Pib2 is a cysteine sensor involved in TORC1 activation in Saccharomyces cerevisiae.

Target of rapamycin complex 1 (TORC1) is a master regulator that monitors the availability of various amino acids to promote cell growth in Saccharomyces cerevisiae. It is activated via two distinct upstream pathways: the Gtr pathway, which corresponds to mammalian Rag, and the Pib2 pathway. This study shows that Ser3 was phosphorylated exclusively in a Pib2-dependent manner. Using Ser3 as an indicator of TORC1 activity, together with the established TORC1 substrate Sch9, we investigated which pathways were employed by individual amino acids. Different amino acids exhibited different dependencies on the Gtr and Pib2 pathways. Cysteine was most dependent on the Pib2 pathway and increased the interaction between TORC1 and Pib2 in vivo and in vitro. Moreover, cysteine directly bound to Pib2 via W632 and F635, two critical residues in the T(ail) motif that are necessary to activate TORC1. These results indicate that Pib2 functions as a sensor for cysteine in TORC1 regulation.

[Development and Evaluation about "2nd-wave" COVID-19 Vaccines].

To tackle the pandemic of the novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2), the international society, including Japan, has been actively promoting vaccination for SARS-CoV-2. To effectively utilize these vaccines, clinical trials have been conducted to evaluate their safety and efficacy. For efficacy evaluation, prevention rate of symptomatic novel coronavirus infections (corona virus disease 2019; COVID-19) between placebo groups and investigational vaccine groups has been the key parameter to evaluate the novel COVID-19 vaccines. This approach is based on a consensus among international regulatory authorities. Compared to several months ago, the public vaccination campaign for COVID-19 has substantially progressed in many countries. This makes it difficult to conduct clinical trials, which have placebo control arms, anywhere in the world because of ethical problems in administering a placebo during a pandemic. Therefore, the new international consensus among regulatory authorities is that immunogenicity bridging studies between the new COVID-19 vaccines that are being developed and approved COVID-19 vaccines may be needed when placebo-controlled studies are no longer feasible. In the future, the number of unvaccinated people worldwide is expected significantly decrease; thus, the issue of how to evaluate additional immunization for those who have completed the initial immunization remains to be addressed. This would require new international convergence. The development of COVID-19 vaccines and their evaluation would have to be updated, considering the social situation and vaccine coverage.

Endoscopic ultrasound-guided fine-needle biopsy for the diagnosis of gastric linitis plastica.

We report two cases of patients with gastric linitis plastica (GLP), in which the histopathological diagnosis was made by endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) using a Franseen-tip needle. Esophagogastroduodenoscopy findings showed mucosal swelling and poor distensibility of the gastric antrum. Abdominal computed tomography findings showed significant thickening of the gastric wall at the antrum. Conventional endoscopic and bite-on-bite biopsy were attempted but resulted in failure to diagnose the lesions. We performed EUS-FNB to obtain histopathological samples from a deeper site, which confirmed the diagnosis. We considered this method safe and effective for the diagnosis of GLP.

Prescription pattern analysis for antibiotics in working-age workers diagnosed with common cold.

Antimicrobial resistance is a major health concern. A primary cause is the inappropriate use of antimicrobials, particularly by patients with upper respiratory tract infection. However, baseline information for antibiotic use for common cold before being applied the National Action Plan on Antimicrobial Resistance in Japan is lacking. Here, we analyzed the inappropriate use of antibiotics in the working-age workers. We used large claims data from an annual health check-up for at least 5 consecutive years. Among 201,223 participants, we included 18,659 working-age workers who were diagnosed with common cold at a clinic/hospital. We calculated the proportion of patients with common cold who were prescribed antibiotics and analyzed predictive factors associated with antibiotics prescription. Antibiotics were prescribed to 49.2% (n = 9180) of patients diagnosed with common cold. In the logistic regression analysis, the group taking antibiotics was predominantly younger, male, without chronic diseases, and diagnosed at a small hospital/clinic (where the number of beds was 0-19). Cephems accounted for the highest proportion of prescribed antibiotics, with 40-45% of patients being prescribed antibiotics. Our data may be applied to prioritize resources such as medical staff-intervention or education of working-age people without chronic diseases who visit clinics for common cold to avoid the potential inappropriate use of antibiotics and prevent antimicrobial resistance acceleration.

