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Purpose: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. Experimental Design: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. Results: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. Conclusions: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

Zusammenfassung Hintergrund Nach der Veröffentlichung der American College of Surgeons Oncology Group Z0011-Studie (ACOSOG Z0011) ist die Analyse der intraoperativen Gefrierschnitte von Wächterlymphknoten zurückgegangen. Aber für Patientinnen, welche die ACOSOG Z0011-Kriterien nicht erfüllen, bleibt die Gefrierschnittanalyse ein wichtiges Instrument für die intraoperative Entscheidungsfindung im Hinblick auf eine Axilladissektion. Ziel dieser Studie war es, eine retrospektive Evaluierung der Vorteile und der diagnostischen Genauigkeit von Gefrierschnittanalysen bei Brustkrebspatientinnen durchzuführen, welche die Kriterien der Z0011-Studie erfüllten oder nicht erfüllten, und dabei mögliche prädiktive Faktoren für falsch negative Ergebnisse festzustellen. Methoden Zwischen 2008 und 2013 wurde eine intraoperative Gefrierschnittanalyse der Wächterlymphknoten bei 522 cT1-T3-Brustkrebspatientinnen durchgeführt. Die klinisch-pathologischen Merkmale wurden retrospektiv mithilfe der Krankenakten evaluiert. Ergebnisse Insgesamt betrug die Sensitivität und Spezifität für alle Gefrierschnittanalysen 67,8 % bzw. 100 %. Generell war die Sensitivität für Makrometastasen höher als für Mikrometastasen. Bei der Gruppe, welche die Kriterien der Z0011-Studie erfüllte, betrugen die Sensitivität und Spezifität 72,7 % bzw. 100 %, verglichen mit 62,1 % bzw. 100 % für die Gruppe, welche die Z0011-Kriterien nicht erfüllte. In der Gruppe, welche die Z0011-Kriterien erfüllte, wurde eine Untergruppenanalyse durchgeführt, und die Ergebnisse für ≤ 2 positiven Wächterlymphknoten wurden mit den Ergebnissen für > 2 verglichen. Bei beiden Untergruppen betrugen Spezifität und Sensitivität jeweils 100 %. In der Patientinnengruppe, welche die Z0011-Kriterien nicht erfüllte, waren mehrere klinisch-pathologische Faktoren mit einer höheren Rate an falsch positiven Ergebnissen assoziiert. Im Hinblick auf die intraoperative Entscheidungsfindung für eine Axilladissektion brachte die Durchführung einer intraoperativen Gefrierschnittanalyse Vorteile für 22,2 % der Patientinnen, welche die Z0011-Kriterien nicht erfüllten, aber nur für 0,6 % der Patientinnen, welche die Z0011-Kriterien erfüllten. Schlussfolgerungen Die Gefrierschnittanalyse ist besonders für die intraoperative Evaluierung von Wächterlymphknoten bei Patientinnen, welche die Z0011-Kriterien nicht erfüllen, vorteilhaft, da dadurch eine Zweitoperation zur Axilladissektion vermieden werden kann. Obwohl die Sensitivität der Gefrierschnittanalyse in der Gruppe, welche die Z0011-Kriterien erfüllte, insgesamt niedriger war, hat die Gefrierschnittanalyse in beiden Gruppen eine vergleichbar hohe Sensitivität und diagnostische Genauigkeit für Makrometastasen.

Supplementary Table from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy.Significance:SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy.This article is highlighted in the In This Issue feature, p. 2007

Supplementary Data from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Migration

Differentially expressed genes in NSD2 p.E1099K RCH-ACV cells with the change of H3K27me3 peaks

Supplementary Table 1: IC20 and IC50 values of ARQ 092 and sorafenib in Hep3B, Huh-7, HepG2, PLC/PRF/5 and HR4 cell lines after 48h of exposure; Supplementary Table 2: List of significantly different phospho-kinases from ARQ 092 treated rats compared to control group.

Supplementary Methods and Figures

Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation.Significance:NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL.This article is highlighted in the In This Issue feature, p. 1

The supplementary information file contains details about primers used for this work, supplementary Figures S1-S8, supplementary Table S1, and results from cell line authentication and from mycoplasma testing.

