Structural differences between the closely related RNA helicases, UAP56 and URH49, fashion distinct functional apo-complexes.

mRNA export is an essential pathway for the regulation of gene expression. In humans, closely related RNA helicases, UAP56 and URH49, shape selective mRNA export pathways through the formation of distinct complexes, known as apo-TREX and apo-AREX complexes, and their subsequent remodeling into similar ATP-bound complexes. Therefore, defining the unidentified components of the apo-AREX complex and elucidating the molecular mechanisms underlying the formation of distinct apo-complexes is key to understanding their functional divergence. In this study, we identify additional apo-AREX components physically and functionally associated with URH49. Furthermore, by comparing the structures of UAP56 and URH49 and performing an integrated analysis of their chimeric mutants, we exhibit unique structural features that would contribute to the formation of their respective complexes. This study provides insights into the specific structural and functional diversification of these two helicases that diverged from the common ancestral gene Sub2.

Relative Adrenal Insufficiency Is a Risk Factor for Pediatric Sepsis: A Proof-of-Concept Study.

Glucocorticoid (GC) therapy had been strongly recommended for pediatric sepsis (grade 1A). However, the recommendation was changed to grade 2C in 2020 due to weak evidence. About 32.8% of patients with pediatric septic develop relative adrenal insufficiency (RAI). But whether GC therapy should be determined by RAI status is controversial. This study utilized 21-day-old SF1CreSRBIfl/fl mice as the first pediatric RAI mouse model to assess the pathogenesis of RAI and evaluate GC therapy. RAI mice exhibited a substantially higher mortality rate in cecal ligation and puncture and cecal slurry-induced sepsis. These mice featured persistent inflammatory responses and were effectively rescued by GC therapy. RNA sequencing analysis revealed persistent inflammatory responses in RAI mice, caused by transcriptional dysregulation of AP-1 and NF-κB, and cytokine-induced secondary inflammatory response. Our findings support a precision medicine approach to guide GC therapy for pediatric patients based on the status of RAI.

Hepcidin expression in the trigeminal ganglion and the oral mucosa in an oral ulcerative mucositis rat model.

Severe intraoral pain induces difficulty in eating and speaking, leading to a decline in the quality of life. However, the molecular mechanisms underlying intraoral pain remain unclear. Here, we investigated gene modulation in the trigeminal ganglion and intraoral pain-related behavior in a rat model of acetic acid-induced oral ulcerative mucositis. Oral ulceration was observed on day 2 after acetic acid treatment to the oral mucosa of male Wistar rats, causing spontaneous pain and mechanical allodynia. Deoxyribonucleic acid microarray analysis of trigeminal ganglion tissue indicated that Hamp (a hepcidin gene that regulates cellular iron transport) was the most upregulated gene. In the oral ulcerative mucositis model, the upregulation of Hamp was also induced in the ulcer region but not in the liver, with no increase in hepcidin levels in the plasma and saliva, indicating that hepcidin was produced locally in the ulcer region in the model. Systemic antibiotic pretreatment did not increase the mRNA levels of Hamp in the trigeminal ganglion and ulcer regions. Hepcidin injection into the oral mucosa enhanced neuronal excitability in response to noxious mechanical stimulation of the oral mucosa in trigeminal spinal subnucleus interpolaris/caudalis neurons. These results imply that oral ulcerative mucositis induces oral mucosal pain because of infectious inflammation of the ulcerative area and potentiates Hamp, which represents anti-bacterial and anti-peptidase gene expression in the ulcer region and trigeminal ganglion. The regulation of cellular iron transport by hepcidin is likely involved in oral ulcerative mucositis-induced pain.

The adrenal stress response is an essential host response against therapy-induced lethal immune activation.

