NMR-based site-resolved profiling of ß-amyloid misfolding reveals structural transitions from pathologically relevant spherical oligomer to fibril.

Increasing evidence highlights the central role of neurotoxic oligomers of the 42-residue-long ß-amyloid (Aß42) in Alzheimer's disease (AD). However, very limited information is available on the structural transition from oligomer to fibril, particularly for pathologically relevant amyloids. To the best of our knowledge, we present here the first site-specific structural characterization of Aß42 misfolding, from toxic oligomeric assembly yielding a similar conformation to an AD-associated Aß42 oligomer, into a fibril. Transmission EM (TEM) analysis revealed that a spherical amyloid assembly (SPA) of Aß42 with a 15.6 ± 2.1-nm diameter forms in a ∼30-µm Aß42 solution after a ∼10-h incubation at 4 °C, followed by a slow conversion into fibril at ∼180 h. Immunological analysis suggested that the SPA has a surface structure similar to that of amylospheroid (ASPD), a patient-derived toxic Aß oligomer, which had a diameter of 10-15 nm in negative-stain TEM. Solid-state NMR analyses indicated that the SPA structure involves a ß-loop-ß motif, which significantly differed from the triple-ß motif observed for the Aß42 fibril. The comparison of the 13C chemical shifts of SPA with those of the fibril prepared in the above conditions and interstrand distance measurements suggested a large conformational change involving rearrangements of intermolecular ß-sheet into in-register parallel ß-sheet during the misfolding. A comparison of the SPA and ASPD 13C chemical shifts indicated that SPA is structurally similar to the ASPD relevant to AD. These observations provide insights into the architecture and key structural transitions of amyloid oligomers relevant for AD pathology.

Principal component analysis of data from NMR titration experiment of uniformly 15N labeled amyloid beta (1-42) peptide with osmolytes and phenolic compounds.

A simple NMR method to analyze the data obtained by NMR titration experiment of amyloid formation inhibitors against uniformly 15N-labeled amyloid-ß 1-42 peptide (Aß(1-42)) was described. By using solution nuclear magnetic resonance (NMR) measurement, the simplest method for monitoring the effects of Aß fibrilization inhibitors is the NMR chemical shift perturbation (CSP) experiment using 15N-labeled Aß(1-42). However, the flexible and dynamic nature of Aß(1-42) monomer may hamper the interpretation of CSP data. Here we introduced principal component analysis (PCA) for visualizing and analyzing NMR data of Aß(1-42) in the presence of amyloid inhibitors including high concentration osmolytes. We measured 1H-15N 2D spectra of Aß(1-42) at various temperatures as well as of Aß(1-42) with several inhibitors, and subjected all the data to PCA (PCA-HSQC). The PCA diagram succeeded in differentiating the various amyloid inhibitors, including epigallocatechin gallate (EGCg), rosmarinic acid (RA) and curcumin (CUR) from high concentration osmolytes. We hypothesized that the CSPs reflected the conformational equilibrium of intrinsically disordered Aß(1-42) induced by weak inhibitor binding rather than the specific molecular interactions.

Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-ß assembly.

Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid ß-protein (Aß) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aß oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na(+)/K(+)-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aß-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.

Nuclear magnetic resonance evidence for the dimer formation of beta amyloid peptide 1-42 in 1,1,1,3,3,3-hexafluoro-2-propanol.

Alzheimer's disease involves accumulation of senile plaques in which filamentous aggregates of amyloid beta (Aß) peptides are deposited. Recent studies demonstrate that oligomerization pathways of Aß peptides may be complicated. To understand the mechanisms of Aß(1-42) oligomer formation in more detail, we have established a method to produce (15)N-labeled Aß(1-42) suited for nuclear magnetic resonance (NMR) studies. For physicochemical studies, the starting protein material should be solely monomeric and all Aß aggregates must be removed. Here, we succeeded in fractionating a "precipitation-resistant" fraction of Aß(1-42) from an "aggregation-prone" fraction by high-performance liquid chromatography (HPLC), even from bacterially overexpressed Aß(1-42). However, both Aß(1-42) fractions after 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) treatment formed amyloid fibrils. This indicates that the "aggregation seed" was not completely monomerized during HFIP treatment. In addition, Aß(1-42) dissolved in HFIP was found to display a monomer-dimer equilibrium, as shown by two-dimensional (1)H-(15)N NMR. We demonstrated that the initial concentration of Aß during the HFIP pretreatment altered the kinetic profiles of Aß fibril formation in a thioflavin T fluorescence assay. The findings described here should ensure reproducible results when studying the Aß(1-42) peptide.

[A Case Report - A Case of a Rapidly Growing Anal Canal Origin Malignant Melanoma].

