Preclinical Results of Camptothecin-Polymer Conjugate (IT-101) in Multiple Human Lymphoma Xenograft Models

Purpose: Camptothecin (CPT) has potent broad-spectrum antitumor activity by inhibiting type I DNA topoisomerase (DNA topo I). It has not been used clinically because it is water-insoluble and highly toxic. As a result, irinotecan (CPT-11), a water-soluble analogue of CPT, has been developed and used as salvage chemotherapy in patients with relapsed/refractory lymphoma, but with only modest activity. Recently, we have developed a cyclodextrin-based polymer conjugate of 20-(S)-CPT (IT-101). In this study, we evaluated the preclinical antilymphoma efficacy of IT-101 as compared with CPT-11. Experimental Design: We determined an in vitro cytotoxicity of IT-101, CPT-11, and their metabolites against multiple human lymphoma cell lines. In human lymphoma xenografts, the pharmacokinetics, inhibitions of tumor DNA topo I catalytic activity, and antilymphoma activities of these compounds were evaluated. Results: IT-101 and CPT had very high in vitro cytotoxicity against all lymphoma cell lines tested. As compared with CPT-11 and SN-38, IT-101 and CPT had longer release kinetics and significantly inhibit higher tumor DNA topo I catalytic activities. Furthermore, IT-101 showed significantly prolonged the survival of animals bearing s.c. and disseminated human xenografts when compared with CPT-11 at its maximum tolerated dose in mice. Conclusions: The promising present results provide the basis for a phase I clinical trial in patients with relapsed/refractory lymphoma

Elucidating the crystal-chemistry of Jbel Rhassoul stevensite (Morocco) by advanced analytical techniques

The composition of Rhassoul clay is controversial regarding the nature of the puremineral clay fraction which is claimed to be stevensite rather than saponite. In this study, the raw and mineral fractions were characterized using various techniques including Fourier transform infrared spectroscopy and magic angle spinning nuclear magnetic resonance (MAS NMR). The isolated fine clay mineral fraction contained a larger amount of Al (>1 wt.%) than that reported for other stevensite occurrences. The 27Al MAS NMR technique confirmed that the mineral is stevensite in which the Al is equally split between the tetrahedral and octahedral coordination sites. The 29Si NMR spectrum showed a single unresolved resonance indicating little or no short-range ordering of silicon. The chemical composition of the stevensite from Jbel Rhassoul was determined to be ((Na0.25K0.20 (Mg5.04Al0.37Fe0.20&0.21)5.61(Si7.76Al0.24)8O20(OH)4). This formula differs from previous compositions described from this locality and shows it to be an Al-bearing lacustrine clay mineral

The shape and erosion of pebbles

The shapes of flat pebbles may be characterized in terms of the statistical distribution of curvatures measured along their contours. We illustrate this new method for clay pebbles eroded in a controlled laboratory apparatus, and also for naturally-occurring rip-up clasts formed and eroded in the Mont St.-Michel bay. We find that the curvature distribution allows finer discrimination than traditional measures of aspect ratios. Furthermore, it connects to the microscopic action of erosion processes that are typically faster at protruding regions of high curvature. We discuss in detail how the curvature may be reliable deduced from digital photographs.Comment: 10 pages, 11 figure

What is in a pebble shape?

We propose to characterize the shapes of flat pebbles in terms of the statistical distribution of curvatures measured along the pebble contour. This is demonstrated for the erosion of clay pebbles in a controlled laboratory apparatus. Photographs at various stages of erosion are analyzed, and compared with two models. We find that the curvature distribution complements the usual measurement of aspect ratio, and connects naturally to erosion processes that are typically faster at protruding regions of high curvature.Comment: Phys. Rev. Lett. (to appear

Phyto-Ruminal-Bioremediation: Grasses to the Rescue

The US EPA has defined Super Fund sites for 594 toxins in the United States. The EPA definition of toxins is one that causes acute or chronic human health effects, or causing significant environmental harm. One of the categories of toxins is nitroaromatic molecules, which are used primarily as munitions. In the United States alone there are 16,000 Department of Defense (DoD) sites. The most common munitions there are TNT, RDX, HMX, and 2,4 DNT. In the world, these sites range historically from World War II residues to those from recent Middle East conflicts. Human health effects from these toxins include damage to the renal, nervous system, reproductive system, dermal, and hepatocarcinoma. In the animal world, a ruminant has four stomachs, one of which the rumen contains obligate ruminant anaerobes. These anaerobic microbes have the unique ability to break down the cellulose bond and other compounds from plant material. An additional fact is that grasses can extract nitroaromatic compounds from the soil and do it far better than dicotyledon plants. A number of people have looked at the uptake of toxins by grasses and other plant material. This paper will present the ability of cool season grasses to remove toxins from the soil and their subsequent degradation by ruminal microbes from sheep

