Necrosensor: a genetically encoded fluorescent sensor for visualizing necrosis in Drosophila.

Historically, necrosis has been considered a passive process, which is induced by extreme stress or damage. However, recent findings of necroptosis, a programmed form of necrosis, shed a new light on necrosis. It has been challenging to detect necrosis reliably in vivo, partly due to the lack of genetically encoded sensors to detect necrosis. This is in stark contrast with the availability of many genetically encoded biosensors for apoptosis. Here we developed Necrosensor, a genetically encoded fluorescent sensor that detects necrosis in Drosophila, by utilizing HMGB1, which is released from the nucleus as a damage-associated molecular pattern (DAMP). We demonstrate that Necrosensor is able to detect necrosis induced by various stresses in multiple tissues in both live and fixed conditions. Necrosensor also detects physiological necrosis that occurs during spermatogenesis in the testis. Using Necrosensor, we discovered previously unidentified, physiological necrosis of hemocyte progenitors in the hematopoietic lymph gland of developing larvae. This work provides a new transgenic system that enables in vivo detection of necrosis in real time without any intervention.

Centrifugal Gel Crushing Tips for Gel-Based Proteome Analysis.

We have developed a centrifugal gel-crushing method using a pipet tip. Polyacrylamide gel slices are extruded from the narrowing cavity of a pipet tip by centrifugation in a few minutes to crush them into pieces of appropriate size. The size of the crushed gel could be controlled by several parameters, including centrifugal force and pipet tip cavity. In shotgun proteomics, gel-based LC/MS/MS, so-called GeLC/MS/MS, involves the essential but tedious processes of prefractionation by SDS-PAGE, followed by dicing the entire gel lane into several parts, fine dicing, and in-gel digestion after the diced gel is manually transferred to a microtube. In this study, we developed an alternative way to crush the prefractionated gel slice into optionally small and irregular-shaped gels by centrifugal extrusion of the sliced gel from the narrow cavity of a pipet tip. As a result, we observed improved recovery and reproducibility of digested proteins compared to the conventional method of manual dicing. We believe that this simple and rapid method of crushing polyacrylamide gels, which allows for parallel operations and automation, is useful for GeLC/MS/MS analysis and applicable to other approaches, including top-down proteomics.

Oncogenic stress-induced Netrin is a humoral signaling molecule that reprograms systemic metabolism in Drosophila.

Cancer exerts pleiotropic, systemic effects on organisms, leading to health deterioration and eventually to organismal death. How cancer induces systemic effects on remote organs and the organism itself still remains elusive. Here we describe a role for NetrinB (NetB), a protein with a particularly well-characterized role as a tissue-level axon guidance cue, in mediating oncogenic stress-induced organismal, metabolic reprogramming as a systemic humoral factor. In Drosophila, Ras-induced dysplastic cells upregulate and secrete NetB. Inhibition of either NetB from the transformed tissue or its receptor in the fat body suppresses oncogenic stress-induced organismal death. NetB from the dysplastic tissue remotely suppresses carnitine biosynthesis in the fat body, which is critical for acetyl-CoA generation and systemic metabolism. Supplementation of carnitine or acetyl-CoA ameliorates organismal health under oncogenic stress. This is the first identification, to our knowledge, of a role for the Netrin molecule, which has been studied extensively for its role within tissues, in humorally mediating systemic effects of local oncogenic stress on remote organs and organismal metabolism.

Extrapyramidal syndromes of chronic kidney disease and dialysis (diabetic uremic syndrome) with reversible parkinsonism and lentiform fork sign: A case report and literature review including metformin-induced encephalopathy.