An 85-year-old woman presented to her local physician in early August 2015 with a new-onset "swelling" of the anal region and was referred to our department for evaluation and treatment at the beginning of September. At our initial evaluation, the rectal examination showed a black mass lesion, approximately 3.0×1.5 cm in size, in the anal canal by November, the mass had grown to approximately 7.0×3.5 cm in size. The lesion was diagnosed as malignant melanoma by colonoscopic biopsy. Abdominal cystography computed tomography showed an area of lymphadenopathy around the rectum, but no distant metastasis was detected. The patient underwent abdominoperineal resection of the rectum in December. Her postoperative course was good, and she was discharged on the 30th postoperative day. Arecurrent lesion was detected at a recent follow-up examination(approximately 5 months post-discharge), and the patient has been scheduled for immunotherapy. Malignant melanoma of the anal canal has a poor prognosis, and no standard therapy has been established. This report includes a brief review of current literature about the disease.

[A Case of Angioimmunoblastic T-Cell Lymphoma Merged with Colorectal Cancer That We Were Able to Resect after a Chemotherapy Response].

The patient was 77-year-old man. He visited our hospital with the chief complaint of an abdominal mass in March 2015. We diagnosed the patient with transverse colon cancer and he was suspected of having malignant lymphoma. In March 2016, we attempted to perform right hemicolectomy for the transverse colon cancer, but it was difficult because swollen lymph nodes had formed a large mass with the surrounding tissue, including vessels of the mesentery. We could only complete the surgery after mesenteric lymph node biopsy. On the basis of the results of this biopsy, we diagnosed angioimmunoblastic Tcell lymphoma. At first, we administered THP-COP therapy for malignant lymphoma. However, after 3 courses of this therapy, the patient suddenly developed ileus due to the aforementioned colon cancer. According to enhanced CT performed at the onset of intestinal obstruction, chemotherapy dramatically reduced the size of the intraperitoneal lymph nodes. We therefore concluded that it was able to treat the colon cancer. We performed right hemicolectomy in June 2015. Angioimmunoblastic T-cell lymphoma comprises only 2-3% of all malignant lymphoma cases. We experienced a rare case of complications from angioimmunoblastic T-cell lymphoma and colorectal cancer.

[Successful Treatment of Local Recurrence of Advanced Gastric Cancer Using Curative Gastrectomy via Distal Pancreatectomy after Chemoradiotherapy].

The patient was a 65-year-old woman. She was diagnosed with advanced gastric cancer with liver invasion. After receiving systemic chemotherapy(S-1 plus PTX)for 3 months, she underwent total gastrectomy and partial hepatectomy in May 2008. Because she developed celiac artery circumference lymph node recurrence in November 2010 during postoperative adjuvant chemotherapy consisting of S-1 plus PTX, we changed her chemotherapy regimen to CPT-11 plus CDDP. We observed an increase in the size of the lymph nodes in August 2013 and the response was poor even after switching to DOC. However, the lymph nodes continued to increase in size and we administered radiotherapy of 60.4 Gy/33 Fr that resulted in shrinkage of the nodes. We observed an increase in lymph node size and pancreas invasion in September 2015, including an expansion of the mid pancreatic duct. We performed distal pancreatectomy without identifying the recurrence observed in November 2015 assuming it was an exacerbation. Six months after the surgery, the recurrence was not apparent. We report an example of long-term survival that was achieved for Stage IV gastric cancer. The patient underwent combined modality therapy for 8 years, and local recurrence was controlled via a primary operation.

[Two Cases of Small Intestinal Metastasis of Lung Cancer].

Case 1: A 66-year-old man who had undergone upper right lobe resection for large cell lung carcinoma 2 months earlier presented with abdominal pain and vomiting. Computed tomography showed intussusception of the small intestine. Partial resection of the small intestine was performed. The tumor was pathologically diagnosed as lung cancer metastasis to the small intestine. The patient died 30 days after surgery. Case 2: A 57-year-old man was admitted to hospital due to appetite loss. Computed tomography showed a large intestinal tumor and a small lung tumor, as well as multiple liver metastases and lymph node metastases. We diagnosed perforation of the small intestine tumor, and partial resection of small intestine was performed. Pathological examination and immunohistochemical staining revealed that the tumor was a metastatic adenocarcinoma, and the patient was diagnosed with small intestine metastasis of lung cancer. He died 75 days after surgery. Although small intestine metastasis of lung cancer is rare, the incidence is increasing. The prognosis of lung cancer metastasis of the small intestine is poor, and therefore, we must urgently decide the appropriate treatment.

Structural Insight into an Alzheimer's Brain-Derived Spherical Assembly of Amyloid ß by Solid-State NMR.