Cytokine release syndrome (CRS) is a systemic inflammatory syndrome associated with infection- or drug-induced T cell activation and can cause multiple organ failure and even death. Because current treatments are ineffective in some patients with severe CRS, we set out to identify risk factors and mechanisms behind severe CRS that might lead to preventive therapies and better clinical outcomes in patients. In mice, we found that deficiency in the adrenal stress response-with similarities to such in patients called relative adrenal insufficiency (RAI)-conferred a high risk for lethal CRS. Mice treated with CD3 antibodies were protected against lethal CRS by the production of glucocorticoids (GC) induced by the adrenal stress response in a manner dependent on the scavenger receptor B1 (SR-BI), a receptor for high-density lipoprotein (HDL). Mice with whole-body or adrenal gland-specific SR-BI deficiency exhibited impaired GC production, more severe CRS, and increased mortality in response to CD3 antibodies. Pretreatment with a low dose of GC effectively suppressed the development of CRS and rescued survival in SR-BI-deficient mice without compromising T cell function through apoptosis. Our findings suggest that RAI may be a risk factor for therapy-induced CRS and that pretreating RAI patients with GC may prevent lethal CRS.

Relative adrenal insufficiency is a risk factor and endotype of sepsis - A proof-of-concept study to support a precision medicine approach to guide glucocorticoid therapy for sepsis.

Introduction: 25-60% of septic patients experience relative adrenal insufficiency (RAI) and glucocorticoid (GC) is frequently used in septic patients. However, the efficacy of GC therapy and whether GC therapy should be based on the status of RAI are highly controversial. Our poor understanding about the pathogenesis of RAI and a lack of RAI animal model present significant barriers to address these critical issues. Methods: Scavenger receptor BI (SR-BI) regulates stress-induced GC (iGC) production in response to stress. We generated SF1CreSR-BIfl/fl mice and utilized the mice as a RAI model to elucidate the pathogenesis of RAI and GC therapy in sepsis. SF1CreSR-BIfl/fl mice did not express SR-BI in adrenal gland and lacked iGC production upon ACTH stimulation, thus, they are RAI. Results and Discussion: RAI mice were susceptible to cecal ligation and puncture (CLP)-induced sepsis (6.7% survival in SF1CreSR-BIfl/fl mice versus 86.4% in SR-BIfl/fl mice; p = 0.0001). Compared to a well-controlled systemic inflammatory response in SR-BIfl/fl mice, SF1CreSR-BIfl/fl mice featured a persistent hyperinflammatory response. Supplementation of a low stress dose of GC to SF1CreSR-BIfl/fl mice kept the inflammatory response under control and rescued the mice. However, SR-BIfl/fl mice receiving GC treatment exhibited significantly less survival compared to SR-BIfl/fl mice without GC treatment. In conclusions, we demonstrated that RAI is a risk factor for death in this mouse model of sepsis. We further demonstrated that RAI is an endotype of sepsis, which features persistent hyperinflammatory response. We found that GC treatment benefits mice with RAI but harms mice without RAI. Our study provides a proof of concept to support a precision medicine approach for sepsis therapy - selectively applying GC therapy for a subgroup of patients with RAI.

Ultrasound Monitoring of Thymus Involution in Septic Mice.

Thymus involution is characterized by a progressive regression of thymus size and contributes to immunosuppression in sepsis. High-frequency ultrasonography is a non-invasive monitoring system in multiple organs, including the thymus, in mice. However, thymus involution has not been studied using ultrasonography in septic mice. This study reports ultrasound approaches to monitoring septic thymus involution in mice. Sepsis was induced by cecum ligation and puncture (CLP). Mice were euthanized at three time points: baseline and days 3 and 10 after CLP. Thymus areas and volumes were measured using 2-D and 3-D ultrasound approaches. Thymus weights were measured ex vivo. Compared with values at baseline, both thymus area and volume decreased significantly at days 3 and 10. In addition, thymus areas and volumes correlated positively with thymus weights. In conclusion, ultrasonography provides reliable thymus measurements and is an optimal technique for monitoring thymus involution in septic mice.

SR-BI (Scavenger Receptor BI), Not LDL (Low-Density Lipoprotein) Receptor, Mediates Adrenal Stress Response-Brief Report.

