Liquidity Is a Critical Determinant for Selective Autophagy of Protein Condensates.

Clearance of biomolecular condensates by selective autophagy is thought to play a crucial role in cellular homeostasis. However, the mechanism underlying selective autophagy of condensates and whether liquidity determines a condensate's susceptibility to degradation by autophagy remain unknown. Here, we show that the selective autophagic cargo aminopeptidase I (Ape1) undergoes phase separation to form semi-liquid droplets. The Ape1-specific receptor protein Atg19 localizes to the surface of Ape1 droplets both in vitro and in vivo, with the "floatability" of Atg19 preventing its penetration into droplets. In vitro reconstitution experiments reveal that Atg19 and lipidated Atg8 are necessary and sufficient for selective sequestration of Ape1 droplets by membranes. This sequestration is impaired by mutational solidification of Ape1 droplets or diminished ability of Atg19 to float. Taken together, we propose that cargo liquidity and the presence of sufficient amounts of autophagic receptor on cargo are crucial for selective autophagy of biomolecular condensates.

Global study of holistic morphological effectors in the budding yeast Saccharomyces cerevisiae.

BACKGROUND: The size of the phenotypic effect of a gene has been thoroughly investigated in terms of fitness and specific morphological traits in the budding yeast Saccharomyces cerevisiae, but little is known about gross morphological abnormalities. RESULTS: We identified 1126 holistic morphological effectors that cause severe gross morphological abnormality when deleted, and 2241 specific morphological effectors with weak holistic effects but distinctive effects on yeast morphology. Holistic effectors fell into many gene function categories and acted as network hubs, affecting a large number of morphological traits, interacting with a large number of genes, and facilitating high protein expression. Holistic morphological abnormality was useful for estimating the importance of a gene to morphology. The contribution of gene importance to fitness and morphology could be used to efficiently classify genes into functional groups. CONCLUSION: Holistic morphological abnormality can be used as a reproducible and reliable gene feature for high-dimensional morphological phenotyping. It can be used in many functional genomic applications.

[Introduction of PBPM and Its Effects for a Home Care Support Clinic].

Medical teams have been promoted in home care. It is possible for pharmacists who are part of a multidisciplinary team to maintain safety and improve the quality of medical care. Protocol-based pharmacotherapy management(PBPM)is recommended for cooperation between the pharmacist and the doctor in the management of pharmacotherapy. In order to introduce PBPM, it is necessary for the pharmacist and the doctor to cooperate and to extract the problems in community medicine. In this study, the clinic pharmacist examined the problem of unnecessary inquiries and proposed PBPM. He suggested that to smoothly introduce PBPM, a protocol creation committee should be set up and an explanation of PBPM should be provided to the Community Pharmacist Association. As a pilot study, we created 5 protocols at Doctor GON Kamakura Clinic with the cooperation of 8 pharmacies. As a result, it became possible to reduce unnecessary inquiries by 46%. Careful coordination is necessary in order to introduce PBPM at clinics and community pharmacies. Moreover, a clinic pharmacist is able to facilitate the introduction of PBPM in the role of coordinator.

Few-Layer Graphene Kills Selectively Tumor Cells from Myelomonocytic Leukemia Patients.

In the cure of cancer, a major cause of today's mortality, chemotherapy is the most common treatment, though serious frequent challenges are encountered by current anticancer drugs. We discovered that few-layer graphene (FLG) dispersions have a specific killer action on monocytes, showing neither toxic nor activation effects on other immune cells. We confirmed the therapeutic application of graphene on an aggressive type of cancer that is myelomonocytic leukemia, where the monocytes are in their malignant form. We demonstrated that graphene has the unique ability to target and boost specifically the necrosis of monocytic cancer cells. Moreover, the comparison between FLG and a common chemotherapeutic drug, etoposide, confirmed the higher specificity and toxicity of FLG. Since current chemotherapy treatments of leukemia still cause serious problems, these findings open the way to new and safer therapeutic approaches.

Visualization of Atg3 during autophagosome formation in Saccharomyces cerevisiae.