Pharmacodynamic response to trametinib/ganitumab in xenograft models does not predict tumor shrinkage

Related to Figure 4

Purpose:PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS.Experimental Design:Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models.Results:Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis.Conclusions:We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

The emotional facial expressions of other individuals are a valuable information source in adapting behaviour to situational demands, and have been found to receive prioritized attention. Yet, enhanced attentional biases, such as a bias to social threat in Social Anxiety Disorder (SAD), or blunted attention to emotional information, as assumed in Autism Spectrum Disorder (ASD), can easily become maladaptive in daily life. In order to investigate individual differences in attentional biases toward different emotional expressions (angry, happy, sad, and fearful versus neutral) and their links to social anxiety and autistic traits, we tested 104 healthy participants with an emotional dot-probe paradigm on a touch screen, and measured clinical trait levels associated with ASD and SAD. While confirming the presence of attentional biases toward all emotional expressions, we did not find robust evidence for systematic links between these biases and either clinical trait dimension. Only an exploratory Bayesian analysis pointed to a less pronounced bias towards happy facial expressions with higher autistic trait levels. Moreover, a closer examination of the attentional bias towards angry facial expressions suggested that alterations in this bias might depend on a complex interplay between both trait dimensions. Novel approaches in the assessment of attentional biases might yield the potential to describe disorder-specific biases in attention to emotions more validly.

Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation. Significance: NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL. This article is highlighted in the In This Issue feature, p. 1

In this paper we describe the performance of a prototype of the High Angle Time Projection Chambers (HA-TPCs) that are being produced for the Near Detector (ND280) upgrade of the T2K experiment. The two HA-TPCs of ND280 will be instrumented with eight Encapsulated Resistive Anode Micromegas (ERAM) on each endplate, for a total of 32 ERAMs. This innovative technique allows the detection of the charge emitted by ionization electrons over several pads, improving the determination of the track position. The TPC prototype has been equipped with the first ERAM module produced for T2K and with the HA-TPC readout electronics chain and it has been exposed to an electron beam at DESY in order to measure spatial and dE/dx resolution. In this paper we characterize the performances of the ERAM and, for the first time, we compare them with a newly developed simulation of the detector response. Spatial resolution better than 800 μm and dE/dx resolution better than 10% are observed for all the incident angles and for all the drift distances of interest. All the main features of the data are correctly reproduced by the simulation and these performances fully fulfill the requirements for the HA-TPCs of T2K. In this paper we describe the performance of a prototype of the High Angle Time Projection Chambers (HA-TPCs) that are being produced for the Near Detector (ND280) upgrade of the T2K experiment. The two HA-TPCs of ND280 will be instrumented with eight Encapsulated Resistive Anode Micromegas (ERAM) on each endplate, thus constituting in total 32 ERAMs. This innovative technique allows the detection of the charge emitted by ionization electrons over several pads, improving the determination of the track position. The TPC prototype has been equipped with the first ERAM module produced for T2K and with the HA-TPC readout electronics chain and it has been exposed to the DESY Test Beam in order to measure spatial and dE/dx resolution. In this paper we characterize the performances of the ERAM and, for the first time, we compare them with a newly developed simulation of the detector response. Spatial resolution better than 800 ${\mu \rm m}$ and dE/dx resolution better than 10% are observed for all the incident angles and for all the drift distances of interest. All the main features of the data are correctly reproduced by the simulation and these performances fully fulfill the requirements for the HA-TPCs of T2K.

