Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules.

The NADase SARM1 (sterile alpha and TIR motif containing 1) is a key executioner of axon degeneration and a therapeutic target for several neurodegenerative conditions. We show that a potent SARM1 inhibitor undergoes base exchange with the nicotinamide moiety of nicotinamide adenine dinucleotide (NAD+) to produce the bona fide inhibitor 1AD. We report structures of SARM1 in complex with 1AD, NAD+ mimetics and the allosteric activator nicotinamide mononucleotide (NMN). NMN binding triggers reorientation of the armadillo repeat (ARM) domains, which disrupts ARM:TIR interactions and leads to formation of a two-stranded TIR domain assembly. The active site spans two molecules in these assemblies, explaining the requirement of TIR domain self-association for NADase activity and axon degeneration. Our results reveal the mechanisms of SARM1 activation and substrate binding, providing rational avenues for the design of new therapeutics targeting SARM1.

Distinct developmental and degenerative functions of SARM1 require NAD+ hydrolase activity.

SARM1 is the founding member of the TIR-domain family of NAD+ hydrolases and the central executioner of pathological axon degeneration. SARM1-dependent degeneration requires NAD+ hydrolysis. Prior to the discovery that SARM1 is an enzyme, SARM1 was studied as a TIR-domain adaptor protein with non-degenerative signaling roles in innate immunity and invertebrate neurodevelopment, including at the Drosophila neuromuscular junction (NMJ). Here we explore whether the NADase activity of SARM1 also contributes to developmental signaling. We developed transgenic Drosophila lines that express SARM1 variants with normal, deficient, and enhanced NADase activity and tested their function in NMJ development. We find that NMJ overgrowth scales with the amount of NADase activity, suggesting an instructive role for NAD+ hydrolysis in this developmental signaling pathway. While degenerative and developmental SARM1 signaling share a requirement for NAD+ hydrolysis, we demonstrate that these signals use distinct upstream and downstream mechanisms. These results identify SARM1-dependent NAD+ hydrolysis as a heretofore unappreciated component of developmental signaling. SARM1 now joins sirtuins and Parps as enzymes that regulate signal transduction pathways via mechanisms that involve NAD+ cleavage, greatly expanding the potential scope of SARM1 TIR NADase functions.

A new mouse model of Charcot-Marie-Tooth 2J neuropathy replicates human axonopathy and suggest alteration in axo-glia communication.

Myelin is essential for rapid nerve impulse propagation and axon protection. Accordingly, defects in myelination or myelin maintenance lead to secondary axonal damage and subsequent degeneration. Studies utilizing genetic (CNPase-, MAG-, and PLP-null mice) and naturally occurring neuropathy models suggest that myelinating glia also support axons independently from myelin. Myelin protein zero (MPZ or P0), which is expressed only by Schwann cells, is critical for myelin formation and maintenance in the peripheral nervous system. Many mutations in MPZ are associated with demyelinating neuropathies (Charcot-Marie-Tooth disease type 1B [CMT1B]). Surprisingly, the substitution of threonine by methionine at position 124 of P0 (P0T124M) causes axonal neuropathy (CMT2J) with little to no myelin damage. This disease provides an excellent paradigm to understand how myelinating glia support axons independently from myelin. To study this, we generated targeted knock-in MpzT124M mutant mice, a genetically authentic model of T124M-CMT2J neuropathy. Similar to patients, these mice develop axonopathy between 2 and 12 months of age, characterized by impaired motor performance, normal nerve conduction velocities but reduced compound motor action potential amplitudes, and axonal damage with only minor compact myelin modifications. Mechanistically, we detected metabolic changes that could lead to axonal degeneration, and prominent alterations in non-compact myelin domains such as paranodes, Schmidt-Lanterman incisures, and gap junctions, implicated in Schwann cell-axon communication and axonal metabolic support. Finally, we document perturbed mitochondrial size and distribution along MpzT124M axons suggesting altered axonal transport. Our data suggest that Schwann cells in P0T124M mutant mice cannot provide axons with sufficient trophic support, leading to reduced ATP biosynthesis and axonopathy. In conclusion, the MpzT124M mouse model faithfully reproduces the human neuropathy and represents a unique tool for identifying the molecular basis for glial support of axons.

