α- and ß-tubulin C-terminal tails with distinct modifications are crucial for ciliary motility and assembly.

α- and ß-tubulin have an unstructured glutamate-rich region at their C-terminal tails (CTTs). The function of this region in cilia and flagella is still unclear, except that glutamates in CTTs act as the sites for post-translational modifications that affect ciliary motility. The unicellular alga Chlamydomonas possesses only two α-tubulin and two ß-tubulin genes, each pair encoding an identical protein. This simple gene organization might enable a complete replacement of the wild-type tubulin with its mutated version. Here, using CRISPR/Cas9, we generated mutant strains expressing tubulins with modified CTTs. We found that the mutant strain in which four glutamate residues in the α-tubulin CTT had been replaced by alanine almost completely lacked polyglutamylated tubulin and displayed paralyzed cilia. In contrast, the mutant strain lacking the glutamate-rich region of the ß-tubulin CTT assembled short cilia without the central apparatus. This phenotype is similar to mutant strains harboring a mutation in a subunit of katanin, the function of which has been shown to depend on the ß-tubulin CTT. Therefore, our study reveals distinct and important roles of α- and ß-tubulin CTTs in the formation and function of cilia.

Gap junction beta-4 accelerates cell cycle progression and metastasis through MET-AKT activation in pancreatic cancer.

Despite continuing advances in the development of effective new therapies, including immunotherapies, the prognosis of pancreatic cancer remains extremely poor. Gap junction proteins have become attractive targets for potential cancer therapy. However, the role of gap junction beta-4 (GJB4) protein remains unexplored in pancreatic cancer. Through bioinformatic analyses we discovered pancreatic cancer tissues showed higher levels of GJB4 transcripts compared to normal pancreatic tissues and this had a negative effect on overall survival in patients that had pancreatic cancer. The high expression of nuclear GJB4 was identified as a negative prognostic factor in such patients. Knockdown of GJB4 in cultured pancreatic cancer cells resulted in G0/G1 arrest followed by decreased cell proliferation and suppression of metastatic potential. The overexpression of GJB4 accelerated cell proliferation, migration, and invasion in a SUIT-2 cell line, whereas MET inhibitor canceled the acceleration. GJB4 suppression with siRNA significantly inhibited tumor growth in a mouse xenograft model. Mechanistically, suppression of GJB4 inhibited MET-AKT activities. Such data suggest that targeting the GJB4-MET axis could represent a promising new therapeutic strategy for pancreatic cancer.

Clinical characterization of patients with gBRCA1/2 mutation-positive unresectable pancreatic cancer: a multicenter prospective study.

BACKGROUND: Accumulating evidence has demonstrated platinum-based chemotherapy followed by maintenance therapy with a poly Adenosine diphosphate (ADP)-ribose polymerase inhibitor (olaparib) show benefits in unresectable pancreatic cancer with a germline (g)BRCA1/2 mutation. Evaluation of the germline BRCA1 and BRCA2 mutation is essential for making decisions on a treatment strategy for patients with unresectable pancreatic cancer. However, the detection rates of germline BRCA1 and BRCA2 mutations and efficacy of maintenance with olaparib remain undetermined, prospectively, in Japan. METHODS & RESULTS: In this prospective analysis, the rate of germline BRCA1 and BRCA2 mutations and efficacy of chemotherapy were analyzed in 136 patients with pancreatic cancer who underwent BRACAnalysis® (85 patients) or FoundationOne® CDx (51 patients) between January 2020 and July 2022. A total of six patients (4.4%) had a germline BRCA1 and BRCA2 mutation. Five patients were treated with modified FOLFIRINOX and one with fluorouracil and oxaliplatin. All patients continued platinum-based chemotherapy for ˃4 months and were subsequently treated with olaparib as a maintenance therapy. The response rate to platinum-based chemotherapy in the germline BRCA1 and BRCA2 mutation-positive group was significantly better than that of the germline BRCA1 and BRCA2 mutation-negative group (66% vs 23%, P = 0.04). All patients harbouring a germline BRCA1 and BRCA2 mutation were able to switch to olaparib. The median progression-free survival using olaparib was 5.7 months (range 3.0-9.2). CONCLUSIONS: The rate of germline BRCA1 and BRCA2 mutations found in patients with unresectable pancreatic cancer was comparable to those of previous studies.An analysis of germline BRCA1 and BRCA2 mutations has benefits for all patients with unresectable pancreatic cancer with regard to decisions on therapeutic strategies in a clinical practice setting.

Chlamydomonas FAP70 is a component of the previously uncharacterized ciliary central apparatus projection C2a.