Diabetic uremic syndrome has been rarely reported in patients on maintenance dialysis for diabetic nephropathy who present subacutely with neurological symptoms and bilateral basal ganglia lesions. There are also a few reports on metformin-induced encephalopathy, which is clinically similar to diabetic uremic syndrome. Because some patients with each of these diseases also have metabolic acidosis, it is speculated that these two diseases may have the same pathology. Recently, the term "extrapyramidal syndromes of chronic kidney disease and dialysis" (EPS-CKDD), with associated diagnostic criteria, has been proposed to describe these conditions, and metformin use is considered a risk factor for developing these syndromes. We report a case of a patient on maintenance hemodialysis for diabetic nephropathy who was taking metformin and developed subacute parkinsonism and bilateral basal ganglia lesions that rapidly improved after discontinuation of metformin with continued maintenance hemodialysis. We should ascertain whether patients with EPS-CKDD are taking metformin because it may be inappropriately prescribed for end-stage renal disease. If metformin has been prescribed, it should be discontinued immediately; its discontinuation may lead to rapid symptom recovery and improved prognosis.

Classification of Extracellular Vesicles Based on Surface Glycan Structures by Spongy-like Separation Media.

Extracellular vesicles (EVs) are lipid bilayer vesicles that enclose various biomolecules. EVs hold promise as sensitive biomarkers to detect and monitor various diseases. However, they have heterogeneous molecular compositions. The compositions of EVs from identical donor cells obtained using the same purification methods may differ, which is a significant obstacle for elucidating objective biological functions. Herein, the potential of a novel lectin-based affinity chromatography (LAC) method to classify EVs based on their glycan structures is demonstrated. The proposed method utilizes a spongy-like monolithic polymer (spongy monolith, SPM), which consists of poly(ethylene-co-glycidyl methacrylate) with continuous micropores and allows an efficient in situ protein reaction with epoxy groups. Two distinct lectins with different specificities, Sambucus sieboldiana agglutinin and concanavalin A, are effectively immobilized on SPM without impacting the binding activity. Moreover, high recovery rates of liposomal nanoparticles as a model of EVs are achieved due to the large flow-through pores (>10 µm) of SPM compared to a typical agarose gel. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling. This is the first approach to clarify the variation of protein contents in EVs by the difference of surface glycans via lectin immobilized media.

Erebosis, a new cell death mechanism during homeostatic turnover of gut enterocytes.

Many adult tissues are composed of differentiated cells and stem cells, each working in a coordinated manner to maintain tissue homeostasis during physiological cell turnover. Old differentiated cells are believed to typically die by apoptosis. Here, we discovered a previously uncharacterized, new phenomenon, which we name erebosis based on the ancient Greek word erebos ("complete darkness"), in the gut enterocytes of adult Drosophila. Cells that undergo erebosis lose cytoskeleton, cell adhesion, organelles and fluorescent proteins, but accumulate Angiotensin-converting enzyme (Ance). Their nuclei become flat and occasionally difficult to detect. Erebotic cells do not have characteristic features of apoptosis, necrosis, or autophagic cell death. Inhibition of apoptosis prevents neither the gut cell turnover nor erebosis. We hypothesize that erebosis is a cell death mechanism for the enterocyte flux to mediate tissue homeostasis in the gut.

Combining failure modes and effects analysis and cause-effect analysis: a novel method of risk analysis to reduce anaphylaxis due to contrast media.

BACKGROUND: Contrast media agents are essential for computed tomography (CT)-based diagnoses. However, they can cause fatal adverse effects such as anaphylaxis in patients. Although it is rare, the chances of anaphylaxis increase with the number of examinations. OBJECTIVE: We aimed to design a quality improvement initiative to reduce patient risk to contrast media agents. METHODS: We analysed CT processes using contrast iodine in a tertiary-care academic hospital that performs approximately 14 000 CT scans per year in Japan. We applied a combination of failure modes and effects analysis (FMEA) and cause-effect analysis to reduce the risk of patients developing allergic reactions to iodine-based contrast agents during CT imaging. RESULTS: Our multidisciplinary team comprising seven professionals analysed the data and designed a 56-process flowchart of CT imaging with iodine. We obtained 177 failure modes, of which 15 had a risk-probability number higher than 100. We identified the two riskiest processes and developed cause-and-effect diagrams for both: one was related to the exchange of information between the radiation and hospital information system regarding the patient's allergy, the other was due to education and structural deficiencies in observation following the exam. CONCLUSION: The combined method of FMEA and cause-and-effect analysis reveals high-risk processes and suggests measures to reduce these risks. FMEA is not well-known in healthcare but has significant potential for improving patient safety. Our findings emphasise the importance of adopting new techniques to reduce patient risk and carry out best practices in radiology.