Macroautophagy (autophagy) is a highly conserved cellular recycling process involved in degradation of eukaryotic cellular components. During autophagy, macromolecules and organelles are sequestered into the double-membrane autophagosome and degraded in the vacuole/lysosome. Autophagy-related 8 (Atg8), a core Atg protein essential for autophagosome formation, is a marker of several autophagic structures: the pre-autophagosomal structure (PAS), isolation membrane (IM), and autophagosome. Atg8 is conjugated to phosphatidylethanolamine (PE) through a ubiquitin-like conjugation system to yield Atg8-PE; this reaction is called Atg8 lipidation. Although the mechanisms of Atg8 lipidation have been well studied in vitro, the cellular locale of Atg8 lipidation remains enigmatic. Atg3 is an E2-like enzyme that catalyzes the conjugation reaction between Atg8 and PE. Therefore, we hypothesized that the localization of Atg3 would provide insights about the site of the lipidation reaction. To explore this idea, we constructed functional GFP-tagged Atg3 (Atg3-GFP) by inserting the GFP portion immediately after the handle region of Atg3. During autophagy, Atg3-GFP transiently formed a single dot per cell on the vacuolar membrane. This Atg3-GFP dot colocalized with 2× mCherry-tagged Atg8, demonstrating that Atg3 is localized to autophagic structures. Furthermore, we found that Atg3-GFP is localized to the IM by fine-localization analysis. The localization of Atg3 suggests that Atg3 plays an important role in autophagosome formation at the IM.

[Hepatocyte nuclear factor 1α-inactivated hepatocellular adenomatosis in a patient with maturity-onset diabetes of the young type 3: case report and literature review].

A 19-year-old Japanese woman had been diagnosed with diabetes at the age of 9 years. She had a strong family history of diabetes, and genetic screening showed she had maturity-onset diabetes of the young type 3 (MODY3). Ultrasonography of the liver and magnetic resonance imaging showed multiple nodules consistent with hepatocellular adenoma (HA). Biopsy of the liver tumors revealed hepatocyte nuclear factor (HNF) 1α-inactivated HA. HA is known as a MODY3-related disease due to mutations in HNF1α. We present the first report of HA associated with MODY3 in Japan.

Near-infrared light-boosted antimicrobial activity of minocycline/hyaluronan/carbon nanohorn composite toward peri-implantitis treatments.

Dental implant therapy is a reliable treatment for replacing missing teeth. However, as dental implants become more widely used, peri-implantitis increasingly has become a severe complication, making successful treatment more difficult. As a result, the development of effective drug delivery systems (DDSs) and treatments for peri-implantitis are urgently needed. Carbon nanohorns (CNHs) are carbon nanomaterials that have shown promise for use in DDSs and have photothermal effects. The present study exploited the unique properties of CNHs to develop a phototherapy employing a near-infrared (NIR) photoresponsive composite of minocycline, hyaluronan, and CNH (MC/HA/CNH) for peri-implantitis treatments. MC/HA/CNH demonstrated antibacterial effects that were potentiated by NIR-light irradiation, a property that was mediated by photothermal-mediated drug release from HA/CNH. These antibacterial effects persisted even following 48 h of dialysis, a promising indication for the clinical use of this material. We propose that the treatment of peri-implantitis using NIR and MC/HA/CNH, in combination with surgical procedures, might be employed to target relatively deep affected areas in a timely and efficacious manner. We envision that this innovative approach will pave the way for future developments in implant therapy.

Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation.

Multi-walled carbon nanotubes (MWCNTs) were functionalized with fibroblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGF-CNT) showed the same effect as FGF alone. In addition, FGF-CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF-CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGF-CNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications.

Liquid droplet formation and cytoplasm to vacuole targeting of aminopeptidase I are temperature sensitive in Saccharomyces cerevisiae.

Aminopeptidase I (Ape1) is one of the major cargoes of the cytoplasm-to-vacuole targeting (Cvt) pathway, which is a kind of selective autophagy, in Saccharomyces cerevisiae. After synthesis, the Ape1 precursor (prApe1) undergoes phase separation to form liquid droplets, termed Ape1 droplets, in the cytoplasm. In this study, we found that cells expressing prApe1-GFP exhibited temperature-sensitive formation of Ape1 droplets, which affected its transport. Moreover, we showed that endogenous Ape1 transport was defective at high temperatures in various laboratory strains due to the defect in the formation of Ape1 droplets at these temperatures. Finally, we found that gene disruptants showing heat-tolerant growth suppressed the temperature sensitivity of the Ape1 transport. The formation of Ape1 droplets might be an indicator of cytoplasmic integrity at high temperature.