Isoflurane induces Art2-Rsp5-dependent endocytosis of Bap2 in yeast.

Although general anesthesia is indispensable during modern surgical procedures, the mechanism by which inhalation anesthetics act on the synaptic membrane at the molecular and cellular level is largely unknown. In this study, we used yeast cells to examine the effect of isoflurane, an inhalation anesthetic, on membrane proteins. Bap2, an amino acid transporter localized on the plasma membrane, was endocytosed when yeast cells were treated with isoflurane. Depletion of RSP5, an E3 ligase, prevented this endocytosis and Bap2 was ubiquitinated in response to isoflurane, indicating an ubiquitin-dependent process. Screening all the Rsp5 binding adaptors showed that Art2 plays a central role in this process. These results suggest that isoflurane affects Bap2 via an Art2-Rsp5-dependent ubiquitination system.

Discrepant Diagnostic Results of Nested Polymerase Chain Reaction-based Genotyping in a Patient with Hepatitis C Virus and Human Immunodeficiency Virus Coinfection.

Accurate genotyping is important to improve the treatment of hepatitis C virus (HCV) infection. We herein report a 44-year-old Japanese man with hemophilia A and coinfection of HCV and human immunodeficiency virus (HIV) who was diagnosed with HCV genotype 4 by direct sequencing. Two genotyping tests based on the nested polymerase chain reaction method that we used misdiagnosed his genotype as 2b and 1b. Although several HCV genotyping tests are available in Japan, it is important to recognize that some cannot detect genotype 4. Care should be taken when genotyping HCV patients who have received non-heated coagulation factor preparations or were infected abroad.

Vacuolar protein Tag1 and Atg1-Atg13 regulate autophagy termination during persistent starvation in S. cerevisiae.

Under starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast Saccharomyces cerevisiae, autophagy is terminated after 10-12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δtag1 mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.

Diagnostic Fine-Needle Biopsy of Small Solid Pancreatic Lesions Using a Franseen Needle during Endoscopic Ultrasound Examination.

BACKGROUND AND AIM: During endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNB), Franseen needles can help collect sufficient tissue to permit histopathological assessment. However, its efficacy might be limited by the size of the targeted lesion. This study aimed to evaluate the feasibility of histopathological assessment of small solid pancreatic lesions using a 22-gauge Franseen needle during EUS-FNB. METHODS: This retrospective study evaluated data from all patients who underwent EUS-FNB using a Franseen needle for solid pancreatic lesions at the University of Toyama Hospital between June 2018 and April 2020. RESULTS: The study included 159 patients who had 152 malignant lesions and 7 benign lesions. The malignant lesions included pancreatic cancers (n = 134), neuroendocrine neoplasms (n = 15), metastatic tumors (n = 2), and a solid pseudopapillary neoplasm (n = 1). The diagnostic accuracy of EUS-FNB (combining histology and cytology) was 98.7%. However, the histopathological diagnosis was only confirmed for 64.3% of small lesions (<10 mm), relative to 97.2% for larger lesions. Multivariate analysis also revealed that lesion size of <10 mm predicted a less accurate histopathological diagnosis (odds ratio: 6.97, 95% confidence interval: 1.02-47.67; p = 0.041). Further analyses revealed a failed histological diagnosis in 4 patients with lesions of <5 mm in size and accurate diagnoses in 9 out of 10 patients with lesions of 5-10 mm in size. CONCLUSIONS: The diagnostic accuracy for small lesions (<10 mm), especially for lesions of <5 mm, based on histological examination alone, was significantly lower than that for others (>10 mm). Furthermore, multivariate analysis revealed that only lesion size was an independent predictor of histopathological diagnosis accuracy.

Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan.

Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca2+ dependent manner. Cytosolic Ca2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca2+ increase, thereby suppressing induction of autophagy.

Low-dose rectal diclofenac does not prevent post-ERCP pancreatitis in low- or high-risk patients.