Accumulating evidence suggests that various neurodegenerative diseases, including Alzheimer's disease (AD), are linked to cytotoxic diffusible aggregates of amyloid proteins, which are metastable intermediate species in protein misfolding. This study presents the first site-specific structural study on an intermediate called amylospheroid (ASPD), an AD-derived neurotoxin composed of oligomeric amyloid-ß (Aß). Electron microscopy and immunological analyses using ASPD-specific "conformational" antibodies established synthetic ASPD for the 42-residue Aß(1-42) as an excellent structural/morphological analogue of native ASPD extracted from AD patients, the level of which correlates with the severity of AD. (13)C solid-state NMR analyses of approximately 20 residues and interstrand distances demonstrated that the synthetic ASPD is made of a homogeneous single conformer containing parallel ß-sheets. These results provide profound insight into the native ASPD, indicating that Aß is likely to self-assemble into the toxic intermediate with ß-sheet structures in AD brains. This approach can be applied to various intermediates relevant to amyloid diseases.

[A Case in Which a Patient Was Operated for Intra-Abdominal Desmoid Tumors after Total Colectomy in FAP].

The patient was a 22-year-old woman with FAP, who had undergone laparoscopic total colectomy 3 years previously. She presented to our hospital with a high fever and abdominal pain. Large hard tumors were palpated in the right lower abdomen and pelvis. Blood examination showed an inflammatory response. CT scan revealed 17 cm diameter solid tumors. At surgery, 2 tumors arising from the mesentery of the small intestine were found, neither of which invaded any organs. We performed tumor extirpation with partial resection of the duodenum, ileum, right fallopian tube and rectum. A jejunal stoma was formed, leaving a length of remnant intestine of about 120 cm. The histopathological diagnosis was given as desmoid tumor with infection. The patient was discharged from the hospital on the 9th postoperative day. Desmoid tumor is the second most common cause of death in FAP patients. Although desmoids can also occur in the extremities, most FAP patients develop intra-abdominal tumors. Despite being histologically benign, they are locally infiltrative and can cause death through invasion and destruction of adjacent vital structures and organs. Here, we report a case of desmoid tumors with FAP with reference to the literature.

[Intraluminal Type Gastrointestinal Stromal Tumor Resected by Single-Incision Percutaneous Endoscopic Intragastric Surgery--A Case Report].

We report a patient with gastric gastrointestinal stromal tumor (GIST) who underwent single-incision percutaneous endoscopic intragastric surgery. The patient was a 70-year-old man. Esophagogastroduodenoscopy and abdominal computed tomography revealed the presence of an intraluminal type gastric submucosal tumor, 4 cm in diameter, in the posterior wall of the gastric body. Laparoscopic partial gastrectomy was performed via a single incision made in the epigastric region. The postoperative course was uneventful. The pathological diagnosis was a low-risk GIST. This method is easy and safe to perform; therefore, we consider it to be an important option for the treatment of intraluminal type gastric GIST.

[Clinical evaluation of the risk factors for liver abscess after TACE or RFA].

Radiofrequency ablation(RFA)and transcatheter arterial chemoembolization (TACE) are widely enforced as a standard combined therapy for liver cancer. Liver abscess occurs occasionally as a complication. This clinical study was conducted to determine risk factors for liver abscess. We investigated the clinical background of 10 cases complicated by liver abscess in 957 cases of patients who underwent TACE or RFA for liver cancer at Minoh City Hospital between April 2002 and March 2012. Risk factors for liver abscess were analyzed statistically in comparison to a control group without liver abscess. Diabetes and a history of biliary tract organic disease were statistically significant independent risk factors determined by multivariate analysis. We consider patients with a history of biliary tract organic disease, or who have a potential biliary tract infection, and diabetes, to be susceptible to infection. A case presenting with diabetes and a history of biliary tract disease is in a high-risk group, so treatment with TACE or RFA for such cases should be considered carefully.

[Recurrent gastric cancer treated with fourth-line chemotherapy consisting of capecitabine and cisplatin leading to partial response].

A 70-year-old man with advanced gastric cancer was treated with neoadjuvant chemotherapy consisting of S-1 plus cisplatin( CDDP). He exhibited symptoms of cerebral infarction during the second course of chemotherapy. Distal gastrectomy was performed and the histological diagnosis was pT3N3aM0, pStage IIIB. Adjuvant chemotherapy was administered; however, after the second course, gastric cancer recurred in the lymph nodes. Second-line chemotherapy with irinotecan (CPT-11) and CDDP was initiated. Thereafter, third-line chemotherapy with docetaxel was performed. However, the response to treatment was progressive disease (PD). Subsequently, fourth-line chemotherapy was performed with capecitabine and CDDP (XP chemotherapy). After the fourth course of XP chemotherapy, the response was partial response (PR). Moreover, PR was maintained after 20 courses of chemotherapy.

[Two cases of liver abscess caused by Clostridium perfringens after transcatheter arterial chemoembolization].