International audience; Cholangiocarcinoma is a deadly cancer comprising very heterogenous subtypes with a limited therapeutic arsenal in all comers. However, recent significant advances were made with immunotherapy in the first-line treatment of advanced cholangiocarcinoma, with the addition of durvalumab to cisplatin–gemcitabine chemotherapy showing a survival benefit. In the second line setting, only FOLFOX (5FU/folinic acid-oxaliplatin) is validated by a phase 3 trial, yet with a very modest benefit on survival; new options using 5FU with nanoliposomal-irinotecan may emerge in the next few years. The advent of molecular profiling in advanced cholangiocarcinoma in the last decade revealed frequent targetable alterations such as IDH1 mutations, FGFR2 fusions or rearrangements, HER2 amplification, BRAF V600E mutation and others. This strategy opened the way to personalised medicine for patients which are still fit after first-line treatment and the use of targeted inhibitors in first line constitutes a huge challenge with many ongoing trials to improve patients’ care. This review exposes the recent clinical trial findings in non-molecularly selected advanced cholangiocarcinoma, offers a focus on how systematic molecular screening should be structured to allow patients to access to personalised medicine, and details which are the therapeutic options accessible in case of actionable alteration.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide, with up to 90% of HCC cases occurring in the setting of underlying cirrhosis. Therapeutic landscape for advanced HCC has dramatically changed in recent years with the advent of immunotherapy, including several combinations. Data suggest that the surrounding liver milieu may influence tumour response. In addition, different aetiologies of HCC and their effects on the host liver may impact response to immunotherapy. However, to date, guidelines do not take into account this parameter to guide therapeutic selection, and phase III trials are likewise performed in patients irrespective of HCC aetiology. Moreover, most clinical trial data are collected in highly selected patients with preserved liver function (defined as Child-Pugh class A) and controlled portal hypertension, which does not accurately reflect routine clinical practice. In this review, we discuss the influence of liver disease aetiology on the response to immunotherapy in patients with advanced HCC. We also discuss the safety and efficacy of various immunotherapeutic agents in Child-Pugh B patients to determine if these treatments are beneficial in this vulnerable patient population.

Attention may be swiftly and automatically tuned to emotional expressions in social primates, as has been demonstrated in humans, bonobos, and macaques, and with mixed evidence in chimpanzees, where rapid detection of emotional expressions is thought to aid in navigating their social environment. Compared to the other great apes, orangutans are considered semi-solitary, but still form temporary social parties in which sensitivity to others’ emotional expressions may be beneficial. The current study investigated whether implicit emotion-biased attention is also present in orangutans (Pongo pygmaeus). We trained six orangutans on the dot-probe paradigm: an established paradigm used in comparative studies which measures reaction time in response to a probe replacing emotional and neutral stimuli. Emotional stimuli consisted of scenes depicting conspecifics having sex, playing, grooming, yawning, or displaying aggression. These scenes were contrasted with neutral scenes showing conspecifics with a neutral face and body posture. Using Bayesian mixed modeling, we found no evidence for an overall emotion bias in this species. When looking at emotion categories separately, we also did not find substantial biases. We discuss the absence of an implicit attention bias for emotional expressions in orangutans in relation to the existing primate literature, and the methodological limitations of the task. Furthermore, we reconsider the emotional stimuli used in this study and their biological relevance.

Background and aims: Pancreatic cancer is highly lethal and often diagnosed at an advanced stage. This cohort study analyzes the impact of care pathways, delays, and socio-spatial determinants on pancreatic cancer patients’ diagnosis, treatment, and prognosis. Method: Patients with pancreatic adenocarcinoma newly diagnosed at all stages between January and June 2016 in the AuRA French region were included. The influence on survival of delays of care, healthcare centers’ expertise, and socio-spatial determinants was evaluated. Results: Here, 538 patients were included in 76 centers including 116 patients (21.8%) with resectable, 64 (12.0%) borderline-resectable, 147 (27.6%) locally-advanced tumors, and 205 (38.5%) with metastatic disease. A delay between first symptoms and CT scans did not statistically influence overall survival (OS). In resected patients, OS was significantly higher in centers with more than 20 surgeries (HR 20 surgeries/year p = 0.0081). Regarding socio-spatial determinants, patients living in municipalities with greater access to a general practitioner (HR = 1.673, p = 0.0153) or with a population density below 795.1 people/km2 (HR = 1.881, p = 0.0057) were significantly more often resectable. Conclusion: This cohort study supports the pivotal role of general practitioner in cancer care and the importance of the centralization of pancreatic surgery to optimize pancreatic cancer patients’ care and outcomes. However, delays of care did not impact patient survival.