Optimisation du système d'édition génique CRISPR-Cas

Développé en 2012, le système CRISPR-Cas a d'ores et déjà révolutionné les sciences du vivant en démocratisant l'édition du génome grâce à sa simplicité d'usage, sa forte efficacité et son adaptabilité. Néanmoins, l'efficacité et la précision de ce système varient grandement ce qui peut freiner ou empêcher sa mise en place. Mes travaux de doctorat se sont articulés autour de ces deux thématiques. L'édition du génome à l'aide de nucléases artificielles repose sur l'activation des voies de réparation de la cellule par induction d'une cassure double brin (DSB) dans l'ADN. Le système CRISPR-Cas est composé d'une nucléase (Cas) associée à un ARN guide qui se lie à la séquence ciblée par appariement de base. Une fois la DSB induite par la nucléase, plusieurs mécanismes de réparation entrent en compétition pour réparer la cassure. La réparation par jonction d'extrémités non-homologues (NHEJ) peut entrainer l'insertion de mutations ce qui permet de réaliser des inactivations de gène alors que la réparation par recombinaison homologue (HDR) permet des corrections ou insertions précises. Les stratégies les plus répandues pour améliorer l'efficacité de l'édition génique reposent sur l'utilisation de marqueurs de sélection. Néanmoins, ces marqueurs peuvent influencer la physiologie des cellules et leur utilisation n'est pas envisageable dans un cadre thérapeutique. Pour y remédier nous avons développé une méthode de cosélection sans marqueur se basant sur la création d'un allèle à gain de fonction. En modifiant le gène ATP1A1 encodant pour la pompe Na+/K+ ATPase par NHEJ et HDR nous avons conféré une résistance à l'ouabaïne aux cellules tout en conservant la fonctionnalité de la pompe. En ciblant simultanément le gène ATP1A1 et un gène d'intérêt, le traitement des cellules à l'ouabaïne permet de sélectionner les cellules résistantes et enrichir la population en cellules génétiquement modifiées dans le gène d'intérêt. Nous avons obtenu des augmentations drastiques de l'efficacité de NHEJ et de HDR et la cosélection à l'aide de Cas12a permet d'enrichir facilement et simultanément de multiples cibles. La méthode est simple et rapide à mettre en place et nous avons démontré sa versatilité en l'appliquant à diverses lignées cellulaires dont les cellules souches et progénitrices hématopoïétiques couramment utilisées en thérapie génique ex vivo, ce qui permet d'envisager de futures applications thérapeutiques. Notre stratégie a été déployée dans de nombreux laboratoires depuis sa publication et, de manière significative, elle a également été utilisée pour enrichir les événements de réparation des éditeurs de base et éditeurs par transcriptase inverse (prime editing) et pourrait aussi être applicable aux futurs outils d'édition du génome. La HDR est la voie privilégiée pour des perspectives thérapeutiques. Néanmoins, la NHEJ est la voie de réparation majoritaire dans les cellules humaines et la recombinaison homologue n'est active que lors des phases S et G2 du cycle cellulaire. La fusion de Cas9 avec le dégron de la géminine a permis de restreindre son activité aux phases S, G2 et M du cycle cellulaire et augmenter sensiblement le ratio de réparation par HDR. Parallèlement à la réplication de l'ADN, la recombinaison homologue présente un pic d'activité en milieu de phase S puis son activité diminue. Nous avons émis l'hypothèse que restreindre l'activité de la nucléase à la phase S permettrait d'augmenter davantage le ratio de réparation par HDR. Néanmoins, aucun dégron existant ne permet une dégradation lors des phases G1, G2 et M. Le système d'identification Fucci se base sur la fusion de dégrons à des protéines fluorescentes pour marquer les différentes phases du cycle cellulaire. Afin de développer un nouveau dégron permettant d'améliorer les systèmes Fucci et CRISPR, nous nous sommes intéressés à SLBP, une protéine active uniquement lors de la phase S. Nous avons caractérisé son dégron et l'avons utilisé afin de développer une sonde fluorescente spécifique de la phase S dont le profil d'expression a été confirmé par cytométrie en flux et microscopie en temps réel. Le marquage précis de la phase S pourrait notamment aider à élucider les voies de réparation de l'ADN. Nous avons également démontré que la fusion d'un de nos dégrons avec SpCas9 permet d'augmenter le taux de réparation par HDR de manière plus significative que le dégron de la géminine. Il sera intéressant d'évaluer sa synergie avec d'autres stratégies d'optimisation du système CRISPR.Developed