Adipocyte-specific inactivation of NAMPT, a key NAD+ biosynthetic enzyme, causes a metabolically unhealthy lean phenotype in female mice during aging.

Nicotinamide adenine dinucleotide (NAD+) is a universal coenzyme regulating cellular energy metabolism in many cell types. Recent studies have demonstrated the close relationships between defective NAD+ metabolism and aging and age-associated metabolic diseases. The major purpose of the present study was to test the hypothesis that NAD+ biosynthesis, mediated by a rate-limiting NAD+ biosynthetic enzyme, nicotinamide phosphoribosyltransferase (NAMPT), is essential for maintaining normal adipose tissue function and whole body metabolic health during the aging process. To this end, we provided in-depth and comprehensive metabolic assessments for female adipocyte-specific Nampt knockout (ANKO) mice during aging. We first evaluated body fat mass in young (≤4-mo-old), middle aged (10-14-mo-old), and old (≥18-mo-old) mice. Intriguingly, adipocyte-specific Nampt deletion protected against age-induced obesity without changing energy balance. However, data obtained from the hyperinsulinemic-euglycemic clamp procedure (HECP) demonstrated that, despite the lean phenotype, old ANKO mice had severe insulin resistance in skeletal muscle, heart, and white adipose tissue (WAT). Old ANKO mice also exhibited hyperinsulinemia and hypoadiponectinemia. Mechanistically, loss of Nampt caused marked decreases in WAT gene expression of lipogenic targets of peroxisome proliferator-activated receptor gamma (PPAR-γ) in an age-dependent manner. In addition, administration of a PPAR-γ agonist rosiglitazone restored fat mass and improved metabolic abnormalities in old ANKO mice. In conclusion, these findings highlight the importance of the NAMPT-NAD+-PPAR-γ axis in maintaining functional integrity and quantity of adipose tissue, and whole body metabolic function in female mice during aging.NEW & NOTEWORTHY Defective NAD+ metabolism is associated with aging and age-associated metabolic diseases. In the present study, we provided in-depth metabolic assessments in female mice with adipocyte-specific inactivation of a key NAD+ biosynthetic enzyme NAMPT and revealed an unexpected role of adipose tissue NAMPT-NAD+-PPAR-γ axis in maintaining functional integrity and quantity of adipose tissue and whole body metabolic health during the aging process.

Multiple domain interfaces mediate SARM1 autoinhibition.

Axon degeneration is an active program of self-destruction mediated by the protein SARM1. In healthy neurons, SARM1 is autoinhibited and, upon injury autoinhibition is relieved, activating the SARM1 enzyme to deplete NAD+ and induce axon degeneration. SARM1 forms a homomultimeric octamer with each monomer composed of an N-terminal autoinhibitory ARM domain, tandem SAM domains that mediate multimerization, and a C-terminal TIR domain encoding the NADase enzyme. Here we discovered multiple intramolecular and intermolecular domain interfaces required for SARM1 autoinhibition using peptide mapping and cryo-electron microscopy (cryo-EM). We identified a candidate autoinhibitory region by screening a panel of peptides derived from the SARM1 ARM domain, identifying a peptide mediating high-affinity inhibition of the SARM1 NADase. Mutation of residues in full-length SARM1 within the region encompassed by the peptide led to loss of autoinhibition, rendering SARM1 constitutively active and inducing spontaneous NAD+ and axon loss. The cryo-EM structure of SARM1 revealed 1) a compact autoinhibited SARM1 octamer in which the TIR domains are isolated and prevented from oligomerization and enzymatic activation and 2) multiple candidate autoinhibitory interfaces among the domains. Mutational analysis demonstrated that five distinct interfaces are required for autoinhibition, including intramolecular and intermolecular ARM-SAM interfaces, an intermolecular ARM-ARM interface, and two ARM-TIR interfaces formed between a single TIR and two distinct ARM domains. These autoinhibitory regions are not redundant, as point mutants in each led to constitutively active SARM1. These studies define the structural basis for SARM1 autoinhibition and may enable the development of SARM1 inhibitors that stabilize the autoinhibited state.

[Analysis of Short-Term Results after Introduction of Robotic Gastrectomy for Gastric Cancer at Our Hospital].