Autorzy: Abe K., Adrich P., Aihara H., Akutsu, R., Alekseev I., Ali A. , Ameli F., Anghel I., Anthony L. H. V., Antonova M. , Araya A., Asaoka Y., Ashida Y., Aushev V., Ballester F., Bandac I., Barbi M., Barker G. J., Barr G., Batkiewicz-Kwasniak M., Bellato M., Berardi V., Bergevin M., Bernard L., Bernardini E., Berns L., Bhadra S., Bian J., Blanchet A., Blaszczyk F. d. M., Blonde A., Boiano A., Bolognesi S., Bonavera L., Booth N., Borjabad S., Boschi, T., Bose D., Boyd S . B., Bozza C., Bravar A., Bravo-Berguño D., Bronner C., Brown L., Bubak Arkadiusz, Buchowicz A., Buizza Avanzini M., Cafagna F. S., Calabria N. F., Calvo-Mozota J. M., Cao S., Cartwright S.L., Carroll A., Catanesi M. G., Cebriàn, S., Chabera M., Chakraborty, S., Checchia C., Choi J.H., Choubey S., Cicerchia M., Coleman J., Collazuol G., Cook L., Cowan G., Cuen-Rochin, S., Danilov M., Díaz López G., De la Fuente E., de Perio P., De Rosa G., Dealtry T., Densham C. J., Dergacheva A., Deshmukh N., Devi M. M., Di Lodovico F., Di Meo, P., Di Palma I., Doyle T. A., Drakopoulou E., Drapier O., Dumarchez J., Dunne P., Dziewiecki M., Eklund L., El Hedri S., Ellis J., Emery S., Esmaili A., Esteve R., Evangelisti A., Feely M., Fedotov S., Feng J., Fernandez P., Fernández-Martinez E., Ferrario P., Ferrazzi,B., Feusels T., Finch A., Finley C., Fiorentini A., Fiorillo G., Fitton M., Frankiewicz K., Friend M., Fujii Y., Fukuda Y., Galinski G., Gao J., Garde C., Garfagnini A., Garode S., Gialanella L., Giganti C., Gomez-Cadenas J.J., Gonin M., González-Nuevo J., Gorin A., Gornea R., Gousy-Leblanc V. Gramegna F. Grassi M. Grella G. Guigue M. Gumplinger P. Hadley D.R. Harada M., Hartfiel B., Hartz M., Hassani S., Hastings N.C., Hayato Y., Hernando-Morata J.A., Herrero V., Hill J., Hiraide K., Hirota S., Holin A., Horiuchi S., Hoshina K., Hultqvist K., Iacob F., Ichikawa A.K., Idrissi Ibnsalih W., Iijima T., Ikeda M., Inomoto M., Inoue K., Insler J., Ioannisian A., Ishida T., Ishidoshiro K., Ishino H., Ishitsuka M., Ito H., Ito S., Itow Y., Iwamoto K., Izmaylov A., Izumi N., Izumiyama S., Jakkapu M., Jamieson B., Jang H.I., Jang J.S., Jenkins S.J., Jeon S.H., Jiang M., Jo H.S., Jonsson P., Joo K.K., Kajita T., Kakuno H., Kameda J., Kano Y., Kalaczynski P., Karlen D., Kasperek J., Kataoka Y., Kato A., Katori T., Kazarian N., Kearns E., Khabibullin M., Khotjantsev A., Kikawa T., Kekic M., Kim J.H., Kim J.Y., Kim S.B., Kim S.Y., King S., Kinoshita T., Kisiel Jan, Klekotko A., Kobayashi T., Koch L., Koga M., Koerich L., Kolev N., Konaka A., Kormos L.L., Koshio Y., Korzenev A., Kotsar Y., Kouzakov K.A., Kowalik K.L., Kravchuk L., Kryukov A.P., Kudenko Y., Kumita T., Kurjata R., Kutter T., Kuze M., Kwak K., La Commara M., Labarga L., Lagoda J., Lamers James J., Lamoureux M., Laveder M., Lavitola L., Lawe M., Learned J.G., Lee J., Leitner R., Lezaun V., Lim I.T., Lindner T., Litchfield R.P., Long K.R., Longhin A., Loverre P., Lu X., Ludovici L., Maekawa Y., Magaletti L., Magar K., Mahn K., Makida Y., Malek M., Malinský M., Marchi T., Maret L., Mariani C., Marinelli A., Martens K., Marti L., Martin J.F. Martin D., Marzec J., Matsubara T., Matsumoto R., Matsuno S., Matusiak M., Mazzucato E., McCarthy M., McCauley N., McElwee J., McGrew C., Mefodiev A., Medhi A., Mehta P., Mellet L., Menjo H., Mermod P., Metelko C., Mezzetto M., Migenda J., Migliozzi P., Mijakowski P., Miki S., Miller E.W., Minakata H., Minamino A., Mine S., Mineev O., Mitra A., Miura M., Moharana R., Mollo C.M., Mondal T., Mongelli M., Monrabal F., Moon D.H., Moon C.S., Mora F.J., Moriyama S., Mueller Th.A., Munteanu L., Murase K., Nagao Y., Nakadaira T., Nakagiri K., Nakahata M., Nakai S., Nakajima Y., Nakamura