Handling unstable analytes: literature review and expert panel survey by the Japan Bioanalysis Forum Discussion Group.

Analyzing unstable small molecule drugs and metabolites in blood continues to be challenging for bioanalysis. Although scientific countermeasures such as immediate cooling, immediate freezing, addition of enzyme inhibitors, pH adjustment, dried blood spot or derivatization have been developed, selecting the best practices has become an issue in the pharmaceutical industry as the number of drugs with such problems is increasing, even for generic drugs. In this study, we conducted a comprehensive literature review and a questionnaire survey to determine a suitable practice for evaluating instability and implementing countermeasures. Three areas of focus, matrix selection, effect of hemolysis and selection of esterase inhibitors, are discussed.

One-Step Isolation of Protein C-Terminal Peptides from V8 Protease-Digested Proteins by Metal Oxide-Based Ligand-Exchange Chromatography.

We have developed a one-step method to isolate protein C-terminal peptides from V8 protease-digested proteins by metal oxide-based ligand-exchange (MOLEX) chromatography. V8 protease cleaves the C-terminal side of Asp and Glu, affording a digested peptide with two carboxy groups at the C-terminus, whereas the protein C-terminal peptide has only one α-carboxy group. In MOLEX chromatography, a stable chelate is formed between dicarboxylates and metal atoms, so that the nonterminal (i.e., internal) peptide is retained, whereas the protein C-terminal peptide flows through the MOLEX column. After the optimization of the MOLEX chromatographic conditions, 1619 protein C-termini were identified from 30 µg of peptides (10 µg each, in triplicate) derived from human HeLa cells by means of nanoLC/MS/MS. When the MOLEX-isolated sample from 200 µg of HeLa peptides was further divided into six fractions by high-pH reversed-phase liquid chromatography (LC) prior to nanoLC/MS/MS, 2203 protein C-termini were identified with less than 3% contamination with internal peptides. We believe that this is the largest coverage with the highest purity reported to date in human protein C-terminomics. This fast, simple, sensitive, and selective method to isolate protein C-terminal peptides should be useful for profiling protein C-termini on a proteome-wide scale.

The Beneficial Effects of Astaxanthin on Glucose Metabolism and Modified Low-Density Lipoprotein in Healthy Volunteers and Subjects with Prediabetes.

Astaxanthin (ASTX) is an antioxidant agent. Recently, its use has been focused on the prevention of diabetes and atherosclerosis. We examined the effects of astaxanthin supplementation for 12 weeks on glucose metabolism, glycemic control, insulin sensitivity, lipid profiles and anthropometric indices in healthy volunteers including subjects with prediabetes with a randomized, placebo-controlled trial. METHODS: We enrolled 53 subjects who met our inclusion criteria and administered them with 12 mg astaxanthin or a placebo once daily for 12 weeks. Subsequently, their HbA1c levels, lipid profiles and biochemical parameters were determined. The participants also underwent a 75 g oral glucose tolerance test (OGTT), vascular endothelial function test and measurement of the visceral fat area. RESULTS: After astaxanthin supplementation for 12 weeks, glucose levels after 120 min in a 75 g OGTT significantly decreased compared to those before supplementation. Furthermore, the levels of HbA1c (5.64 ± 0.33 vs. 5.57 ± 0.39%, p < 0.05), apo E (4.43 ± 1.29 vs. 4.13 ± 1.24 mg/dL, p < 0.05) and malondialdehyde-modified low-density lipoprotein (87.3 ± 28.6 vs. 76.3 ± 24.6 U/L, p < 0.05) were also reduced, whereas total cholesterol (TC), triglyceride (TG) and high-density lipoprotein-C (HDL-C) levels were unaltered. The Matuda index, which is one of the parameters of insulin resistance, was improved in the ASTX group compared to that before supplementation. CONCLUSIONS: our results suggest that ASTX may have preventive effects against diabetes and atherosclerosis and may be a novel complementary treatment option for the prevention of diabetes in healthy volunteers, including subjects with prediabetes, without adverse effects.