BACKGROUND: Robotic gastrectomy(RG)for gastric cancer(GC)has been covered by health insurance since 2018. In this study, we examined the results of RG for GC at our hospital during the initial period of its introduction. MATERIALS AND METHOD: From August 2022 to May 2023, we retrospectively examined the surgical outcomes and short-term postoperative outcomes of the first 9 patients who underwent RG for GC at our hospital. RESULTS: The median patient age was 77(67-82) years, gender was 4 males and 5 females, and distal gastrectomy was performed in all patients. The median operative time was 410(323-486)min, blood loss was 5(1-140)mL, postoperative hospital stay was less than 9 days in all patients, and there was no conversion to laparoscopic or open surgery. There were no postoperative complications of Clavien-Dindo Grade Ⅱ or above. CONCLUSION: In this study, RG for GC was performed safely without intraoperative or postoperative complications.

Adipocyte NMNAT1 expression is essential for nuclear NAD+ biosynthesis but dispensable for regulating thermogenesis and whole-body energy metabolism.

Nicotinamide adenine dinucleotide (NAD+) functions as an essential cofactor regulating a variety of biological processes. The purpose of the present study was to determine the role of nuclear NAD+ biosynthesis, mediated by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), in thermogenesis and whole-body energy metabolism. We first evaluated the relationship between NMNAT1 expression and thermogenic activity in brown adipose tissue (BAT), a key organ for non-shivering thermogenesis. We found that reduced BAT NMNAT1expression was associated with inactivation of thermogenic gene program induced by obesity and thermoneutrality. Next, we generated and characterized adiponectin-Cre-driven adipocyte-specific Nmnat1 knockout (ANMT1KO) mice. Loss of NMNAT1 markedly reduced nuclear NAD+ concentration by approximately 70% in BAT. Nonetheless, adipocyte-specific Nmnat1 deletion had no impact on thermogenic (rectal temperature, BAT temperature and whole-body oxygen consumption) responses to ß-adrenergic ligand norepinephrine administration and acute cold exposure, adrenergic-mediated lipolytic activity, and metabolic responses to obesogenic high-fat diet feeding. In addition, loss of NMNAT1 did not affect nuclear lysine acetylation or thermogenic gene program in BAT. These results demonstrate that adipocyte NMNAT1 expression is required for maintaining nuclear NAD+ concentration, but not for regulating BAT thermogenesis or whole-body energy homeostasis.

Computational reduction of the spectral division method for synthesizing moving sources by source trajectory approximation.

This paper proposes a method to reduce the computational cost of the spectral division method that synthesizes moving sources. The proposed method consists of two approximations: that of the secondary source driving function and that of the trajectory of the moving sources. Combining these two approximations simplifies the integral calculations that traditionally appear in the driving functions, replacing them with a correction of the frequency magnitude and phase of the source signals. Numerical simulations and subjective experiments show that the computational cost can be reduced by a factor of 50-100 compared to the conventional method without significantly affecting the synthesized sound field and the sense of localization.

Synthesis of sound field from moving complex sources with arbitrary trajectories by linear and spherical loudspeaker arrays.

The spectral division method (SDM) and near-field compensated higher order ambisonics (NFC-HOA) are sound field synthesis techniques based on the spatial Fourier representation of sound fields. Previous studies have derived the driving functions of SDM for sound field synthesis with consideration to uniformly moving point sources and moving point sources with arbitrary trajectories. However, the driving functions of NFC-HOA for synthesizing sound fields from moving sound sources have not been proposed to date. For a more realistic auditory experience, the synthesis of a sound field produced by a moving sound source with a complex radiation property is required. This study focused on deriving the driving functions for synthesizing sound fields produced by moving sound sources with arbitrary trajectories and radiation properties. Sound fields were formulated in the angular spectrum and spherical harmonic domains and applied to SDM and NFC-HOA, respectively. Numerical and measurement experiments were conducted to evaluate the proposed method. The results reveal that the proposed method can synthesize the desired sound fields.

[A Case of Hepatocellular Carcinoma with Haemobilia after Transcatheter Hepatic Arterial Embolization for Tumor Rupture].