BACKGROUND AND AIM: The most common adverse event following an endoscopic retrograde cholangiopancreatography (ERCP) procedure is post-ERCP pancreatitis (PEP). Rectal nonsteroidal anti-inflammatory drug (NSAID) administration has shown promise to reduce the risk of PEP in high-risk patients. However, in contrast to high-risk patients, the role of NSAID administration in patients with low risk remains controversial. METHODS: We performed a prospective, single-center, single-blinded, two-arm parallel group, randomized controlled trial to clarify the efficacy of low dose (50 mg) rectal NSAID administration for preventing PEP in at-risk patients. Patients scheduled to undergo ERCP were randomized into two groups, those with and without rectal administration of diclofenac. Patients in the diclofenac group received 50 mg of rectal diclofenac 30 min before undergoing ERCP. The primary endpoint was rate of PEP. RESULTS: A total of 303 were randomized into the study groups. Four patients declined participation following randomization, and another two were withdrawn. As a result, a total of 147 patients were assigned to the diclofenac group and 150 to the control group. The baseline and procedural characteristics were similar in both groups. The primary endpoint of PEP occurrence was seen in 13 of 297 patients (4.4%), including eight (5.4%) in the diclofenac group and five (3.3%) in the control group (P = 0.286). Additionally, those results were not significantly different when patients were classified as low or high risk. CONCLUSIONS: Prophylactic low-dose rectal diclofenac did not reduce the incidence of PEP following ERCP in patients classified as low or high risk.

Gtr/Ego-independent TORC1 activation is achieved through a glutamine-sensitive interaction with Pib2 on the vacuolar membrane.

TORC1 is a central regulator of cell growth in response to amino acids. The role of the evolutionarily conserved Gtr/Rag pathway in the regulation of TORC1 is well-established. Recent genetic studies suggest that an additional regulatory pathway, depending on the activity of Pib2, plays a role in TORC1 activation independently of the Gtr/Rag pathway. However, the interplay between the Pib2 pathway and the Gtr/Rag pathway remains unclear. In this study, we show that Pib2 and Gtr/Ego form distinct complexes with TORC1 in a mutually exclusive manner, implying dedicated functional relationships between TORC1 and Pib2 or Gtr/Rag in response to specific amino acids. Furthermore, simultaneous depletion of Pib2 and the Gtr/Ego system abolishes TORC1 activity and completely compromises the vacuolar localization of TORC1. Thus, the amino acid-dependent activation of TORC1 is achieved through the Pib2 and Gtr/Ego pathways alone. Finally, we show that glutamine induces a dose-dependent increase in Pib2-TORC1 complex formation, and that glutamine binds directly to the Pib2 complex. These data provide strong preliminary evidence for Pib2 functioning as a putative glutamine sensor in the regulation of TORC1.

Phenanthrene Synthesis by Palladium-Catalyzed Benzannulation with o-Bromobenzyl Alcohols through Multiple Carbon-Carbon Bond Formations.

A palladium-catalyzed benzannulation with o-bromobenzyl alcohols enabled the facile construction of phenanthrene skeletons via the sequential multiple carbon-carbon bond formations. A variety of multisubstituted phenanthrenes were synthesized by the reaction of (Z)-ß-halostyrenes with o-bromobenzyl alcohols as well as by the three-component coupling of alkynes, aryl bromides, and o-bromobenzyl alcohols. The electron-deficient phosphine ligand played an important role to control the sequential oxidative addition of two different organic halides employed, which realized the selective formation of the desired phenanthrenes in good yields. This synthetic protocol was also applicable to the synthesis of the highly fused polycyclic aromatic hydrocarbons such as tetraphenes.

Atg9A trafficking through the recycling endosomes is required for autophagosome formation.