Case 1 involved a 74-year-old man. After transcatheter arterial chemoembolization( TACE) for hepatocellular carcinoma (HCC), abdominal computed tomography (CT) revealed a gas-containing lesion in the liver. The patient was diagnosed as having a gas-containing liver abscess, necessitating emergency drainage under laparotomy. Blood culture revealed Clostridium perfringens. He was discharged on day 63 after surgery. Case 2 involved a 70-year-old man who was admitted to our hospital for obstructive jaundice caused by HCC. He was treated with TACE after endoscopic retrograde biliary tract drainage (ERBD). On the second day, he was diagnosed as having a ruptured gas-containing liver abscess with massive hemolysis, necessitating emergency drainage under laparotomy. He died the next day after surgery. The clinical course of liver abscess caused by Clostridium perfringens can be fulminant and fatal with massive hemolysis.

[A case of superior sulcus tumor treated with carbon ion radiotherapy].

Superior sulcus tumor( SST) is a rare type of lung cancer. Treatment usually consists of surgical resection after chemoradiotherapy. We report a case of a woman in her fifties who underwent carbon ion radiotherapy for SST. The patient complained of left shoulder pain, and imaging studies revealed a 5.2×3.5-cm local solid tumor at the apex of the left lung, invasion to the ribs, and no lymph node swelling. The level of tumor marker, carcinoembryonic antigen (CEA), was 5.7 ng/mL. Needle biopsy specimen revealed adenocarcinoma. The diagnosis was SST, T3N0M0, stage IIB. We did not detect Horner syndrome. Carbon ion radiotherapy at 66 Gy equivalent dose per 10 fractions was administered to the SST site. Subsequently, the tumor size decreased to 4.5×1.9-cm. The adverse effect was Grade 1 skin and pulmonary toxicity. Six months later, Grade 2 left shoulder connective tissue toxicity was observed; it was difficult to differentiate this from tumor recurrence. After 2.5 years from radiotherapy, the patient is free from recurrence. Carbon ion radiotherapy is effective and safe and can be considered as an important treatment option for SST.

Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses.

Nonfibrillar assemblies of amyloid ß-protein (Aß) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aß labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aß assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ∼330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ∼128 kDa (∼32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aß(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aß assembly steps at which inhibition may be beneficial.

[Fluoropyrimidines with oxaliplatin(L-OHP) as an adjuvant chemotherapy for Stage III colon cancer].

As an adjuvant treatment for Dukes B2 and C colon cancer, adding oxaliplatin (L-OHP) to a regimen of fluorouracil and Leucovorin improved disease-free survival in Western countries. In Japan, however, adjuvant chemotherapy with L-OHP is not commonly used to treat Stage III colon cancer. We report the present condition of adjuvant treatment for colon cancer in our hospital. Between September 2009 and December 2011, 66 patients with Stage III colon cancer were enrolled after curative surgery. The details of adjuvant therapy with fluoropyrimidines with and without L-OHP were explained to the patients. After the explanation, 33.3% of the patients(IIIa: 18.9%, IIIb: 55.5%) selected L-OHP chemotherapy. Regardless of the side effects, adjuvant chemotherapy including L-OHP is expected to protect against cancer recurrence in patients with Stage IIIb colon cancer.

[A case of advanced gastric cancer with Trousseau syndrome and pulmonary embolism].

A 60-year-old man showed symptoms associated with pulmonary embolism and anemia in June 2011, and was subsequently diagnosed with stage IV gastric cancer. Following frequent multiple cerebral infarctions and the development of symptoms, the patient was diagnosed with Trousseau syndrome. A total gastrectomy was performed to control bleeding. After the surgery, oral ingestion became possible. The patient was discharged and a hypodermic injection of heparin was given by the home doctor.

[Chemotherapy-mediated tumor regression in a patient with stage IV stomach small cell cancer].

Gastrointestinal tract endoscopy revealed the presence of a 5-cm-diameter type 3 tumor in the cardiac portion of the stomach. The tumor was chromogranin positive, and stomach small cell cancer was diagnosed by immunostaining and biopsy pathology. S-1+CDDP therapy was administered as the first-line treatment. A switch to S-1 monotherapy was made after the patient experienced grade 4 hyponatremia. However, following 7 courses of therapy the disease had progressed. Second-line chemotherapy of CPT-11+CDDP was initiated and after 2 courses the disease stabilized.

High-throughput screening for agonists of ROS production in live human vascular endothelial cells.

Reactive oxygen species (ROS) are important physiological molecules, and identifying agonists for ROS production can yield useful tools for future research. Here we present an optimized protocol for high-throughput screening for agonists that induce ROS production. We describe the use of a fluorescent probe in human vascular endothelial cells, which can establish whether ROS production occurs in mitochondria or in the plasma membrane of live cells. For complete details on the use and execution of this profile, please refer to Sasahara et al. (2021).