A 79-year-old man was scheduled for surgery for hepatocellular carcinoma(HCC)after transcatheter hepatic arterial embolization for rupture. Two weeks before surgery, the patient came to our hospital with a chief complaint of back pain. First, we performed biliary drainage, under the diagnosis of HCC with obstructive jaundice due to haemobilia. Hepatectomy was performed when the patient's condition stabilized. It should be kept in mind that haemobilia may occur after TAE for HCC with bile duct tumor thrombus, and appropriate treatment should be performed when bleeding occurs.

[A Case of Colon Metastasis from Gastric Cancer Treated by Laparoscopic-Assisted Segmental Colectomy].

We report a case of colon metastasis from gastric cancer treated by laparoscopic-assisted segmental colectomy. An 81-year-old man was undergone distal gastrectomy, D2 dissection and Billroth Ⅰ reconstruction for gastric cancer 3 years previously, with a final diagnosis of gastric cancer L, Post, Type 2, sig/por2, pT4a(SE), pN3b(30/56), H0, P0, M0, pStage ⅢC. Three years after gastrectomy, CT scan showed an elevated lesion in the transverse colon, which was suspected to be metastatic colorectal cancer on closer examination. As no metastatic lesions were found other than the tumor of transverse colon, we performed laparoscopic-assisted segmental colon resection. A small incision was placed in the umbilical region, and the transverse colon was extracted from the umbilical region after dissection of the adhesions by single-incision laparoscopic surgery. The transverse colon containing the mass lesion was partially resected extracorporeally and reconstructed with a functional end-to-end anastomosis. The postoperative pathological findings revealed tumor cells predominantly below the submucosal layer and partly showing the signet ring cell carcinoma, and the transvers colon tumor was diagnosed as a metastasis from gastric cancer. The postoperative course was uneventful and the patient was discharged 8 days after surgery, and is alive for 10 months after the segmental colon resection followed by chemotherapy.

[A Case of Recurrent Esophageal Cancer with Long-Term Survival Treated by S-1 Monotherapy].

We report a case of recurrent esophageal cancer with long-term survival treated by S-1 monotherapy. A 66-year-old man underwent subtotal esophagectomy, two-field lymphadenectomy after 2 courses of DCF chemotherapy for esophageal cancer 1 year earlier. The final diagnosis was esophageal cancer, Lt, CT-Type 2, ypT3, ypN0(0/62), M0, ypStage Ⅲ. At 6 months after esophagectomy, CT scan revealed mediastinal lymph node metastasis and pleural dissemination, and paclitaxel monotherapy was performed, but lymph node re-enlargement was observed on CT at 12 months after esophagectomy. Chemotherapy with S-1 monotherapy was performed, and 3 months after initiation of S-1 monotherapy, CT showed reduced lymph node metastases and pleural dissemination remained reduced. Adverse events were CTCAE v5.0 Grade 2 thrombocytopenia and diarrhoea, but no Grade 3 or higher adverse events were observed. Long-term survival was achieved with no disease progression for more than 2.5 years after initiation of S-1 monotherapy.

[A Rare Case of Advanced Gastric Cancer with Duodenal Intramural Metastasis].

Here we report the case of a patient with advanced gastric cancer who presented with duodenal intramural metastasis based on the pathological results after surgery. The patient was 78-year-old female, who was referred to our department for further evaluation and treatment of upper abdominal pain. An upper gastrointestinal series demonstrated a tumor occupying the lesser curvature of the gastric body. Biopsy specimens from the tumor demonstrated moderately to poorly differentiated adenocarcinoma. A computed tomography scan showed thickening of the gastric wall and swelling of the regional lymph nodes. The patient underwent distal gastrectomy and D2 lymph node dissection for gastric cancer. A histopathological examination disclosed that the gastric tumor was poorly differentiated adenocarcinoma with severe lymphatic permeation and also demonstrated the other poorly differentiated adenocarcinoma occupying the part of the muscularis propria layer of the duodenum. The gastric tumor was not contiguous with the duodenal tumor, and the duodenal cancer cells had the same pathological characteristics as the primary gastric cancer cells; therefore, we diagnosed the duodenal tumor as an intramural metastasis from gastric cancer. The patient's disease was staged as pT4aN3bM1, Stage Ⅳ according to the TNM classification. We report this rare case along with a discussion of the literature.