K., Nakamura KI., Nakamura H., Nakano Y., Nakaya T., Nakayama S., Nakayoshi K., Nascimento Machado L., Naseby C.E.R., Navarro-Garcia B., Needham M., Nicholls T., Niewczas K., Nishimura Y., Noah E., Nova F., Nugent J.C., Nunokawa H., Obrebski W., Ochoa-Ricoux J.P., O’Connor E., Ogawa N., Ogitsu T., Ohta K., Okamoto K., O’Keeffe H.M., Okumura K., Onishchuk Y., Orozco-Luna F., Oshlianskyi A., Ospina N., Ostrowski M., O’Sullivan E., O’Sullivan L., Ovsiannikova T., Oyama Y., Ozaki H., Pac M.Y., Paganini P., Palladino V., Paolone V., Pari M., Parsa S., Pasternak J., Pastore C., Pastuszak G., Patel D.A., Pavin M., Payne D., Peña-Garay C., Pidcott C., Pinzon Guerra E., Playfer S., Pointon B.W., Popov A., Popov B., Porwit Kamil, Posiadala-Zezula M., Poutissou J.M., Pozimski J., Pronost G., Prouse N.W., Przewlocki P., Quilain B., Quiroga A.A., Radicioni E., Radics B., Rajda P.J., Renner J., Rescigno M., Retiere F., Ricciardi G., Riccio C., Richards B., Rondio E., Rose H.J., Roskovec B., Roth S., Rott C., Rountree S.D., Rubbia A., Ruggeri A.C., Ruggles C., Russo S., Rychter A., Ryu D., Sakashita K., Samani S., Sánchez F., Sánchez M.L., Sanchez M.C., Sano S., Santos J.D., Santucci G., Sarmah P., Sashima I., Sato K., Scott M., Seiya Y., Sekiguchi T., Sekiya H., Seo J.W., Seo S.H., Sgalaberna D., Shaikhiev A., Shan Z., Shaykina A., Shimizu I., Shin C.D., Shinoki M., Shiozawa M., Sinnis G., Skrobova N., Skwarczynski K., Smy M.B., Sobczyk J., Sobel H.W., Soler F. J. P., Sonoda Y., Spina R., Spisso B., Spradlin B., Stankevich K.L., Stawarz L., Stellacci S.M., Stopa K., Studenikin A.I., Suárez Gómez S.L., Suganuma T., Suvorov S., Suwa Y., Suzuki A.T., Suzuki S.Y., Suzuki Y., Svirida D., Svoboda R., Taani M., Tada M., Takeda A., Takemoto Y., Takenaka A., Taketa A., Takeuchi Y., Takhistov V., Tanaka H., Tanaka H.A., Tanaka H.I., Tanaka M., Tashiro T., Thiesse M., Thompson L.F., Toledo J., Tomatani-Sánchez A.K., Tortone G., Tsui K.M., Tsukamoto T., Tzanov M., Uchida Y., Vagins M.R., Valder S., Valentino V., Vasseur G., Vijayvargi A., Vilela C., Vinning W. G. S., Vivolo D., Vladisavljevic T., Vogelaar R.B., Vyalkov M.M., Wachala T., Walker J., Wark D., Wascko M.O., Wendell R.A., Wilkes R.J., Wilking M.J., Wilson M.R., Wronka S., Xia J., Xie Z., Xin T., Yamaguchi Y., Yamamoto K., Yanagisawa C., Yano T., Yen S., Yershov N., Yeum D.N., Yokoyama M., Yonenaga M., Yoo J., Yu I., Yu M., Zakrzewski T., Zaldivar B., Zalipska J., Zaremba K., Zarnecki G., Ziembicki M., Zietara K., Zito M., Zsoldos S., Core-collapse supernovae are among the most magnificent events in the observable universe. They produce many of the chemical elements necessary for life to exist and their remnants-neutron stars and black holes-are interesting astrophysical objects in their own right. However, despite millennia of observations and almost a century of astrophysical study, the explosion mechanism of core-collapse supernovae is not yet well understood. Hyper-Kamiokande is a next-generation neutrino detector that will be able to observe the neutrino flux from the next galactic core-collapse supernova in unprecedented detail. We focus on the first 500 ms of the neutrino burst, corresponding to the accretion phase, and use a newly-developed, high-precision supernova event generator to simulate Hyper-Kamiokandeʼs response to five different supernova models. We show that Hyper-Kamiokande will be able to distinguish between these models with high accuracy for a supernova at a distance of up to 100 kpc. Once the next galactic supernova happens, this ability will be a powerful tool for guiding simulations toward a precise reproduction of the explosion mechanism observed in nature.