OBJECTIVE: Adrenal gland secretes stress-induced glucocorticoids (iGCs) to coping with stress. Previous study showed that SR-BI (scavenger receptor BI) null (SR-BI-/-) mice failed to generate iGC in stress conditions, suggesting that SR-BI-mediated cholesterol uptake from HDL (high-density lipoprotein) is a key regulator for iGC production. However, the LDL (low-density lipoprotein)/LDLr (LDL receptor) pathway can also provide cholesterol for iGC synthesis, but rodents have limited LDL levels in circulation. Here, we generated SR-BI-/-ApoBtg (apolipoprotein B transgenic) mice with normal LDL levels in circulation to determine the relative contribution of the HDL/SR-BI and LDL/LDLr pathways to iGC production in stress conditions. Approach and Results: To obtain mouse models with normal LDL levels, SR-BI-/- mice were bred to ApoBtg mice. Then, the F1 SR-BI±ApoBtg mice were backcrossed to SR-BI-/- to obtain SR-BI-/-ApoBtg, SR-BI-/-ApoBwt (apolipoprotein B wild type), and SR-BI+/+ApoBtg mice. We first examined the lipoprotein profile, which shows a 6.5-fold increase in LDL levels in SR-BI-/-ApoBtg mice compared with SR-BI-/-ApoBwt mice. Then, we induced stress with adrenocorticotropic hormone and cecal ligation and puncture. One hour after adrenocorticotropic hormone stimulation, SR-BI+/+ApoBtg control mice produced iGC (14.9-fold), but both SR-BI-/-ApoBwt and SR-BI-/-ApoBtg showed no iGC production (P<0.001). Three hours after cecal ligation and puncture treatment, SR-BI+/+ApoBtg control mice showed iGC production (6.4-fold), but both SR-BI-/-ApoBwt and SR-BI-/-ApoBtg mice showed no iGC production (P<0.001). CONCLUSIONS: SR-BI-/-ApoBtg mice fail to produce iGC in stress conditions even though with restored LDL levels in circulation. These findings clarify that the HDL/SR-BI, not LDL/LDLr, pathway is responsible for iGC production in stress conditions.

Hyposalivation due to chemotherapy exacerbates oral ulcerative mucositis and delays its healing.

OBJECTIVES: Cancer therapy including chemotherapy causes gland atrophy, resulting in low salivary secretion in cancer patients. Since saliva plays an important role in oral health, the dysfunction may exacerbate oral ulcerative mucositis (OUM), which is another side effect. Here, we investigated the effect of hyposalivation on OUM using sialoadenectomized rats and examined the effects of anticancer drugs on the salivary glands. DESIGN: As models for hyposalivation, the bilateral submandibular and sublingual glands except (2EXT) or together with (3EXT) the parotid glands were extracted. At 16 days after the procedure, OUM was experimentally developed by topical acetic acid treatment on the labial fornix region of the inferior incisors, and the severity and bacterial loading level were evaluated. The salivary gland weights and histology were analyzed after administration of the representative anticancer drugs 5-fluorouracil or cisplatin. RESULTS: The severity of OUM was greater in both the 3EXT and 2EXT rats and delayed the healing process compared with that in sham rats without salivary gland extraction. The healing process in the 3EXT rats was longer than that in the 2EXT rats. The number of colony-forming units in the ulcerative region from the 3EXT rats was 10-fold greater than that in the sham rats. Both 5-fluorouracil and cisplatin reduced glands weights and damaged the salivary glands. CONCLUSIONS: These results suggest that chemotherapy-induced hyposalivation exacerbates OUM and delays healing, most likely due to loss of salivary clearance and antimicrobial functions. This study illustrates the significance of oral health care for cancer patients undergoing chemotherapy.

Disseminated Cryptococcosis with Adrenal Insufficiency and Meningitis in an Immunocompetent Individual.

We present a case of cryptococcosis with adrenal insufficiency and meningitis in a healthy host without any risk factors. Antifungal therapy did not reduce the cryptococcal antigen titers of the cerebrospinal fluid and serum or the bilateral adrenal gland enlargement. It was suggested that the adrenal glands were the focus of persistent fungemia. Removal of both adrenal glands brought about a response to antifungal therapy. We conclude that if antifungal therapy is ineffective, bilateral adrenalectomy is an effective measure for treatment of such patients. Cryptococcosis is a possible cause of primary adrenal insufficiency in immunocompetent patients.

