Eluent and ligand effects in cation chromatography

Low-capacity surface-sulfonated cation exchange resins were prepared from high-performance gel beads of moderate cross- linking. Conditions necessary to control the reaction and obtain high efficiency cation-exchange material of a low-capacity were explored. Effects of reaction parameters on the depth and degree of sulfonation are discussed. An alternate method of determining total exchange capacity is also described;Selectivity of polyvalent metal cations versus hydrogen, sodium, magnesium, and ethylenediammonium cations is examined for the low-capacity sulfonated resins. Effects of complexing agents in the eluent are also examined for their effects on selectivity. Elution behavior with both complexing and non-complexing eluents are found to be predicted readily by use of the mass-action equilibrium expression for ion-exchange systems;The addition of complexing agents to the sample are shown to selectively complex certain metals as strong, stable complexes while leaving other metals essentially unbound. When injected, the masked ions do not compete for exchange sites on the column and are eluted in the void volume. The uncomplexed metal cations are chromatographed as usual. Analysis of alkaline earths in up to a 100-fold excess of interferent is carried out with reasonable recoveries;A method for speciation of iron by cation chromatography is described. Iron(II) is determined directly and iron(III) is detected after reduction to iron(II) just before injection. Choice of reducing agents and characteristics of the procedure are discussed. The;method shows favorable comparison to the spectrophotometric determination using 1,10-phenanthroline; (\u271)DOE Report IS-T 1170. This work was performed under Contract W-7405-Eng-82 with the Department of Energy

Analyse chromosomaler Translokationen und potenziell Leukämie-relevanter Mutationen einer mixed phenotype acute leukemia im Kindesalter (Fallstudie)

Einleitung: Akute mixed phenotype Leukämien (MPAL), welche einen kombinierten myeloischen und lymphatisches B-/T-Zell-Phänotyp aufweisen, zeigen mehrheitlich komplexe chromosomale Aberrationen. In dieser Dissertation wird eine MPAL im Kindesalter zum Zeitpunkt der Ersterkrankung und der Folgerezidive mit verschiedenen Methoden detailliert untersucht und umfassend charakterisiert. Die Aufklärung molekularer Mechanismen in Leukämien mit modernen Untersuchungsmethoden eröffnet neue Möglichkeiten der targeted Therapie. Insbesondere Analysen der chromosomalen Imbalancen und der Genexpressionsveränderungen können Einblicke in die molekularen Netzwerke und dysregulierten Signaltransduktionswege geben. Material und Methoden: In Ergänzung zur Routinediagnostik wurden molekulare (zyto)genetische Analyseverfahren wie Fluoreszenz-in-situ-Hybridisierung (FISH), Spectral Karyotyping (SKY), Microarray-basierte komparative genomische Hybridisierung (aCGH), Gensequenzierung, quantitative PCR und genomweite Microarray-Genexpressionsanalysen angewendet, um leukämische Blasten zu den Zeitpunkten der Diagnose der Ersterkrankung und zweier Rezidive zu charakterisieren. Zudem erfolgte zur Expansion der Probenmenge eine whole genome amplification sowie das Engraftment im murinen Xenograftmodell. Ergebnisse und Diskussion: Die MPAL zeigt eine ausgeprägte intraleukämische klo- nale Diversität und genetische Instabilität im Krankheitsverlauf, gemeinsames Merkmal scheint eine TCF3-ZNF384-Fusion als potenzielle founder Mutation und eine Amplifikation von KRAS zu sein. Nach Literaturrecherche ist dies die erste Beschreibung einer Wildtyp-KRAS Amplifikation bei Leukämien, welche in der MPAL u.a. auch für die gene- tische Instabilität ursächlich verantwortlich zu sein scheint. Weitere Aberrationen mit potenzieller Aktivierung des RAS-Signaltransduktionsweges wurden nachgewiesen. Basierend auf den molekulargenetischen Ergebnissen wurde eine second-line Therapie mit dem Multikinase-Inhibitor Sorafenib empfohlen, weitere Ansatzpunkte für Inhibitoren des gesamten MAPK-Signalweges werden diskutiert. Zudem offenbarten die Analysen weitere Kandidatengene: So liegt wahrscheinlich eine Dysregulation des Zellzyklus- (u.a. mit Nachweis einer CDKN2A-Deletion) und des Apoptose-Signalweges (insbesondere bei BCL2-Überexpression und Kopienzahlzugewinn im Rezidiv) vor, der Einsatz von MDM2- und BCL2-Inhibitoren zur targeted therapy wird diskutiert.Introduction: Acute mixed phenotype leukemias (MPAL) expressing a combined mye- loid and lymphatic B-/T-cell phenotype, show in their majority complex chromosomal aberrations. In this doctoral thesis, a case of pediatric MPAL was analyzed at initial presentation and at subsequent relapses in detail using multiple techniques. Uncovering molecular mechanisms in leukemia with modern analytical tools opens up new possibilities for targeted therapy. In particular, analyses defining chromosomal imbalances and gene expression changes can provide insights into molecular networks and dysregulated signal transduction pathways. Material and Methods: In addition to routine diagnostics, molecular (cyto)genetic tech- niques such as fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), microarray-based comparative genomic hybridization (aCGH), gene sequencing, quantitative PCR (qPCR) and genome-wide oligonucleotide microarray analysis were used to characterize leukemic blasts at the points of initial disease and two relapse stages. To expand the sample sizes, whole genome amplification and engraftment in murine xeno- graft model was performed. Results and Discussion: Pronounced intra-tumour clonal diversity and genetic instabil- ity in the course of disease were found, common traits appear to be a TCF3-ZNF384 fusion and a KRAS amplification. According to literature, this is the first report showing wild-type KRAS amplification in a leukemia, which seems to be causally responsible for genetic instability in MPAL. Further aberrations with potential activation of the RAS signal transduction pathway were detected. Based on these results, second-line therapy with the multikinase inhibitor sorafenib was recommended, further targets for inhibitors of the entire MAPK signaling pathway are discussed. In addition, the analyses revealed further candidate genes: there is probably a dysregulation of the cell cycle (including deletion of CDKN2A) and the apoptosis pathway (especially with BCL2 overexpression and copy number gain in relapse), the use of MDM2- and BCL2-inhibitors for targeted therapy is discussed

