Characterization of antimutagenic mechanism of 3-allyl-5-substituted 2-thiohydantoins against 2-amino-3-methylimidazo[4,5-f]quinoline.

3-Allyl-5-substituted 2-thiohydantoins (ATH-amino acids) derived from allyl isothiocyanate and amino acids can inhibit the mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the Salmonella assay. In this report, we studied possible mechanisms for the inhibition using rat liver S9 in assays for ethoxyresorufin O-deethylase (EROD), a marker activity for cytochrome P450 1A (CYP1A), which activates heterocyclic amines, and the Salmonella assays with the direct-acting mutagen 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline (N-hydroxy-IQ). Quantitative analysis of ATH-amino acids and IQ during incubation with rat liver S9 fraction by HPLC showed that ATH-amino acids could act as S9-inhibitors, thereby inhibiting metabolic activation of IQ. Among the tested ATH-amino acids, ATH-Phe, ATH-Trp, ATH-Leu and ATH-Val showed a dose-dependent inhibition of EROD activity. ATH-Gly, ATH-Glu, and ATH-Asp behaved as blocking agents toward N-hydroxy-IQ, but exhibited no inhibition of EROD activity.     

Proton beam therapy for hepatocellular carcinoma: a retrospective review of 162 patients.

PURPOSE: We present results of patients with hepatocellular carcinoma (HCC) treated with proton beam therapy. EXPERIMENTAL DESIGN: We reviewed 162 patients having 192 HCCs treated from November 1985 to July 1998 by proton beam therapy with or without transarterial embolization and percutaneous ethanol injection. The patients in the present series were considered unsuitable for surgery for various reasons, including hepatic dysfunction, multiple tumors, recurrence after surgical resection, and concomitant illnesses. The median total dose of proton irradiation was 72 Gy in 16 fractions over 29 days. RESULTS: The overall survival rate for all of the 162 patients was 23.5% at 5 years. The local control rate at 5 years was 86.9% for all 192 tumors among the 162 patients. The degree of impairment of hepatic functions attributable to coexisting liver cirrhosis and the number of tumors in the liver significantly affected patient survival. For 50 patients having least impaired hepatic functions and a solitary tumor, the survival rate at 5 years was 53.5%. The patients had very few acute reactions to treatments and a few late sequelae during and after the treatments. CONCLUSIONS: Proton beam therapy for patients with HCC is effective, safe, well tolerable, and repeatable. It is the useful treatment mode for either cure or palliation for patients with HCC irrespective of tumor size, tumor location in the liver, insufficient feeding of the tumor with arteries, presence of vascular invasion, impaired hepatic functions, and coexisting intercurrent diseases.     

Improved trends in survival and engraftment after single cord blood transplantation for adult acute myeloid leukemia

Unrelated cord blood transplantation (CBT) is an alternative curative option for adult patients with acute myeloid leukemia (AML) who need allogeneic hematopoietic cell transplantation (HCT) but lack an HLA-matched related or unrelated donor. However, large-scale data are lacking on CBT outcomes for unselected adult AML. To investigate the trends of survival and engraftment after CBT over the past 22 years, we retrospectively evaluated the data of patients with AML in Japan according to the time period of CBT (1998–2007 vs 2008–2013 vs 2014–2019). A total of 5504 patients who received single-unit CBT as first allogeneic HCT for AML were included. Overall survival (OS) at 2 years significantly improved over time. The improved OS among patients in ≥ complete remission (CR)3 and active disease at CBT was mainly due to a reduction of relapse-related mortality, whereas among patients in first or second CR at CBT, this was due mainly to a reduction of non-relapse mortality. The trends of neutrophil engraftment also improved over time. This experience demonstrated that the survival and engraftment rate after CBT for this group has improved over the past 22 years.Subject terms: Acute myeloid leukaemia, Cancer immunotherapy     

Simple smoke ventilation method for uniportal video-assisted thoracoscopic surgery

Fogging of the thoracoscopic lens affects a surgeon’s ability to maintain a clear operating field. In uniportal video-assisted thoracoscopic surgery, the thoracoscopic lens tends to fog when the surgeon does not hold a suction instrument. Thus, a suction instrument needs to be held by the surgeon’s nondominant hand to remove surgical smoke, mist, and moisture. Here, we describe a simple, easy and cost-effective surgical smoke ventilation technique for uniportal video-assisted thoracoscopic surgery using a suction catheter to solve the problem. We present this technique and comment on its advantages, including decreased cost and improved surgical visualization.     

