å resolution. A water molecule was observed near the active-site zinc ion. Since this water was not observed in the product (GPO)-complexed Ghcol, it was hypothesized that the GPOGPO-complexed Ghcol structure reflects a Michaelis complex, providing a structural basis for understanding the catalytic mechanism. Analyses of the active-site geometry and site-directed mutagenesis of the active-site tyrosine residues revealed that Glu493 and Tyr564 were essential for catalysis, suggesting that Glu493 functions as an acid and base catalyst while Tyr564 stabilizes the tetrahedral complex in the transition state. These results shed light on the catalytic mechanism of bacterial collagenase.

Bivalent structure of a TNFR2-selective and agonistic TNF-α mutein Fc-fusion protein enhances the expansion activity of regulatory T cells.

Recently, TNF receptor type 2 (TNFR2) signaling was found to be involved in the proliferation and activation of regulatory T cells (Tregs), a subpopulation of lymphocytes that suppress immune responses. Tregs mediate peripheral immune tolerance, and the disruption of their functions causes autoimmune diseases or allergy. Therefore, cell expanders or regulators of Tregs that control immunosuppressive activity can be used to treat these diseases. We focused on TNFR2, which is preferentially expressed on Tregs, and created tumor necrosis factor-α (TNF-α) muteins that selectively activate TNFR2 signaling in mice and humans, termed R2agoTNF and R2-7, respectively. In this study, we attempted to optimize the structure of muteins to enhance their TNFR2 agonistic activity and stability in vivo by IgG-Fc fusion following single-chain homo-trimerization. The fusion protein, scR2agoTNF-Fc, enhanced the expansion of CD4+CD25+ Tregs and CD4+Foxp3+ Tregs and contributed to their immunosuppressive activity ex vivo and in vivo in mice. The prophylactic administration of scR2agoTNF-Fc suppressed inflammation in contact hypersensitivity and arthritis mouse models. Furthermore, scR2-7-Fc preferentially expanded Tregs in human peripheral blood mononuclear cells via TNFR2. These TNFR2 agonist-Fc fusion proteins, which have bivalent structures, are novel Treg expanders.