Cilia are essential organelles required for cell signaling and motility. Nearly all motile cilia have a '9+2' axoneme composed of nine outer doublet microtubules plus two central microtubules; the central microtubules together with their projections are termed the central apparatus (CA). In Chlamydomonas reinhardtii, a model organism for studying cilia, 30 proteins are known CA components, and ∼36 more are predicted to be CA proteins. Among the candidate CA proteins is the highly conserved FAP70 (CFAP70 in humans), which also has been reported to be associated with the doublet microtubules. Here, we determined by super-resolution structured illumination microscopy that FAP70 is located exclusively in the CA, and show by cryo-electron microscopy that its N-terminus is located at the base of the C2a projection of the CA. We also found that fap70-1 mutant axonemes lack most of the C2a projection. Mass spectrometry revealed that fap70-1 axonemes lack not only FAP70 but two other conserved candidate CA proteins, FAP65 (CFAP65 in humans) and FAP147 (MYCBPAP in humans). Finally, FAP65 and FAP147 co-immunoprecipitated with HA-tagged FAP70. Taken together, these data identify FAP70, FAP65 and FAP147 as the first defining components of the C2a projection.

Effect of Oligomers Derived from Biodegradable Polyesters on Eco- and Neurotoxicity.

Biodegradable polymers are eco-friendly materials and have attracted attention for use in a sustainable society because they are not accumulated in the environment. Although the characteristics of biodegradable polymers have been assessed well, the effects of their degradation products have not. Herein, we comprehensively evaluated the chemical toxicities of biodegradable polyester, polycaprolactone (PCL), and synthetic oligocaprolactones (OCLs) with different degrees of polymerization. While the PCL did not show any adverse effects on various organisms, high levels of shorter OCLs and the monomer (1 µg/mL for freshwater microorganisms and 1 mg/mL for marine algae and mammalian cells) damaged the tested organisms, including freshwater microorganisms, marine algae, and mammalian cells, which indicated the toxicities of the degradation products under unnaturally high concentrations. These results highlight the need for a further understanding of the effects of the degradation products resulting from biodegradable polyesters to ensure a genuinely sustainable society.

Addition-Fragmentation Ring-Opening Polymerization of Bio-Based Thiocarbonyl l-Lactide for Dual Degradable Vinyl Copolymers.

This study is designed to synthesize novel degradable polymers by radical addition-fragmentation ring-opening copolymerization of bio-based thiocarbonyl compounds with various vinyl monomers. Thiocarbonyl l-lactide is capable of radical copolymerization with acrylates and styrene via radical addition to the carbon-sulfur double bonds followed by ring-opening as well as controlled copolymerization in conjunction with the reversible addition-fragmentation chain transfer (RAFT) process. The obtained polymers possess ring-opened thioester and ring-retained thioacetal functionalities in the backbone, both of which could be cleaved under appropriate conditions with different chemical stimuli.

Diffusion rather than intraflagellar transport likely provides most of the tubulin required for axonemal assembly in Chlamydomonas.

Tubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using Chlamydomonas, we attenuated IFT-based tubulin transport of GFP-ß-tubulin by altering the IFT74N-IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP-ß-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by ∼90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP-ß-tubulin in the axoneme was only moderately reduced. In vivo imaging showed more GFP-ß-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth.

[A case of intraductal tubulopapillary adenocarcinoma with bone metastases].

A woman in her 70s with main pancreatic duct dilatation was referred to our hospital. Various imaging examinations showed an extensive mass within the lumen of the main pancreatic duct in the head and body of the pancreas. The microscopic examination of a biopsy specimen revealed an adenocarcinoma. She was diagnosed with intraductal tubulopapillary adenocarcinoma of the pancreas;a pylorus-preserving total pancreatectomy was subsequently performed. However, 30 days after surgery, the patient presented with neck pain and left upper arm numbness. Results of magnetic resonance imaging and bone scintigraphy revealed a cervical spinal tumor that was subsequently biopsied. The patient was diagnosed with intraductal tubulopapillary adenocarcinoma with bone metastasis.

Synthesis of Multifunctional Homopolymers through Using Thiazolidine Chemistry and Post-Polymerization Modification.

Multifunctional homopolymers, defined here as polymers that contain multiple reactive functional groups per repeat unit, are versatile scaffolds for preparing complex macromolecules via post-polymerization modification. However, there are limited methods for preparing multifunctional homopolymers that contain more than one nucleophilic site per repeat unit. Herein, a strategy to synthesize a multifunctional homopolymer using thiazolidine chemistry is demonstrated. Controlled radical polymerization of a thiazolidine-containing acrylamido monomer allows for the synthesis of a polymer with pendent latent nucleophiles. Ring-opening of the thiazolidine affords a homopolymer with two side-chain reactive sites, an amine and a thiol. One-pot functionalization via disulfide formation and acyl substitution is performed to introduce two distinct groups in each repeat unit.

Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin.

The assembly and maintenance of most cilia and flagella rely on intraflagellar transport (IFT). Recent in vitro studies have suggested that, together, the calponin-homology domain within the IFT81 N-terminus and the highly basic N-terminus of IFT74 form a module for IFT of tubulin. By using Chlamydomonas mutants for IFT81 and IFT74, we tested this hypothesis in vivo. Modification of the predicted tubulin-binding residues in IFT81 did not significantly affect basic anterograde IFT and length of steady-state flagella but slowed down flagellar regeneration, a phenotype similar to that seen in a strain that lacks the IFT74 N-terminus. In both mutants, the frequency of tubulin transport by IFT was greatly reduced. A double mutant that combined the modifications to IFT81 and IFT74 was able to form only very short flagella. These results indicate that, together, the IFT81 and IFT74 N-termini are crucial for flagellar assembly, and are likely to function as the main module for IFT of tubulin.

Hepatitis C virus reactivation due to antiemetic steroid therapy during treatment of hepatocellular carcinoma.

Hepatitis C virus (HCV) reactivation is relatively rare compared with hepatitis B reactivation in patients treated with immunosuppressive or anticancer drugs. Here, we present the first case of genotype 2 HCV reactivation due to antiemetic steroid therapy during chemotherapy for hepatocellular carcinoma (HCC), which was verified by not only increased viral load but also pathological exacerbation of liver injury during HCV reactivation. Further chemotherapy for HCC could be continued without steroid therapy. This present case highlights the awareness of HCV reactivation and the management of complex situation.

Introducing "Ynene" Metathesis: Ring-Expansion Metathesis Polymerization Leads to Highly Cis and Syndiotactic Cyclic Polymers of Norbornene.

Tungsten alkylidynes [CF3-ONO]W≡CC(CH3)3(THF)2 (1) and [(t)BuOCO]W≡CC(CH3)3(THF)2 (3) react with ethylene. Complex 1 reacts reversibly with ethylene to give the metallacyclobutene (2). Complex 3 reacts with ethylene to form the tethered alkylidene (4) featuring a tetraanionic pincer ligand. Complexes 1 and 3 initiate the polymerization of norbornene at room temperature. The polymerization of norbornene by 1 is not stereoselective, whereas 3 generates a highly cis and syndiotactic cyclic polynorbornene. Comparison of the intrinsic viscosity, radius of gyration, and elution time of the synthesized cyclic polynorbornene with those of linear analogues provides conclusive evidence for a cyclic topology.

Highly Tactic Cyclic Polynorbornene: Stereoselective Ring Expansion Metathesis Polymerization of Norbornene Catalyzed by a New Tethered Tungsten-Alkylidene Catalyst.

The tungsten alkylidyne [(t)BuOCO]W≡C((t)Bu) (THF)2 (1) reacts with CO2, leading to complete cleavage of one C═O bond, followed by migratory insertion to generate the tungsten-oxo alkylidene 2. Complex 2 is the first catalyst to polymerize norbornene via ring expansion metathesis polymerization to yield highly cis-syndiotactic cyclic polynorbornene.

Increased duodenal iron absorption through up-regulation of divalent metal transporter 1 from enhancement of iron regulatory protein 1 activity in patients with nonalcoholic steatohepatitis.

UNLABELLED: Increased hepatic iron accumulation is thought to be involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). Hepatic iron accumulation, as well as oxidative DNA damage, is significantly increased in NASH livers. However, the precise mechanism of iron accumulation in the NASH liver remains unclear. In this study, 40 cases with a diagnosis of NASH (n = 25) or simple steatosis (SS; n = 15) by liver biopsy were enrolled. An oral iron absorption test (OIAT) was used, in which 100 mg of sodium ferrous citrate was administered to each individual. The OIAT showed that absorption of iron from the gastrointestinal (GI) tract was increased significantly in NASH patients, compared to SS and control subjects. Iron reduction therapy was effective in patients with NASH, who exhibited iron deposition in the liver and no alanine aminotransferase improvement after other therapies (n = 9). Serum hepcidin concentration and messenger RNA (mRNA) levels of divalent metal transporter 1 (DMT1) also were significantly elevated in patients with NASH. OIAT results were correlated with grade of liver iron accumulation and DMT1 mRNA levels. Then, we demonstrated that DMT1 mRNA levels increased significantly in Caco-2/TC7 cell monolayers cultured in transwells with serum from NASH patients. An electrophoresis mobility shift assay showed activation of iron regulatory protein (IRP) in those cells, and IRP1 small interfering RNA clearly inhibited the increase of DMT1 mRNA levels. CONCLUSION: In spite of elevation of serum hepcidin, iron absorption from the GI tract increased through up-regulation of DMT1 by IRP1 activation by humoral factor(s) in sera of patients with NASH.