Tribute to Dr. Takuo Aoyagi, inventor of pulse oximetry.

INTRODUCTION: Dr. Takuo Aoyagi invented pulse oximetry in 1974. Pulse oximeters are widely used worldwide, most recently making headlines during the COVID-19 pandemic. Dr. Aoyagi passed away on April 18, 2020, aware of the significance of his invention, but still actively searching for the theory that would take his invention to new heights. METHOD: Many people who knew Dr. Aoyagi, or knew of him and his invention, agreed to participate in this tribute to his work. The authors, from Japan and around the world, represent all aspects of the development of medical devices, including scientists and engineers, clinicians, academics, business people, and clinical practitioners. RESULTS: While the idea of pulse oximetry originated in Japan, device development lagged in Japan due to a lack of business, clinical, and academic interest. Awareness of the importance of anesthesia safety in the US, due to academic foresight and media attention, in combination with excellence in technological innovation, led to widespread use of pulse oximetry around the world. CONCLUSION: Dr. Aoyagi's final wish was to find a theory of pulse oximetry. We hope this tribute to him and his invention will inspire a new generation of scientists, clinicians, and related organizations to secure the foundation of the theory.

Combinatorial analysis of translation dynamics reveals eIF2 dependence of translation initiation at near-cognate codons.

Although ribosome-profiling and translation initiation sequencing (TI-seq) analyses have identified many noncanonical initiation codons, the precise detection of translation initiation sites (TISs) remains a challenge, mainly because of experimental artifacts of such analyses. Here, we describe a new method, TISCA (TIS detection by translation Complex Analysis), for the accurate identification of TISs. TISCA proved to be more reliable for TIS detection compared with existing tools, and it identified a substantial number of near-cognate codons in Kozak-like sequence contexts. Analysis of proteomics data revealed the presence of methionine at the NH2-terminus of most proteins derived from near-cognate initiation codons. Although eukaryotic initiation factor 2 (eIF2), eIF2A and eIF2D have previously been shown to contribute to translation initiation at near-cognate codons, we found that most noncanonical initiation events are most probably dependent on eIF2, consistent with the initial amino acid being methionine. Comprehensive identification of TISs by TISCA should facilitate characterization of the mechanism of noncanonical initiation.

Methionine restriction breaks obligatory coupling of cell proliferation and death by an oncogene Src in Drosophila.

Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. Here, we reveal that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. Our findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. We also exemplify a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism.

Evidence-based clinical practice guidelines for management of colorectal polyps.

BACKGROUND: The Japanese Society of Gastroenterology (JSGE) published ''Daicho Polyp Shinryo Guideline 2014'' in Japanese and a part of this guideline was published in English as "Evidence-based clinical practice guidelines for management of colorectal polyps" in the Journal of Gastroenterology in 2015. A revised version of the Japanese-language guideline was published in 2020, and here we introduce a part of the contents of revised version. METHODS: The guideline committee discussed and drew up a series of clinical questions (CQs). Recommendation statements for the CQs were limited to items with multiple therapeutic options. Items with established conclusions that had 100% agreement with previous guidelines (background questions) and items with no (or old) evidence that are topics for future research (future research questions: FRQs) were given descriptions only. To address the CQs and FRQs, PubMed, ICHUSHI, and other sources were searched for relevant articles published in English from 1983 to October 2018 and articles published in Japanese from 1983 to November 2018. The Japan Medical Library Association was also commissioned to search for relevant materials. Manual searches were performed for questions with insufficient online references. RESULTS: The professional committee created 18 CQs and statements concerning the current concept and diagnosis/treatment of various colorectal polyps, including their epidemiology, screening, pathophysiology, definition and classification, diagnosis, management, practical treatment, complications, and surveillance after treatment, and other colorectal lesions (submucosal tumors, nonneoplastic polyps, polyposis, hereditary tumors, ulcerative colitis-associated tumors/carcinomas). CONCLUSIONS: After evaluation by the moderators, evidence-based clinical practice guidelines for management of colorectal polyps were proposed for 2020. This report addresses the therapeutic related CQs introduced when formulating these guidelines.