Autophagy is an intracellular degradation pathway conserved in eukaryotes. Among core autophagy-related (Atg) proteins, mammalian Atg9A is the sole multi-spanning transmembrane protein, and both of its N- and C-terminal domains are exposed to the cytoplasm. It is known that Atg9A travels through the trans-Golgi network (TGN) and the endosomal system under nutrient-rich conditions, and transiently localizes to the autophagosome upon autophagy induction. However, the significance of Atg9A trafficking for autophagosome formation remains elusive. Here, we identified sorting motifs in the N-terminal cytosolic stretch of Atg9A that interact with the adaptor protein AP-2. Atg9A with mutations in the sorting motifs could not execute autophagy and was abnormally accumulated at the recycling endosomes. The combination of defects in autophagy and Atg9A accumulation in the recycling endosomes was also found upon the knockdown of TRAPPC8, a specific subunit of the TRAPPIII complex. These results show directly that the trafficking of Atg9A through the recycling endosomes is an essential step for autophagosome formation.

Synthesis of Multisubstituted Triphenylenes and Phenanthrenes by Cascade Reaction of o-Iodobiphenyls or (Z)-ß-Halostyrenes with o-Bromobenzyl Alcohols through Two Sequential C-C Bond Formations Catalyzed by a Palladium Complex.

o-Bromobenzyl alcohol has been developed as a novel annulating reagent, bearing both nucleophilic and electrophilic substituents, for the facile synthesis of polycyclic aromatic hydrocarbons. A palladium/electron-deficient phosphine catalyst efficiently coupled o-iodobiphenyls or (Z)-ß-halostyrenes with o-bromobenzyl alcohols to afford triphenylenes and phenanthrenes, respectively. The present cascade reaction proceeded through deacetonative cross-coupling and sequential intramolecular cyclization. An array of experimental data suggest that the reaction mechanism involves the equilibrium of 1,4-palladium migration.

Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity.

Autophagy is a conserved eukaryotic process of protein and organelle self-degradation within the vacuole/lysosome. Autophagy is characterized by the formation of an autophagosome, for which Vps34-dervied phosphatidylinositol 3-phosphate (PI3P) is essential. In yeast, Vps34 forms two distinct protein complexes: complex I, which functions in autophagy, and complex II, which is involved in protein sorting to the vacuole. Here we identify and characterize Atg38 as a stably associated subunit of complex I. In atg38Δ cells, autophagic activity was significantly reduced and PI3-kinase complex I dissociated into the Vps15-Vps34 and Atg14-Vps30 subcomplexes. We find that Atg38 physically interacted with Atg14 and Vps34 via its N terminus. Further biochemical analyses revealed that Atg38 homodimerizes through its C terminus and that this homodimer formation is indispensable for the integrity of complex I. These data suggest that the homodimer of Atg38 functions as a physical linkage between the Vps15-Vps34 and Atg14-Vps30 subcomplexes to facilitate complex I formation.

The CENP-A chaperone Scm3 becomes enriched at kinetochores in anaphase independently of CENP-A incorporation.

Centromeres are specialized chromatin domains where kinetochores assemble. Centromeres contain as a conserved feature nucleosomes that are composed of the canonical histones H2A, H2B and H4 and a centromere-specific histone H3 variant, known as CENP-A in humans and Cse4 in budding yeast. The incorporation of CENP-A homologues into centromeric chromatin is cell cycle regulated and is assisted by related assembly factors named Scm3 in yeast and HJURP in human cells. Here we describe that the budding yeast Scm3 binds weakly to centromeres during interphase including S phase when Cse4 assembles into centromeres. In anaphase Scm3 then becomes 2.5-fold enriched at kinetochores where it is dynamic with a half recovery time t(1/2) of 36 s. In contrast, Cse4 is stably integrated into kinetochores. In addition, ten Scm3 molecules bind to a cluster of 16 kinetochores with 32 Cse4 molecules suggesting a 1:3 ratio at kinetochores between the two proteins. Analysis of conditional lethal scm3-1 mutant cells indicated that Scm3 participates in maintaining Cse4 at centromeres in anaphase. Thus, Scm3 interacts transiently with kinetochores in anaphase where it safeguards Cse4 even after its S phase incorporation into centromeres.

N-terminal regions of Mps1 kinase determine functional bifurcation.