Phase IIb study of pembrolizumab combined with S‐1 + oxaliplatin or S‐1 + cisplatin as first‐line chemotherapy for gastric cancer

The KEYNOTE‐659 study evaluated the efficacy and safety of first‐line pembrolizumab plus S‐1 and oxaliplatin (SOX) (cohort 1) or S‐1 and cisplatin (SP) (cohort 2) for advanced gastric/gastroesophageal junction (G/GEJ) cancer in Japan. Herein, we update the results of cohort 1 and describe the results of cohort 2. This open‐label phase IIb study enrolled patients with advanced programmed death‐ligand 1 (PD‐L1)‐positive (combined positive score ≥ 1) human epidermal growth factor receptor 2 (HER2)‐negative G/GEJ adenocarcinoma. The primary end‐point was the objective response rate (ORR). Other end‐points were duration of response (DOR), disease control rate (DCR), progression‐free survival (PFS), overall survival (OS), and safety. One hundred patients were enrolled. In cohorts 1 and 2, median follow‐up time was 16.9 and 17.1 months; ORR (central review), 72.2% and 80.4%; DOR, 10.6 and 9.5 months; DCR (central review), 96.3% and 97.8%; median PFS (central review), 9.4 and 8.3 months; and median OS, 16.9 and 17.1 months, respectively. Treatment‐related adverse events (TRAEs) occurred in all patients, including peripheral sensory neuropathy (94.4%, cohort 1), decreased neutrophil count (82.6%, cohort 2), nausea (59.3% and 60.9% in cohorts 1 and 2), and decreased appetite (61.1% and 60.9% in cohorts 1 and 2). Grade 3 or higher TRAEs were reported by 59.3% (cohort 1) and 78.3% (cohort 2), including decreased platelet count (14.8%, cohort 1) and decreased neutrophil count (52.2%, cohort 2). Pembrolizumab in combination with SOX or SP showed favorable efficacy and safety in patients with PD‐L1‐positive, HER2‐negative G/GEJ adenocarcinoma.     

Japanese trial for classification of gestational trophoblastic disease

OBJECTIVE: To evaluate the clinical usefulness of the Japanese Diagnostic Score to differentiate choriocarcinoma clinically without histologic findings from persistent gestational trophoblastic disease (GTD). STUDY DESIGN: We reviewed the clinical records and histologic reports on all 809 patients with persistent GTD treated with surgery and chemotherapy in Japan. There were 347 cases of choriocarcinoma and 462 cases of invasive mole with histologic confirmation. We retrospectively applied the Japanese Diagnostic Score to all patients for detection of choriocarcinoma in persistent trophoblastic disease. RESULTS: The sensitivity of the score for choriocarcinoma was 92.2%. The specificity was 93.5%. This retrospective study showed that the accuracy of this scoring system to differentiate true malignant choriocarcinoma clinically from both low risk and high risk gestational trophoblastic neoplasia without histologic findings was 92.9%. CONCLUSION: Our trial to differentiate choriocarcinoma clinically from persistent GTD without histologic findings using a unique scoring system was successful. Proper management in the early stages strongly influences the outcome of these diseases. This scoring system should be very useful in comparing the incidence and survival rate of choriocarcinoma between nations.     

Inadequate tolerance induction may induce pre-eclampsia

The fetus is semi-allograft to the maternal host; therefore, a system of tolerance must be present during pregnancy. Epidemiological findings support a relationship between pre-eclampsia and the failure of tolerance induction. For induction of major histocompatibility complex (MHC) class I-specific tolerance, long-term exposure to seminal fluid, which contains paternal soluble MHC class I antigens, may induce paternal MHC class I-specific tolerance. Furthermore, soluble HLA-G1, which induces the deletion of CD8(+) T-cells, and the combination of maternal killer-immunoglobulin-like receptors (KIR) on NK cells and fetal HLA-C, which affects the balance between inhibition and activation signals of NK cells, regulatory CD8(+) T cells, and regulatory NK cells, may play very important roles in the induction of MHC class I-specific tolerance. On the other hand, exposure to sperm, which express paternal HLA-DR, and trophoblastic debris, which contain intracellular fetal HLA-DR, may induce paternal MHC class II-specific tolerance. In this process, CD4(+)CD25(+) regulatory T (Treg)-cells play central roles. In this review, we discuss the relationship between the risk of pre-eclampsia and tolerance induction.     