BAK is a predictive and prognostic biomarker for the therapeutic effect of docetaxel treatment in patients with advanced gastric cancer.

BACKGROUND: Preoperative chemotherapy is a promising strategy for downstaging advanced gastric cancer before radical resection, although severe adverse events can occur and clinical outcomes are often unsatisfactory. To identify predictive biomarkers of drug sensitivity, we used a well-designed functional apoptosis assay and assessed the correlations between chemosensitivity and clinical outcomes. METHODS: Drug sensitivity to docetaxel, cisplatin, and 5-fluorouracil was examined in 11 gastric cancer cell lines. BCL2-homology domain 3 (BH3) profiling was performed and assessed for correlations with drug sensitivity. Immunohistochemical staining of clinical gastric cancer specimens was performed before preoperative chemotherapy, and correlations with histopathological responses and clinical outcomes were assessed. RESULTS: BIM (BCL2L11)-BH3 profiling results correlated with docetaxel sensitivity and BAK protein expression, whose knockdown caused docetaxel resistance. The BAK expression indexes of 69 gastric cancer specimens before preoperative chemotherapy (including docetaxel treatment) were determined by multiplying numerical values describing the degrees of BAK positivity and staining intensity observed. Patients whose specimens showed good chemotherapeutic histopathological responses had higher BAK indexes than those with poor responses. Patients with BAK index values ≥3 showed improved progression-free survival (HR, 2.664; 95 % CI, 1.352-5.248; P = 0.005) and overall survival (HR, 3.390; 95 % CI, 1.549-7.422; P = 0.002). CONCLUSIONS: BH3 profiling clearly showed that BIM expression, which depends on BAK expression, correlated with docetaxel sensitivity. BAK expression in gastric cancer is thus predictive of chemotherapeutic responses to docetaxel and clinical prognosis in patients treated with preoperative chemotherapy.

Tubulin glycylation controls ciliary motility through modulation of outer-arm dyneins.

Tubulins undergo several kinds of posttranslational modifications (PTMs) including glutamylation and glycylation. The contribution of these PTMs to the motilities of cilia and flagella is still unclear. Here, we investigated the role of tubulin glycylation by examining a novel Chlamydomonas mutant lacking TTLL3, an enzyme responsible for initiating glycylation. Immunostaining of cells and flagella revealed that glycylation is only restricted to the axonemal tubulin composing the outer-doublet but not the central-pair microtubules. Furthermore, the flagellar localization of TTLL3 was found to be dependent on intraflagellar transport. The mutant, ttll3(ex5), completely lacks glycylation and consequently exhibits slower swimming velocity compared with the wild-type strain. By combining the ttll3(ex5) mutation with multiple axonemal dynein-deficient mutants, we found that the lack of glycylation does not affect the motility of the outer-arm dynein lacking mutations. Sliding disintegration assay using isolated axonemes revealed that the lack of glycylation decreases microtubule sliding velocity in the normal axoneme but not in the axoneme lacking the outerarm dyneins. Based on our recent study that glycylation occurs exclusively on ß-tubulin in Chlamydomonas, these findings suggest that tubulin glycylation controls flagellar motility through modulating outer-arm dyneins, presumably by neutralizing the negative charges of glutamate residues at the C-terminus region of ß-tubulin.

Pulmonary Metastasectomy after Radiofrequency Ablation of Hepatocellular Carcinoma.

Introduction: Single distant metastases after radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC) are rare. There are no guidelines for treating patients without liver tumors after resecting lung metastases. Case Presentation: Here, we report a patient with HCC recurring as a single lung metastasis 14 months after RFA. A 76-year-old woman with primary biliary cholangitis without hepatitis B virus or hepatitis C virus infection had been treated by RFA for a single 16-mm-sized HCC lesion in liver S8. Fourteen months thereafter, despite lack of intrahepatic recurrence, a single new 26-mm-sized mass was found in S10 of the right lung. The patient underwent right lower lobectomy. The histopathological diagnosis was HCC metastasis. Because no residual disease could be found, she was followed up without any additional treatment after surgery. She remains alive with no signs of recurrence 3 years later. Conclusion: HCC patients who relapse with lung metastases but without intrahepatic recurrence after RFA are extremely rare, especially when RFA is used to treat HCC lesions <30 mm. However, it should be noted that, although rare, HCC may recur in the form of extrahepatic metastases after RFA. Furthermore, it is suggested that, as in the presently-described case, at least some patients without intrahepatic recurrence whose lung metastases are completely resected have a good prognosis even without additional treatment for HCC.