[A Case of Sigmoid Rectal Cancer with a Vesicoconstrictor Fistula Curatively Resected after Neoadjuvant Chemotherapy].

A 50-year-old man presented with fecaluria and was diagnosed with sigmoid colon cancer with a colovesical fistula. Total bladder resection was determined to be necessary for curative resection at the time of diagnosis. In anticipation of bladder preservation, 6 courses of mFOLFOX6 plus panitumumab were administered after transverse colostomy, resulting in marked tumor regression and a decision to proceed with surgery. The patient underwent robotic-assisted low anterior resection of the rectum and partial cystectomy, which yielded pathological radical treatment. We report a case of sigmoid colon cancer with a colovesical fistula complicated by bladder invasion, in which preoperative chemotherapy was effective and total cystectomy was avoided, allowing bladder preservation.

Age- and sex-based changes in spike protein antibody status after SARS-CoV-2 vaccination and effect of past-infection in healthcare workers in Osaka.

OBJECTIVE: We aimed to compare the changes in SARS-CoV-2 spike protein antibody titres based on age group and sex using paired blood sampling after vaccination in association with the presence of nucleocapsid protein antibody. METHODS: All participants were healthcare workers at Yao Municipal Hospital in Osaka who voluntarily provided peripheral blood samples (n = 636, men/women 151/485, mean age 45 years). We investigated the serial changes in SARS-CoV-2 spike protein antibody titres at 1 and 7 months after the second vaccination regarding their relationship with sex and age group. At 7 months, we also examined anti-nucleocapsid assays. Antibody titres were shown as logarithmic values and the differences were assessed using a paired or unpaired student's t-test as appropriate. RESULTS: Among participants younger than 30 years, the antibody titres of spike protein were significantly higher in women one (p = 0.005) and seven (p = 0.038) months after vaccination. However, among those aged 30-49 years, the antibody titres were not different between the sexes at either follow-up time point. In contrast, among those aged 50-59 years, between-sex differences in antibody titres were observed only at 7 months, which was associated with a significant reduction in men. A significant negative correlation was observed between the antibody titres for spike protein at both time points in participants with positive nucleocapsid protein antibody at 7 months (r = - 0.467, p = 0.043), although a significant positive correlation was observed in those with negative results (r = 0.645, p < 0.001), CONCLUSIONS: Between-sex differences in SARS-CoV-2 spike protein antibody titres by paired blood sampling at different time points after vaccination depended on age group. The presence of nucleocapsid protein antibody was associated with changes in spike protein antibody titres after vaccination.

Adipose tissue NAD+ biosynthesis is required for regulating adaptive thermogenesis and whole-body energy homeostasis in mice.

Nicotinamide adenine dinucleotide (NAD+) is a critical coenzyme for cellular energy metabolism. The aim of the present study was to determine the importance of brown and white adipose tissue (BAT and WAT) NAD+ metabolism in regulating whole-body thermogenesis and energy metabolism. Accordingly, we generated and analyzed adipocyte-specific nicotinamide phosphoribosyltransferase (Nampt) knockout (ANKO) and brown adipocyte-specific Nampt knockout (BANKO) mice because NAMPT is the rate-limiting NAD+ biosynthetic enzyme. We found ANKO mice, which lack NAMPT in both BAT and WAT, had impaired gene programs involved in thermogenesis and mitochondrial function in BAT and a blunted thermogenic (rectal temperature, BAT temperature, and whole-body oxygen consumption) response to acute cold exposure, prolonged fasting, and administration of ß-adrenergic agonists (norepinephrine and CL-316243). In addition, the absence of NAMPT in WAT markedly reduced adrenergic-mediated lipolytic activity, likely through inactivation of the NAD+-SIRT1-caveolin-1 axis, which limits an important fuel source fatty acid for BAT thermogenesis. These metabolic abnormalities were rescued by treatment with nicotinamide mononucleotide (NMN), which bypasses the block in NAD+ synthesis induced by NAMPT deficiency. Although BANKO mice, which lack NAMPT in BAT only, had BAT cellular alterations similar to the ANKO mice, BANKO mice had normal thermogenic and lipolytic responses. We also found NAMPT expression in supraclavicular adipose tissue (where human BAT is localized) obtained from human subjects increased during cold exposure, suggesting our finding in rodents could apply to people. These results demonstrate that adipose NAMPT-mediated NAD+ biosynthesis is essential for regulating adaptive thermogenesis, lipolysis, and whole-body energy metabolism.