A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the ∼200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and crosslinking through a Grb2-Grb2 binding interface. The Grb2 Y160 residue plays a structurally critical role in the Grb2-Grb2 interaction, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that the condensation state of the EGFR tail controls the ability of SOS, recruited via Grb2, to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

Recently, a class of phenomena known as protein condensation phase transitions has begun to emerge in biology. Originally identified in the context of nuclear organization (1) and gene expression (2), a distinct two-dimensional protein condensation on the cell membrane has now been discovered in the T cell receptor (TCR) signaling system involving the scaffold protein LAT (3–5). TCR activation results in phosphorylation of LAT on at least four distinct tyrosine sites, which subsequently recruit the adaptor protein Grb2 and the signaling molecule PLCγ via selective binding interactions with their SH2 domains. Additional scaffold and signaling molecules, including SOS, GADS, and SLP76, are recruited to Grb2 and PLCγ through further specific protein–protein interactions (6, 7). Multivalency among some of these binding interactions can crosslink LAT molecules in a two-dimensional bond percolation network on the membrane surface. The resulting LAT protein condensate resembles the nephrin:NCK:N-WASP condensate (8) in that both form on the membrane surface under control of tyrosine phosphorylation and exert at least one aspect of functional control over signaling output via a distinct type of kinetic regulatory mechanism (9–11). The basic molecular features controlling the LAT and nephrin protein condensates are common among biological signaling machinery, and other similar condensates continue to be discovered (12, 13). The LAT condensation shares downstream signaling molecules with the EGF-receptor (EGFR) signaling system, raising the question if EGFR may participate in a signaling-mediated protein condensation itself.EGFR signals to the mitogen-activated protein kinase (MAPK) pathway and controls key cellular functions, including growth and proliferation (14–16). EGFR is a paradigmatic model system in studies of signal transduction, and immense, collective scientific effort has revealed the inner workings of its signaling mechanism down to the atomic level (17). EGFR is autoinhibited in its monomeric form. Ligand-driven activation is achieved through formation of an asymmetric receptor dimer in which one kinase activates the other to phosphorylate the nine tyrosine sites in the C-terminal tails (17, 18). There is an obvious conceptual connection between EGFR and the LAT signaling system in T cells. The ∼200-residue–long cytoplasmic tail of EGFR resembles LAT in that both are intrinsically disordered and contain multiple sites of tyrosine phosphorylation that recruit adaptor proteins, including Grb2, upon receptor activation (19). Phosphorylation at tyrosine residues Y1068, Y1086, Y1148, and Y1173 in the EGFR tail creates sites to which Grb2 can bind via its SH2 domain. EGFR-associated Grb2 subsequently recruits SOS, through binding of its SH3 domains to the proline-rich domain of SOS. Once at the membrane, SOS undergoes a multistep autoinhibition-release process and begins to catalyze nucleotide exchange of RasGDP to RasGTP, activating Ras and the MAPK pathway (20).While these most basic elements of the EGFR activation mechanism are widely accepted, larger-scale features of the signaling complex remain enigmatic. A number of studies have reported higher-ordered multimers of EGFR during activation, including early observations by Förster Resonance Energy Transfer and fluorescence lifetime studies (21–23), as have more recent studies using single molecule (24, 25) and computational methods (26). Structural analyses and point mutation studies on EGFR have identified a binding interface enabling EGFR asymmetric dimers to associate (27), but the role of these higher-order assemblies remains unclear. At the same time, many functional properties of the signaling system remain unexplained as well. For example, EGFR is a frequently altered oncogene in human cancers, and drugs (including tyrosine kinase inhibitors) targeting EGFR signaling have produced impressive initial patient responses (28). All too often, however, these drugs fail to offer sustained patient benefits, in large part because of poorly understood resistance mechanisms (29). Physical aspects of the cellular microenvironment have been implicated as possible contributors to resistance development (30), and there is a growing realization that EGFR possesses kinase-independent (e.g., signaling independent) prosurvival functions in cancer cells (31). These points fuel speculation that additional layers of regulation over the EGFR signaling mechanism exist, including at the level of the receptor signaling complex itself.Here we report that EGFR undergoes a protein condensation-phase transition upon activation. We reconstituted the cytoplasmic tails of EGFR on supported bilayers and characterized the system behavior upon interaction with Grb2 and SOS, using total internal reflection fluorescence (TIRF) imaging. This experimental platform has been highly effective for revealing both phase-transition characteristics and functional signaling aspects of LAT protein condensates (4, 5, 10, 32–34). Published reports on the LAT system to date have emphasized SOS (or the SOS proline-rich [PR] domain) as a critical crosslinking element. Titrating the SOS PR domain into an initially homogeneous mixture of phosphorylated LAT and Grb2 revealed a sharp transition to the condensed phase, which we have also observed with the EGFR:Grb2:SOS system. Under slightly different conditions, however, we report observations of an EGFR:Grb2 condensation-phase transition without any SOS or other crosslinking molecule. We show that crosslinking is achieved through a Grb2–Grb2 binding interface. Phosphorylation on Grb2 at Y160 as well as a Y160E mutation [both reported to disrupt Grb2–Grb2 binding (35, 36)] were observed to prevent formation of EGFR condensates. We note that the evidence of Grb2–Grb2 binding we observed occurred in the context of EGFR-associated Grb2, which is localized to the membrane surface; free Grb2 dimers are not necessary.The consequence of EGFR condensation on downstream signaling is characterized by mapping the catalytic efficiency of SOS to activate Ras as a function of the EGFR condensation state. SOS is the primary Ras guanine nucleotide exchange factor (GEF) responsible for activating Ras in the EGFR-to-MAPK signaling pathway (37–40). At the membrane, SOS undergoes a multistep process of autoinhibition release before beginning to activate Ras. Once fully activated, SOS is highly processive, and a single SOS molecule can activate hundreds of Ras molecules before disengaging from the membrane (41–43). Autoinhibition release in SOS is a slow process, which necessitates that SOS be retained at the membrane for an extended time in order for Ras activation to begin (5, 10). This delay between initial recruitment of SOS and subsequent initiation of its Ras GEF activity provides a kinetic proofreading mechanism that essentially requires SOS to achieve multivalent engagement with the membrane (e.g., through multiple Grb2 or other interactions) in order for it to activate any Ras molecules.Experimental results described here reveal that Ras activation by SOS is strongly enhanced by EGFR condensation. Calibrated measurements of both SOS recruitment and Ras activation confirmed enhanced SOS catalytic activity on a per-molecule basis, in addition to enhanced recruitment to the condensates. These results suggest that a Grb2-mediated EGFR protein condensation-phase transition is a functional element controlling signal propagation from EGFR downstream to the MAPK signaling pathway.     