Toluene o-xylene monooxygenase (ToMO) was found to oxidize chlorobenzene to form 2-chlorophenol (2-CP, 4%), 3-CP (12%), and 4-CP (84%) with a total product formation rate of 1.2 ± 0.17 nmol/min/mg protein. It was also discovered that ToMO forms 4-chlorocatechol (4-CC) from 3-CP and 4-CP with initial rates of 0.54 ± 0.10 and 0.40 ± 0.04 nmol/min/mg protein, respectively, and chlorohydroquinone (CHQ, 13%), 4-chlororesorcinol (4-CR, 3%), and 3-CC (84%) from 2-CP with an initial product formation rate of 1.1 ± 0.32 nmol/min/mg protein. To increase the oxidation rate and alter the oxidation regiospecificity of chloroaromatics, as well as to study the roles of active site residues L192 and A107 of the alpha hydroxylase fragment of ToMO (TouA), we used the saturation mutagenesis approach of protein engineering. Thirteen TouA variants were isolated, among which some of the best substitutions uncovered here have never been studied before. Specifically, TouA variant L192V was identified which had 1.8-, 1.4-, 2.4-, and 4.8-fold faster hydroxylation activity toward chlorobenzene, 2-CP, 3-CP, and 4-CP, respectively, compared to the native ToMO. The L192V variant also had the regiospecificity of chlorobenzene changed from 4% to 13% 2-CP and produced the novel product 3-CC (4%) from 3-CP. Most of the isolated variants were identified to change the regiospecificity of oxidation. For example, compared to the native ToMO, variants A107T, A107N, and A107M produced 6.3-, 7.0-, and 7.3-fold more 4-CR from 2-CP, respectively, and variants A107G and A107G/L192V produced 3-CC (33 and 39%, respectively) from 3-CP whereas native ToMO did not. IMPORTANCE Chlorobenzene is a commonly used toxic solvent and listed as a priority environmental pollutant by the US Environmental Protection Agency. Here, we report that Escherichia coli TG1 cells expressing toluene o-xylene monooxygenase (ToMO) can successfully oxidize chlorobenzene to form dihydroxy chloroaromatics, which are valuable industrial compounds. ToMO performs this at room temperature in water using only molecular oxygen and a cofactor supplied by the cells. Using protein engineering techniques, we also isolated ToMO variants with enhanced oxidation activity as well as fine-tuned regiospecificities which make direct microbial oxygenations even more attractive. The significance of this work lies in the ability to degrade environmental pollutants while at the same time producing valuable chemicals using environmentally benign biological methods rather than expensive, complex chemical processes.