Mps1 is a conserved kinase that in budding yeast functions in duplication of the spindle pole body (SPB), spindle checkpoint activation, and kinetochore biorientation. The identity of Mps1 targets and the subdomains that convey specificity remain largely unexplored. Using a novel combination of systematic deletion analysis and chemical biology, we identified two regions within the N terminus of Mps1 that are essential for either SPB duplication or kinetochore biorientation. Suppression analysis of the MPS1 mutants defective in SPB duplication and biochemical enrichment of Mps1 identified the essential SPB components Spc29 and the yeast centrin Cdc31 as Mps1 targets in SPB duplication. Our data suggest that phosphorylation of Spc29 by Mps1 in G1/S recruits the Mps2-Bbp1 complex to the newly formed SPB to facilitate its insertion into the nuclear envelope. Mps1 phosphorylation of Cdc31 at the conserved T110 residue controls substrate binding to Kar1 protein. These findings explain the multiple SPB duplication defects of mps1 mutants on a molecular level.

Upf1 potentially serves as a RING-related E3 ubiquitin ligase via its association with Upf3 in yeast.

Three Upf proteins are essential to the nonsense-mediated mRNA decay (NMD) pathway. Although these proteins assemble on polysomes for recognition of aberrant mRNAs containing premature termination codons, the significance of this assembly remains to be elucidated. The Cys- and His-rich repeated N terminus (CH domain) of Upf1 has been implicated in its binding to Upf2. Here, we show that CH domain also plays a RING-related role for Upf1 to exhibit E3 ubiquitin ligase activity in yeast. Despite the sequence divergence from typical E3-RING fingers, the CH domain of yeast Upf1 specifically and directly interacted with the yeast E2 Ubc3. Interestingly, Upf1 served as a substrate for the in vitro self-ubiquitination, and the modification required its association with Upf3 rather than Upf2. Substitution of the coordinated Cys and His residues in the CH domain impaired not only self-ubiquitination of Upf1 but also rapid decay of aberrant mRNAs. These results suggest that Upf1 may serve as an E3 ubiquitin ligase upon its association with Upf3 and play an important role in signaling to the NMD pathway.

Caf1 regulates translocation of ribonucleotide reductase by releasing nucleoplasmic Spd1-Suc22 assembly.

Appropriate supply of deoxyribonucleotides by the ribonucleotide reductase (RNR) complex is essential for DNA replication and repair. One recent model for the RNR activation in Schizosaccharomyces pombe is translocation of the regulatory subunit Suc22 from the nucleoplasm to the cytoplasm. The RNR inhibitory protein Spd1, which retains Suc22 in the nucleoplasm, is rapidly degraded upon DNA-replication stress, resulting in release of Suc22 to form the active RNR complex in the cytoplasm. Here, we show that Caf1, a component of the Ccr4-Not complex, is responsible for resistance of the replication stress and control of the Suc22 translocation. Caf1 is required not only for the stress-induced translocation of Suc22 from nucleoplasm to cytoplasm but also for the degradation of nucleoplasmic Spd1. DNA-replication stress appears to allow Caf1 to interact with Suc22, resulting in release of the nucleoplasmic Spd1-Suc22 assembly. Taken together, these results suggest a novel function of Caf1 as a key regulator in the stress-induced RNR activation.

The decapping enzyme Dcp1 participates in translation termination through its interaction with the release factor eRF3 in budding yeast.

One of the rate-limiting steps in messenger RNA decay pathway is the 5'-cap cleavage of mRNAs, decapping reaction, which is conducted by the protein complex of Dcp1 and Dcp2. We find here that Dcp1p can interact with the release factor eRF3p (Sup35p) in Saccharomyces cerevisiae. Knockout of DCP1 caused not only the accumulation of nonsense mRNAs possibly due to the impaired decapping activity but also the enhancement of the read-through of nonsense codon. To examine the relationship between the two DCP1-knockout phenotypes, we produced DCP1 point mutants that lack the ability to support the translation termination. Interestingly, decapping activity of Dcp1p was still intact, but its interaction with eRF3p was abolished in the DCP1 mutants, indicating that the two functions originated from different entities of Dcp1p. These results suggest that the decapping enzyme Dcp1p may have an additional role in the translation termination through its interaction with eRF3p.