Turning “Cold” Into “Hot” Tumors—Opportunities and Challenges for Radio-Immunotherapy Against Primary and Metastatic Brain Cancers

The development of immunotherapies has revolutionized intervention strategies for a variety of primary cancers. Despite this promising progress, treatment options for primary brain cancer and brain metastasis remain limited and still largely depend on surgical resection, radio- and/or chemotherapy. The paucity in the successful development of immunotherapies for brain cancers can in part be attributed to the traditional view of the brain as an immunologically privileged site. The presence of the blood-brain barrier and the absence of lymphatic drainage were believed to restrict the entry of blood-borne immune and inflammatory cells into the central nervous system (CNS), leading to an exclusion of the brain from systemic immune surveillance. However, recent insight from pre-clinical and clinical studies on the immune landscape of brain cancers challenged this dogma. Recruitment of blood-borne immune cells into the CNS provides unprecedented opportunities for the development of tumor microenvironment (TME)-targeted or immunotherapies against primary and metastatic cancers. Moreover, it is increasingly recognized that in addition to genotoxic effects, ionizing radiation represents a critical modulator of tumor-associated inflammation and synergizes with immunotherapies in adjuvant settings. This review summarizes current knowledge on the cellular and molecular identity of tumor-associated immune cells in primary and metastatic brain cancers and discusses underlying mechanisms by which ionizing radiation modulates the immune response. Detailed mechanistic insight into the effects of radiation on the unique immune landscape of brain cancers is essential for the development of multimodality intervention strategies in which immune-modulatory effects of radiotherapy are exploited to sensitize brain cancers to immunotherapies by converting immunologically “cold” into “hot” environments

Exploring glutathione lyases as biocatalysts: paving the way for enzymatic lignin depolymerization and future stereoselective Applications

Glutathione-dependent β-etherases and glutathione lyases are key-enzymes for the biocatalytic depolymerization of lignin. In the first step, the nucleophilic attack of glutathione to the common β-O-4-aryl-ether motif in lignin is catalyzed by β-etherases and afterwards the glutathione is removed again by the action of glutathione lyases. Given their potential impact for lignin valorization, in this paper novel glutathione lyases are reported and biocatalytically characterized based on lignin model compounds. As a result, an enzyme exhibiting increased thermostability and lowered enantioselectivity - key features for implementation of glutathione lyases in enzymatic lignin depolymerization processes - was identified. Furthermore, first mutational studies of these enzymes revealed the possibility to further alter the activity as well as enantioselectivity of glutathione lyases by means of protein engineering. From a practical perspective, one-pot multi-step processes combining β-etherases and glutathione lyases are successfully set-up, giving hints on the potential that the implementation of these biocatalysts may bring for biorefinery purposes

Impact of different water activities (aw) adjusted by solutes on high pressure high temperature inactivation of Bacillus amyloliquefaciens spores