Prostanoid-dependent spontaneous pain and PAR2-dependent mechanical allodynia following oral mucosal trauma: involvement of TRPV1, TRPA1 and TRPV4.

Abstract: During dental treatments, intraoral appliances frequently induce traumatic ulcers in the oral mucosa. Such mucosal injury-induced mucositis leads to severe pain, resulting in poor quality of life and decreased cooperation in the therapy. To elucidate mucosal pain mechanisms, we developed a new rat model of intraoral wire-induced mucositis and investigated pain mechanisms using our proprietary assay system for conscious rats. A thick metal wire was installed in the rats between the inferior incisors for one day. In the mucosa of the mandibular labial fornix region, which was touched with a free end of the wire, traumatic ulcer and submucosal abscess were induced on day 1. The ulcer was quickly cured until next day and abscess formation was gradually disappeared until five days. Spontaneous nociceptive behavior was induced on day 1 only, and mechanical allodynia persisted over day 3. Antibiotic pretreatment did not affect pain induction. Spontaneous nociceptive behavior was sensitive to indomethacin (cyclooxygenase inhibitor), ONO-8711 (prostanoid receptor EP1 antagonist), SB-366791, and HC-030031 (TRPV1 and TRPA1 antagonists, respectively). Prostaglandin E2 and 15-deoxyΔ12,14-prostaglandin J2 were upregulated only on day 1. In contrast, mechanical allodynia was sensitive to FSLLRY-NH2 (protease-activated receptor PAR2 antagonist) and RN-1734 (TRPV4 antagonist). Neutrophil elastase, which is known as a biased agonist for PAR2, was upregulated on days 1 to 2. These results suggest that prostanoids and PAR2 activation elicit TRPV1- and TRPA1-mediated spontaneous pain and TRPV4-mediated mechanical allodynia, respectively, independently of bacterial infection, following oral mucosal trauma. The pathophysiological pain mechanism suggests effective analgesic approaches for dental patients suffering from mucosal trauma-induced pain.

Distinct TRPV1- and TRPA1-based mechanisms underlying enhancement of oral ulcerative mucositis-induced pain by 5-fluorouracil.

In many patients with cancer, chemotherapy-induced severe oral ulcerative mucositis causes intractable pain, leading to delays and interruptions in therapy. However, the pain mechanism in oral ulcerative mucositis after chemotherapy has not been extensively studied. In this study, we investigated spontaneous pain and mechanical allodynia in a preclinical model of oral ulcerative mucositis after systemic administration of the chemotherapy drug 5-fluorouracil, using our proprietary pain assay system for conscious rats. 5-Fluorouracil caused leukopenia but did not induce pain-related behaviors. After 5-fluorouracil administration, oral ulcers were developed with topical acetic acid treatment. Compared with saline-treated rats, 5-fluorouracil-exposed rats showed more severe mucositis with excessive bacterial loading due to a lack of leukocyte infiltration, as well as enhancements of spontaneous pain and mechanical allodynia. Antibacterial drugs, the lipid A inhibitor polymyxin B and the TRPV1/TRPA1 channel pore-passing anesthetic QX-314, suppressed both the spontaneous pain and the mechanical allodynia. The cyclooxygenase inhibitor indomethacin and the TRPV1 antagonist SB-366791 inhibited the spontaneous pain, but not the mechanical allodynia. In contrast, the TRPA1 antagonist HC-030031 and the N-formylmethionine receptor FPR1 antagonist Boc MLF primarily suppressed the mechanical allodynia. These results suggest that 5-fluorouracil-associated leukopenia allows excessive oral bacterial infection in the oral ulcerative region, resulting in the enhancement of spontaneous pain through continuous TRPV1 activation and cyclooxygenase pathway, and mechanical allodynia through mechanical sensitization of TRPA1 caused by neuronal effects of bacterial toxins. These distinct pain mechanisms explain the difficulties encountered with general treatments for oral ulcerative mucositis-induced pain in patients with cancer and suggest more effective approaches.