Phospholamban p.Arg14del Cardiomyopathy: A Japanese Case Series

Phospholamban p.Arg14del is reported to cause hereditary cardiomyopathy with malignant ventricular tachycardia (VT) and advanced heart failure. However, the clinical courses of Japanese cardiomyopathy patients with phospholamban p.Arg14del remain uncharacterized. We identified five patients with this variant. All patients were diagnosed with dilated cardiomyopathy (DCM), developed end-stage heart failure and experienced VT requiring implantable cardioverter defibrillator discharge. Four patients survived after implantation of a left ventricular assist device (LVAD), while one patient who refused LVAD implantation died of heart failure. Based on the severe course of the disease, we propose genetic screening for phospholamban p.Arg14del in DCM patients.     

Usefulness of gastroesophageal reflux scintigraphy using the knee-chest position for the diagnosis of gastroesophageal reflux disease

OBJECTIVES: The aim of this study is to evaluate the usefulness of gastroesophageal reflux (GER) scintigraphy using the knee-chest (KC) position for the diagnosis of gastroesophageal reflux disease (GERD). METHODS: The study subjects were 37 patients with GERD and 8 healthy volunteers (control group). Endoscopically observed esophageal mucosal breaks were evaluated with the Los Angeles classification. For GER scintigraphy, the subjects ingested liquid yogurt labeled with 99mTc-diethylene triamine pentaacetic acid (99mTc-DTPA) and water. Imaging was performed in the supine and KC position, and GER was graded as 1-4 according to the extent of GER assessed by scintigraphy. RESULTS: GER scintigraphy revealed no reflux in the control group (specificity: 100%). In the supine position, gastroesophageal reflux was observed in 49% of the patients with GERD, compared to 76% in the KC position. 21 of 23 (91%) patients with erosive esophagitis were shown to have GER with scintigraphy. GER scintigraphy revealed severe reflux (grade 3 or 4) (83%, 10/12) in the patients who had severe mucosal breaks (LA grade C or D). GER scintigraphy detected grade 1 or 2 reflux in 7 of the 14 patients who were endoscopically negative. There was a correlation between the endoscopically determined severity of mucosa and the reflux grade which was determined with GER scintigraphy. CONCLUSION: GER scintigraphy can detect gastroesophageal reflux with a high sensitivity in the KC position and might be a useful method in the screening and assessment of the severity of this disease. This method would be useful for the diagnosis of GERD in endoscopically negative patients.