Advancements in minimal residual disease detection: a practical approach using single-cell droplet PCR for comprehensive monitoring in hematological malignancy.

The identification of chromosomal abnormalities accompanied by copy number alterations is important for understanding tumor characteristics. Testing methodologies for copy number abnormality have limited sensitivity, resulting in their use only for the sample provided at the time of diagnosis or recurrence of malignancy, but not for the monitoring of minimal residual disease (MRD) during and after therapy. We developped the "DimShift" technology which enable to measure the copy number of target gene/chromosome in each cell, which is given by the single cell droplet PCR. Qualitative result of DimShift given by peripheral blood was perfectly concordant with that of bone marrow. These findings and performances are promising to be the new methodology for MRD detection in malignant diseases utilizing bone marrow as well as peripheral blood.

Germline BRCA1-Mutated Synchronous and Metachronous Pancreatic Acinar Cell Carcinoma With Long-Term Survival.

BACKGROUND: Pancreatic acinar cell carcinoma (PACC) is a rare pancreatic neoplasm. Recently, molecular analysis revealed that PACC shows a high frequency of the BRCA1/2 mutation and is likely to be considered a cancer associated with hereditary breast and ovarian cancer (HBOC). Hereditary cancers, including HBOC, are characterized by multifocal and/or metachronous tumors. However, no case reports exist of germline BRCA1-mutated synchronous and metachronous PACC. CASE: A 58-year-old man was diagnosed with synchronous and metachronous PACC at the age of 56 and underwent two surgeries. Ten months after the second surgery, the patient developed multiple liver metastases. Gemcitabine plus nab-paclitaxel therapy was administered as first-line chemotherapy. After seven cycles, computed tomography examination revealed progressive disease (PD). Therefore, modified FOLFIRINOX (mFFX) was administered as second- line chemotherapy. After 19 cycles of mFFX, comprehensive cancer genomic profiling (CGP) identified a BRCA1 pathogenic variant that was confirmed to be germline origin. Accordingly, we treated the patient with olaparib; however, he was diagnosed with PD after 4 months. He subsequently died 5 years and 9 months after the initial surgery, and 3 years and 10 months after chemotherapy. Based on the genetic data of the patients, his family members received genetic counseling followed by cascade testing. Consequently, the same gBRCA1 pathogenic variant was detected in the son and his surveillance for HBOC-related cancers was initiated. CONCLUSION: We diagnosed a 58-year-old man with a synchronous and metachronous PACC with germline BRCA1 pathogenic variant. Considering that PACC is likely to have BRCA1/2 mutations responsible for HBOC, we need to be aware of the possible presence of multifocal and/or metachronous tumors in patients with PACC. Additionally, patients with PACC should undergo genetic examinations, which would be beneficial in determining treatment strategies and health care for blood relatives.

Marked Response to Nivolumab by a Patient With SMARCA4-Deficient Undifferentiated Urothelial Carcinoma Showing High PD-L1 Expression: A Case Report.

BACKGROUND: SMARCA4 is a component gene of the SWI/SNF (SWItch/Sucrose NonFermentable) chromatin remodeling complex; undifferentiated tumors associated with its functional deletion have been described in several organs. However, no established treatment for these tumors currently exists. CASE: In this study, we report a case of a SMARCA4-deficient undifferentiated urothelial carcinoma with high PD-L1 expression that was effectively treated with nivolumab after early relapse following treatment for non-invasive bladder cancer. The histological morphology of the rhabdoid-like undifferentiated tumor of unknown primary led us to suspect a SWI/SNF-deficient tumor, and subsequent immunostaining led to the diagnosis of a SMARCA4-deficient undifferentiated tumor. This effort also led to the identification of the developmental origin of this SMARCA4-deficient undifferentiated tumor as a non-invasive bladder cancer. We also carried out a detailed immune phenotypic assay on peripheral T cells. In brief, a phenotypic change of CD8+T cells from naive to terminally differentiated effector memory cells was observed. CONCLUSION: Regardless of the organ of cancer origin or cancer type, SWI/SNF-deficient tumors should be suspected in undifferentiated and dedifferentiated tumors, and immune checkpoint inhibitors may be considered as a promising treatment option for this type of tumor. The pathogenesis of SMARCA4-deficient anaplastic tumors awaits further elucidation for therapeutic development.