[A Case of Sigmoid Colon Cancer with Bladder Fistula Treated by Laparoscopic Radical Resection].

The patient was a woman in her 70 s. Computed tomography(CT)showed a sigmoid colon tumor invading the uterus and ovaries, and a fistula to the bladder. The patient was scheduled to receive neoadjuvant chemotherapy(NAC), but while waiting for treatment, generalized peritonitis due to perforation of the tumor was observed, and a laparoscopic transverse colostomy was performed. After NAC with CAPOX and FOLFIRI plus panitumumab, the tumor was found to have shrunk, and a laparoscopic posterior pelvic exenteration was performed. The bladder including the fistula was partially resected, and the tumor, uterus, and right ovary were resected in combination as R0, besides the ureter and remaining bladder could be preserved. The postoperative course was uneventful, and the patient is alive without recurrence to date. In this article, we report a case of a patient with sigmoid colon cancer with a bladder fistula who underwent laparoscopic surgery after NAC, and bladder function could be preserved, with some discussion of the literature.

[Analysis of Chemotherapy-Induced Nausea and Vomiting during Chemotherapy for Gastric Cancer].

BACKGROUND: Chemotherapy-induced nausea and vomiting(CINV)are typical side effects caused by chemotherapy. We analyzed CINV during first-line chemotherapy for gastric cancer. MATERIALS AND METHOD: Thirty-one patients who received first-line chemotherapy for gastric cancer were retrospectively assessed for CINV. RESULTS: The median age was 70 years, and the gender(male/female)was 23/8 cases. NK1 receptor antagonist, 5-HT3 receptor antagonist, and dexamethasone were used as antiemetic agents in 29 patients(94%). Sixteen patients(52%)had Grade 1 or higher nausea, and 6 patients (19%)had Grade 1 or higher vomiting, and complete control of nausea and vomiting was achieved in 21 patients(68%). Nausea was significantly more frequent in patients with liver metastasis(p=0.0008), but there was no significant difference in vomiting(p=1.0000). There was no significant difference in the occurrence of CINV between chemotherapy regimens or combination of olanzapine. CONCLUSION: During first-line chemotherapy for gastric cancer, 3 antiemetic agents were used in 94% of cases, and the complete control rate of CINV was 67.8%.

[A Case of Early Gastric Cancer with Adachi Type â ¥ Vascular Anomaly Treated by Laparoscopic Distal Gastrectomy].

We report a case of early gastric cancer with Adachi Type Ⅵ vascular anomaly treated by laparoscopic distal gastrectomy. An 81-year-old woman was admitted because of anorexia, and was diagnosed with early gastric cancer. Preoperative MDCT revealed Adachi Type Ⅵ vascular anomaly, where the hepatic artery does not appear at the superior border of the pancreas. The patient was treated successfully with laparoscopic distal gastrectomy with D1+lymph node dissection. At surgery, we identified the portal vein, then, dissection of No. 8a lymph nodes was performed. The postoperative course was uneventful and the patient was discharged 10 days after surgery. The final pathology result showed gastric cancer, M, Less, Type 0-Ⅱc+Ⅲ, 58×50 mm, tub1>pap, pT1a(M), Ly0, V0, pN0(0/40), H0, P0, M0, pStage ⅠA. We understand the arterial running pattern before surgery by using MDCT, and performed laparoscopic surgery safely.

[A Case of Laparoscopic Resection Using the ICG Fluorescence for Peritoneal Disseminations of Hepatocellular Carcinoma].

A 71-year-old male had repeated resection and transcatheter arterial chemo-embolization(TACE)for hepatocellular carcinoma(HCC). Treatment with lenvatinib was started due to multiple liver recurrences and peritoneal disseminations. Since only the disseminated lesion had increased, it was decided to perform laparoscopic resection. Indocyanine green(ICG) was intravenously injected the day before surgery. Disseminated lesions could be easily detected with intraoperative fluorescence imaging, and we could completely resect disseminated lesions. The ICG fluorescence could be considered to be useful in laparoscopic resection for peritoneal dissemination of HCC.