Umbilical Complications That Require Surgical Intervention after Gynecologic Laparoscopic Surgery.

Background: In laparoscopic surgery, the trocar is often inserted through the umbilicus because of the ease of insertion and inconspicuous postoperative scar formation. However, postoperative complications that require plastic surgical intervention may occur to the umbilicus. Methods: We reviewed 14 patients who received plastic surgery for umbilical issues following gynecologic laparoscopic surgery in our department from January 2015 to September 2021. Results: Most complications requiring umbilical surgery post gynecologic laparoscopic surgery include local infections, scar contractures, ectopic endometriosis, and umbilical necrosis. Mass resection and umbilical formation procedures were performed under general or local anesthesia. After a follow-up period of 6 months following surgery, no incidences of tumor development or recurrence of infection were seen, and the hypertrophic scar at the wound site gradually healed after the complete removal of the tumor and adequate suturing. Pathologically, 90% of the cases with keloid-like collagen disorder had concomitant inflammatory diseases such as epidermal cysts and abscesses. Conclusions: The majority of umbilical complications associated with laparoscopic surgery were predicted to be due to implantation of epithelial and tumor components during laparoscopic surgery and delayed postoperative inflammation. Therefore, it is necessary to educate surgeons about general measures of local infection control and careful surgical manipulation to prevent umbilical problems associated with laparoscopic surgery.

An antibacterial conjugate of carbon nanohorns for NIR-light mediated peri-implantitis treatment.

This study developed a novel antibacterial conjugate based on carbon nanohorns for peri-implantisis, an inflammatory disease around dental implants, which may result in failing implants by bone loss around them. The conjugate demonstrates much better photodurability than commonly used indocyanine green and a significant antibacterial effect under NIR illumination.

Single-Walled Carbon Nanotube Membranes Accelerate Active Osteogenesis in Bone Defects: Potential of Guided Bone Regeneration Membranes.

Carbon nanotubes (CNTs) are potentially important biomaterials because of their chemical, physical, and biological properties. Our research indicates that CNTs exhibit high compatibility with bone tissue. The guided bone regeneration (GBR) technique is commonly applied to reconstruct alveolar bone and treat peri-implant bone defects. In GBR, bone defects are covered with a barrier membrane to prevent the entry of nonosteogenic cells such as epithelial cells and fibroblasts. The barrier membrane also maintains a space for new bone formation. However, the mechanical and biological properties of materials previously used in clinical practice sometimes delayed bone regeneration. In this study, we developed a CNT-based membrane for GBR exhibiting high strength to provide a space for bone formation and provide cellular shielding to induce osteogenesis. The CNT membrane was made via the dispersion of single-walled CNTs (SWCNTs) in hyaluronic acid solution followed by filtration. The CNT membrane assumed a nanostructure surface due to the bundled SWCNTs and exhibited high strength and hydrophilicity after oxidation. In addition, the membrane promoted the proliferation of osteoblasts but not nonosteogenic cells. CNT membranes were used to cover experimental bone defects made in rat calvaria. At 8 weeks after surgery, more extensive bone formation was observed in membrane-covered defects compared with bone defects not covered with membrane. Almost no diffusion of CNTs was observed around the membrane. These results indicate that the CNT membrane has adequate strength, stability, and surface characteristics for osteoblasts, and its shielding properties promote bone formation. Demonstration of the safety and osteogenic potential of the CNT membranes through further animal studies should facilitate their clinical application in GBR.

An effective in vivo mitochondria-targeting nanocarrier combined with a π-extended porphyrin-type photosensitizer.