in 2012, the CRISPR-Cas system has rapidly revolutionized life sciences and is routinely used in research laboratories worldwide. Its efficiency, simplicity and versatility greatly facilitate gene editing and functional genomics. However, the variability of its precision and efficiency is a major concern since it restrains its implementation, especially for therapeutic use. My PhD investigations revolves around these challenges. Gene editing through artificial nucleases relies on inducing a double-strand break (DSB) in the DNA to activate cellular repair pathways. For CRISPR-Cas systems, targeting is realised through base pairing between the targeted sequence and a guide RNA that associates with the Cas nuclease, making the design of new guides a simple process. Once the nuclease has elicited the DSB, several repair mechanisms compete to repair the break. Non-homologous end joining (NHEJ) can lead to mutations in the targeted sequence and allows gene knock-out while homology-directed repair (HDR) permits precise corrections or insertions. The most common strategy to enrich for cells that have undergone the desired genetic modification relies on the use of selection markers. However, since these markers can impact cell physiology, they are not suitable for therapeutic use. To address this issue, we have developed a marker free co-selection method based on the creation of a gain of function allele. By targeting ATP1A1, the gene encoding for the Na+/K+ ATPase pump, we conferred resistance to ouabain to the cells by either NHEJ or HDR while conserving the pump properties. Simultaneous targeting of ATP1A1 and a gene of interest followed by cell treatment with ouabain allows enrichment for cells genetically modified in the gene of interest. We observed a drastic improvement in efficiency for both NHEJ and HDR events and several targets can be enriched simultaneously and easily by exploiting Cas12a multiplexing capabilities. It's a simple and fast strategy and we have demonstrated its versatility by modifying various cell lines including hematopoietic and progenitor stem cells, commonly used in ex vivo gene therapy, demonstrating therapeutic potential. Since its publication, the ATP1A1 co-selection strategy has been exploited in numerous laboratories and successfully applied to enrich for base and prime editors' modifications and it could as well be applied to future genome editing tools, further demonstrating its versatility. Due to its fidelity, HDR is the preferred pathway for potential therapeutic use. Nevertheless, NHEJ is the major repair mechanism in human cells and homologous recombination is only active during S and G2 cell cycle phases. Although inhibiting NHEJ or promoting HDR by targeting proteins involved in these pathways is greatly efficient, the efficiency variability between cell lines and toxicity is considerable. Fusing Cas9 to the geminin degron restricts its activity to the S, G2 an M phases and slightly improves the HDR ratio. Alongside DNA replication, homologous recombination activity is thought to peak in the mid S phase and decline during G2 phase. We hypothesized that restricting Cas9 nuclease expression to the S phase will further bias repair towards HDR. However, no degron allowing G1, G2 and M phases degradation has been developed yet. The Fucci system is based on the fusion between degrons and fluorescent proteins to distinguish the different cell cycle phases but lack an S-phase specific probe. To improve cell cycle identification and HDR ratio, we decided to develop a degron allowing such a regulation. In that order, we studied the stem-loop binding protein (SLBP) which bind histone mRNAs and is only active during S phase and is degraded in other phases. We analysed SLBP endogenous expression pattern, characterised its degron, and used it to engineer an S-phase specific probe that we named Fucci-S. K562 and HeLa S3 cells constitutively expressing Fucci-S probe were created and their fluorescence expression pattern were analysed by FACS and live cell microscopy to confirm its S-phase specificity. Combined with the Fucci probes it allows to differentiate all the cell cycles phases and could be used in developmental and DNA repair studies. Fusing one of our newly developed degrons to SpCas9 increases HDR ratio more than the geminin degron. Additional studies would allow to establish its range of use and how it synergizes with other CRISPR-Cas optimisation strategies

Detection of Endophyte Mycotoxins by Service Laboratories: Providing Answers for Safe Feed