Much research has been conducted to comprehend the mechanisms of high pressure (HP) inactivation of spores in aqueous systems but for food model systems these information are scarce. In these systems spores can interact with ingredients which then could possibly lead to retarded or reduced inactivation, which can cause a problem for the sterilization process. The protective mechanism of a reduced aw-value is still unclear. HP processing might prove valuable to overcome protective effects of solutes and achieve shorter process times for sterilization under HP. To gain insight into the underlying mechanisms five aw-values (0.9, 0.92, 0.94, 0.96, 1) were adjusted with two different solutes (NaCl, sucrose). Solutions were inoculated with spores of Bacillus amyloliquefaciens and treated at 105, 110, and 115°C at 600 MPa. Further a thermal inactivation was conducted at the same temperatures for a comparison with the HP data. Afterward, the influence of HP high temperature treatment on the inactivation, the dipicolinic acid (DPA)-release and membrane constitution was assessed by plate count, HPLC and flow cytometry (FCM). The results show that during HP treatments sucrose and salt both have a protective effect, in which the influence of sucrose on the retarded inactivation is higher. The threshold water activities (aw), which is 0.94, here salt and sucrose have a significant influence on the inactivation. The comparison of thermal (105–115°C) and HP and high temperature (600 MPa, 105–115°C) treated samples showed that the time needed to achieve a 4–5 log10 inactivation is reduced from 45 (aw = 1) to 75 (aw = 0.9) min at 105°C to 3 (aw = 1) to 15 (aw = 0.9) minutes at 600 MPa and 105°C. The release of DPA is the rate limiting step of the inactivation and therefore monitoring the release is of great interest. The DPA-release is slowed down in high concentrated solutions (e.g., sucrose, salt) in comparison to aw 1. Since there is a difference in the way the solutes protect the spore it could be seen as an inner spore membrane effect. Maybe as shown for vegetative microorganism the solutes can interact with membranes, e.g., the inner spore membrane. Flow cytometry (FCM) measurement data show a similar trend

Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice

Cathepsin B (CTSB) and cathepsin L (CTSL) are two widelyexpressed cysteine proteases thought to predominantly reside withinlysosomes. Functional analysis of CTSL in humans is complicated by theexistence of two CTSL-like homologues (CTSL and CTSL2), in contrast tomice which contain only one CTSL enzyme. Thus transgenic expression ofhuman CTSL in CTSL deficient mice provides an opportunity to study the invivo functions of this human protease without interference by its highlyrelated homologue. While mice with single gene deficiencies for murineCTSB or CTSL survive without apparent neuromuscular impairment, murineCTSB/CTSL double deficient mice display degeneration of cerebellarPurkinje cells and neurons of the cerebral cortex, resulting in severehypotrophy, motility defects, and lethality during their third to fourthweek of life. Here we show that expression of human CTSL through agenomic transgene results in widespread expression of human CTSL in themouse which is capable of rescuing the lethality found in CTSB/CTSLdouble-deficient animals. Human CTSL is expressed in the brain of thesecompound mutants predominantly in neurons of the cerebral cortex and inPurkinje cells of the cerebellum, where it appears to prevent neuronalcell death

Extracellular matrix signatures of human mammary carcinoma identify novel metastasis promoters

The extracellular matrix (ECM) is a major component of tumors and a significant contributor to cancer progression. In this study, we use proteomics to investigate the ECM of human mammary carcinoma xenografts and show that primary tumors of differing metastatic potential differ in ECM composition. Both tumor cells and stromal cells contribute to the tumor matrix and tumors of differing metastatic ability differ in both tumor- and stroma-derived ECM components. We define ECM signatures of poorly and highly metastatic mammary carcinomas and these signatures reveal up-regulation of signaling pathways including TGFβ and VEGF. We further demonstrate that several proteins characteristic of highly metastatic tumors (LTBP3, SNED1, EGLN1, and S100A2) play causal roles in metastasis, albeit at different steps. Finally we show that high expression of LTBP3 and SNED1 correlates with poor outcome for ER−/PR−breast cancer patients. This study thus identifies novel biomarkers that may serve as prognostic and diagnostic tools.National Cancer Institute (U.S.) (Tumor Microenvironment Network, grant no. U54 CA126515/CA163109)National Cancer Institute (U.S.) (David H. Koch Institute Support Grant P30-CA14051)Howard Hughes Medical InstituteBroad Institute of MIT and HarvardNational Institutes of Health (U.S.) (NIH/National Research and Service Award)Virginia and D. K. Ludwig Fund for Cancer ResearchNational Cancer Cente