A photochemical reaction mediated by light-activated molecules (photosensitizers) in photodynamic therapy (PDT) causes molecular oxygen to be converted into highly reactive oxygen species (ROS) that are beneficial for cancer therapy. As the active oxygen consumer and the primary regulator of apoptosis, mitochondria are known as an important target for optimizing PDT outcomes. However, most of the clinically used photosensitizers exhibited a poor tumor accumulation profile as well as lack of mitochondria targeting ability. Therefore, by applying a nanocarrier platform, mitochondria-specific delivery of photosensitizers can be materialized. The present research develops an effective mitochondria-targeting liposome-based nanocarrier system (MITO-Porter) encapsulating a π-extended porphyrin-type photosensitizer (rTPA), which results in a significant in vivo antitumor activity. A single PDT treatment of the rTPA-MITO-Porter resulted in a dramatic tumor inhibition against both human and murine tumors that had been xenografted in a mouse model. Furthermore, depolarization of the mitochondrial membrane was observed, implying the damage of the mitochondrial membrane due to the photochemical reaction that occurred specifically in the mitochondria of tumor cells. The findings presented herein serve to verify the significance of the mitochondria-targeted nanocarrier system for advancing the in vivo PDT effectivity in cancer therapy regardless of tumor type.

Atg15 in Saccharomyces cerevisiae consists of two functionally distinct domains.

Autophagy is a cellular degradation system widely conserved among eukaryotes. During autophagy, cytoplasmic materials fated for degradation are compartmentalized in double membrane-bound organelles called autophagosomes. After fusing with the vacuole, their inner membrane-bound structures are released into the vacuolar lumen to become autophagic bodies and eventually degraded by vacuolar hydrolases. Atg15 is a lipase that is essential for disintegration of autophagic body membranes and has a transmembrane domain at the N-terminus and a lipase domain at the C-terminus. However, the roles of the two domains in vivo are not well understood. In this study, we found that the N-terminal domain alone can travel to the vacuole via the multivesicular body pathway, and that targeting of the C-terminal lipase domain to the vacuole is required for degradation of autophagic bodies. Moreover, we found that the C-terminal domain could disintegrate autophagic bodies when it was transported to the vacuole via the Pho8 pathway instead of the multivesicular body pathway. Finally, we identified H435 as one of the residues composing the putative catalytic triad and W466 as an important residue for degradation of autophagic bodies. This study may provide a clue to how the C-terminal lipase domain recognizes autophagic bodies to degrade them.

Carbon nanohorn coating by electrodeposition accelerate bone formation on titanium implant.

Direct contact between bone and implant materials is required for dental implants. Titanium is used for the implant material owing to its mechanical and biological properties. The anodisation as the surface treatment was employed to enhance osteogenesis around titanium. Moreover, carbon nanohorn (CNH), a type of nanometer-sized carbon material, was reported to promote the bone formation. Thus, it is expected that if the surface of anodised Ti (AnTi) is modified with CNHs, Ti-bone contact would be enhanced. In this study, the Ti surface was modified with CNHs by electrophoresis and obtained anodised titanium coated with CNHs (CNH/AnTi). In vitro, CNH/AnTi attracted osteoblastic cells more than AnTi, thereby the proliferation of osteoblastic cell was enhanced by CNH/AnTi more than by AnTi. In vivo, at 7 and 28 days after implantation of CNH/AnTi or AnTi into the rat femur, more aggressive bone formation was observed on the surface of CNH/AnTi than on AnTi. More importantly, the area where newly formed bone tissue directly attached to CNH/AnTi was significantly larger than that for AnTi, suggesting that "contact osteogenesis" was accelerated on CNH/AnTi during the early post-implantation period. CNH/AnTi would be advantageous especially for the early stages of bone regeneration after surgery.

Transmission electron microscopic observation of cells cultured on multiwalled carbon nanotube-coated sponges.

The cell structure and interface between cultured cells and a multiwalled carbon nanotube (MWCNT)-coated sponge (MWCNT-coated sponge) were observed by transmission electron microscopy (TEM). Moreover, the atomic structure of MWCNTs that entered the cells was also examined by means of high-resolution TEM (HRTEM). MWCNTs were observed in the cytoplasm, and a few MWCNTs were recognized in the cell nuclei. Those MWCNTs maintained their structure there. Subcellular organelles did not appear to be different from those on the collagen sponge despite the cellular uptake of MWCNTs.