. A global network of service laboratories exists to test livestock feed materials (typically grass hay and pellets) for ergovaline, ergot alkaloids and lolitrem B to ensure ‘safe feeds’ are being given to livestock. These compounds are mycotoxins produced by endophytic fungi that naturally reside in feed material. They have been purposely bred into grass species, as they enhance the plant’s survival from drought and insect predation. Unfortunately, ergovaline and other ergot alkaloids also cause vasoconstrictive effects and reproductive difficulties in livestock, resulting in a $1 million annual loss in production for the cattle industry in the USA alone. Lolitrem B is a neurotoxicant that causes a syndrome known as ‘ryegrass staggers,’ which involves a tremoring response in the animal. Clients of these service laboratories include hay farmers who want to be confident that the product they are selling is safe to feed to livestock; veterinarians who are trying to rule out causes of disease in clinical cases; individuals wanting to check personal feed sources; and researchers investigating innovative solutions to these feed contaminants. Each year, approximately 33,000 containers of hay are shipped overseas from the USA, including Pacific Rim and Middle Eastern countries, bringing in an estimated$130 million annually. If the importing country requires it, the material in these containers must be tested for the appropriate mycotoxin(s) and have a certificate stating that the level found was below the established threshold of toxicity. Discussion of sample submission, analysis and result receipt will be compared amongst international laboratories known to perform analyses for these mycotoxins

Fossil Carder Bee's nest from the Hominin locality of Taung, South Africa

The Buxton-Norlim Limeworks southwest of Taung, South Africa, is renowned for the discovery of the first Australopithecus africanus fossil, the ‘Taung Child’. The hominin was recovered from a distinctive pink calcrete that contains an abundance of invertebrate ichnofauna belonging to the Coprinisphaera ichnofacies. Here we describe the first fossil bee’s nest, attributed to the ichnogenus Celliforma, from the Plio-Pleistocene of Africa. Petrographic examination of a cell lining revealed the preservation of an intricate organic matrix lined with the calcitic casts of numerous plant trichomes–a nesting behaviour unique to the modern-day carder bees (Anthidiini). The presence of Celliforma considered alongside several other recorded ichnofossils can be indicative of a dry, savannah environment, in agreement with recent work on the palaeoenvironment of Plio-Pleistocene southern Africa. Moreover, the occurrence of ground-nesting bees provides further evidence that the pink calcrete deposits are of pedogenic origin, rather than speleogenic origin as has previously been assumed. This study demonstrates the potential value of insect trace fossils as palaeoenvironmental indicators

Metabolism of toxic plant alkaloids in livestock : comparative studies on the hepatic metabolism of pyrrolizidine alkaloids in sheep and cattle and of ergot alkaloids in an endophyte-resistant mouse model

The pyrrolizidine alkaloids (PAs) and ergot alkaloids are known natural toxicants found in livestock forage. These alkaloids contribute to large economic losses in livestock throughout the world. An understanding of the mechanisms of toxicity and development of better diagnostic tools for better management practices was investigated. Variability exists in the toxicity of PAs in ruminants where cattle are more susceptible and sheep are more resistant. The mechanism of PA resistance in sheep has been attributed to hepatic metabolism or rumen microbial degradation of PAs to non-toxic moieties. The hepatic metabolism of the PA senecionine was investigated in cattle and sheep liver microsomes. The level of a toxic pyrrole metabolite 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine pyrrole (DHP) formed in cattle and sheep were similar. However, the level of a non-toxic N-oxide metabolite was greater in sheep than in cattle. Cytochrome P450 and flavin monooxygenases (FMOs) responsible for PA oxidative metabolism were similar in both ruminant species. Therefore, hepatic metabolism of PAs is not solely responsible for resistance observed in sheep versus cattle. Ergot alkaloids present in endophyte-infected plants also cause toxicity in livestock. HPLC is the typical method used to quantify ergot alkaloid content; however, it is costly and time-consuming. An enzyme-linked immunosorbent assay (ELISA) developed with lysergol as the hapten was evaluated to ascertain its feasibility as an analytical tool for the ergot alkaloids found in forage plants. The ELISA detected the presence of lysergic acid but was not a reliable assay for the ergopeptine alkaloids such as ergovaline. The genetic divergence in mice previously selected into ergot alkaloid susceptible and resistant lines was studied after ten generations of relaxed selection. Physiologically no difference was seen between the susceptible and resistant line for average daily weight gain. However, hepatic metabolism of the ergot alkaloid ergotamine showed differences between genders and between animals on diets containing no ergot alkaloids or a high concentration of ergot alkaloids. Four major biotransformation products were identified as hydroxylated ergotamine isomers based on mass spectroscopic analysis