Chemotherapy-induced Nasopharyngeal Stenosis Treated with Bilateral Facial Artery Musculomucosal Flaps.

We report the first case of a 50-year-old woman who developed nasopharyngeal stenosis (NPS) after chemotherapy for malignant lymphoma. The chemotherapy was effective, but NPS developed following treatment. The tumors of the pharynx and soft palate became necrotic and turned into scar tissue, which caused NPS, especially in the caudal part of the soft palate. The patient developed nasal obstruction and obstructive sleep apnea due to the stenosis. The patient underwent 2 surgeries to resolve the NPS: the first was a simple incision of the stenosis, and the second was Z-plasty and mucous membrane transplantation from the posterior pharyngeal wall. However, the NPS recurred soon after these 2 surgeries. We used bilateral inferior-based facial artery musculomucosal (FAMM) flaps as a solution for recurrent NPS, and it was effective in preventing further stenosis. The blood supply to the flaps was stable, and the size of the flaps was enough to compensate for the area of tissue deficit. The use of bilateral FAMM flaps allowed both sides of the NPS to be corrected, and the flaps provided sufficient retracting strength to keep expanding the nasopharyngeal space by pulling from both sides. After the operation, nasal obstruction was decreased, and the sleep quality of the patient improved significantly. The velopharyngeal function was maintained, and there was no symptom of nasopharyngeal insufficiency. Our results suggest that the bilateral FAMM flap is a suitable method to rescue intractable cases of NPS.

Atg2 mediates direct lipid transfer between membranes for autophagosome formation.

A key event in autophagy is autophagosome formation, whereby the newly synthesized isolation membrane (IM) expands to form a complete autophagosome using endomembrane-derived lipids. Atg2 physically links the edge of the expanding IM with the endoplasmic reticulum (ER), a role that is essential for autophagosome formation. However, the molecular function of Atg2 during ER-IM contact remains unclear, as does the mechanism of lipid delivery to the IM. Here we show that the conserved amino-terminal region of Schizosaccharomyces pombe Atg2 includes a lipid-transfer-protein-like hydrophobic cavity that accommodates phospholipid acyl chains. Atg2 bridges highly curved liposomes, thereby facilitating efficient phospholipid transfer in vitro, a function that is inhibited by mutations that impair autophagosome formation in vivo. These results suggest that Atg2 acts as a lipid-transfer protein that supplies phospholipids for autophagosome formation.

Atg4 plays an important role in efficient expansion of autophagic isolation membranes by cleaving lipidated Atg8 in Saccharomyces cerevisiae.

Autophagy, an intracellular degradation system, is highly conserved among eukaryotes from yeast to mammalian cells. In the yeast Saccharomyces cerevisiae, most Atg (autophagy-related) proteins, which are essential for autophagosome formation, are recruited to a restricted region close to the vacuole, termed the vacuole-isolation membrane contact site (VICS), upon induction of autophagy. Subsequently, the isolation membrane (IM) expands and sequesters cytoplasmic materials to become a closed autophagosome. In S. cerevisiae, the ubiquitin-like protein Atg8 is C-terminally conjugated to the phospholipid phosphatidylethanolamine (PE) to generate Atg8-PE. During autophagosome formation, Atg8-PE is cleaved by Atg4 to release delipidated Atg8 (Atg8G116) and PE. Although delipidation of Atg8-PE is important for autophagosome formation, it remains controversial whether the delipidation reaction is required for targeting of Atg8 to the VICS or for subsequent IM expansion. We used an IM visualization technique to clearly demonstrate that delipidation of Atg8-PE is dispensable for targeting of Atg8 to the VICS, but required for IM expansion. Moreover, by overexpressing Atg8G116, we showed that the delipidation reaction of Atg8-PE by Atg4 plays an important role in efficient expansion of the IM other than supplying unlipidated Atg8G116. Finally, we suggested the existence of biological membranes at the Atg8-labeled structures in Atg8-PE delipidation-defective cells, but not at those in atg2Δ cells. Taken together, it is likely that Atg2 is involved in localization of biological membranes to the VICS, where Atg4 is responsible for IM expansion.