A simplified genomic profiling approach predicts outcome in metastatic colorectal cancer

The response of metastatic colorectal cancer (mCRC) to the first-line conventional combination therapy is highly variable, reflecting the elevated heterogeneity of the disease. The genetic alterations underlying this heterogeneity have been thoroughly characterized through omic approaches requiring elevated efforts and costs. In order to translate the knowledge of CRC molecular heterogeneity into a practical clinical approach, we utilized a simplified Next Generation Sequencing (NGS) based platform to screen a cohort of 77 patients treated with first-line conventional therapy. Samples were sequenced using a panel of hotspots and targeted regions of 22 genes commonly involved in CRC. This revealed 51 patients carrying actionable gene mutations, 22 of which carried druggable alterations. These mutations were frequently associated with additional genetic alterations. To take into account this molecular complexity and assisted by an unbiased bioinformatic analysis, we defined three subgroups of patients carrying distinct molecular patterns. We demonstrated these three molecular subgroups are associated with a different response to first-line conventional combination therapies. The best outcome was achieved in patients exclusively carrying mutations on TP53 and/or RAS genes. By contrast, in patients carrying mutations in any of the other genes, alone or associated with mutations of TP53/RAS, the expected response is much worse compared to patients with exclusive TP53/RAS mutations. Additionally, our data indicate that the standard approach has limited efficacy in patients without any mutations in the genes included in the panel. In conclusion, we identified a reliable and easy-to-use approach for a simplified molecular-based stratification of mCRC patients that predicts the efficacy of the first-line conventional combination therapy

Highly twisted carbazole-borane derivatives: B\u2013N stereodynamic analysis and consequences on their emission properties

The stereodynamic properties of amino bis-mesityl-boranes bearing carbazole and benzocarbazole as donor heterocycles have been investigated by dynamic NMR analysis and simulated by DFT calculations. The \u3c0-contribution to the B\u2013N bond has been estimated to be 24 kcal mol 121 when carbazole is the donor heterocycle, while a value of 21.7 kcal mol 121 has been found for the benzocarbazole series. Two rotational barriers were determined for the B\u2013N bond, the lower one (11.1\u201316.9 kcal mol 121) leading to conformational enantiomers, and the higher one (21.0\u201324.0 kcal mol 121) likely being responsible for the E-Z isomerization in compounds bearing different aryl rings bound to the boron atom. It has been shown that both kinds of dynamic rearrangements involve a correlated motion of all the three rings. The difference in the ground state geometries and the different \u3c0-contributions led to pronounced variations in the fluorescence spectra, due to different geometric rearrangements in the TICT excited state. Stokes shifts larger than 10\u2006000 cm 121 were observed in the carbazole series, with quantum yields up to 50%. It has been found that the \u3c0-contribution to the B\u2013N bond in the excited state is still significant, with B\u2013N isomerism likely not taking place on the ns scale

The archaeal "7kDa DNA-binding" proteins: extended characterization of an old gifted family

International audienceThe " 7 kDa DNA-binding " family, also known as the Sul7d family, is composed of chromatin proteins from the Sulfolobales archaeal order. Among them, Sac7d and Sso7d have been the focus of several studies with some characterization of their properties. Here, we studied eleven other proteins alongside Sac7d and Sso7d under the same conditions. The dissociation constants of the purified proteins for binding to double-stranded DNA (dsDNA) were determined in phosphate-buffered saline at 25 °C and were in the range from 11 μM to 22 μM with a preference for G/C rich sequences. In accordance with the extremophilic origin of their hosts, the proteins were found highly stable from pH 0 to pH 12 and at temperatures from 85.5 °C to 100 °C. Thus, these results validate eight putative " 7 kDa DNA-binding " family proteins and show that they behave similarly regarding both their function and their stability among various genera and species. As Sac7d and Sso7d have found numerous uses as molecular biology reagents and artificial affinity proteins, this study also sheds light on even more attractive proteins that will facilitate engineering of novel highly robust reagents. In living organisms, the long genomic DNA has to be packed in order to fit into cells, while the genetic information must stay accessible for replication and transcription events. To this aim, organisms have developed different compaction systems, such as the wrapping of DNA around histones to form the chromatin in Eukarya, and the supercoiling of DNA with the help of non-histone proteins to form the nucleoid in Bacteria. Archaea often live in extreme environments and have the additional challenge to protect their genomic DNA from extreme conditions, such as high temperatures. Many Archaea contain homologs of eukaryotic histones, but Desulfurococcales, Thermoplasmatales and Sulfolobales use a different kind of packaging proteins 1,2. Hyperthermophile and acidophile archaea of the Sulfolobales order from the Crenarchaeota kingdom express small basic DNA-binding proteins, which represent about 5% of the total soluble cellular proteins, sufficient to coat the entire genome of a Sulfolobus cell 3. These proteins constitute the family called " 7 kDa DNA-binding " or Sul7d 4. They were first isolated from Sulfolobus acido-caldarius which produces five of them, named Sac7a, b, c, d, and e. Sac7d and Sac7e are encoded by distinct genes, while Sac7a and b are truncated versions of Sac7d 5–7. Highly similar homologs have been found in all Sulfolobus species, such as Sso7d from Sulfolobus solfataricus 8 , and Ssh7a and Ssh7b from Sulfolobus shibatae-two proteins encoded by two distinct genes 3,9. Sac7d and Sso7d are the two most studied proteins of this family. They have been characterized for their structure, function, chemical stability and biophysical properties 7. Sac7d and Sso7d are hyperthermostable (T m = 90.4 °C and 100.2 °C, respectively) 10,11 and are resistant from pH 0 up to at least pH 12 12,13. Although Sac7d and Sso7d sequences show only few differences, Sso7d is more stable than Sac7d. Their three-dimensional structures show that they both fold as an SH3-like domain capped by a C-terminal α-helix 14,15 and that they sharply kink the double DNA helix upon binding into the minor groove 16,17. It has been shown that Sac7d and Sso7d are general dsDNA binders with K D values varying in a salt dependent manner from 20 nM (low salt) to 3.8 μ M (high salt) for Sac7d, and from 116 nM to 12.8 μ M for Sso7d, and with a preference for G/C rich sequences 18,19. Sac7d has the property to increase the thermal stability of DNA duplexes by as much as 43.5 °C 6,15. Furthermore, Ssh7a and Ssh7b have been partially characterized and an affinity for dsDNA of about 100 n

Le Parmigiano Reggiano : évolution de la microflore lactique cultivable et totale et des activités peptidasiques pendant la fabrication et l'affinage

Parmigiano Reggiano is a Protected Designation of Origin, long-ripened cheese, made from cow's milk supplemented with natural whey starter, which thus contains a large microbial biodiversity. The aim of this study was to understand the population dynamics of the total lactic microflora throughout the manufacture and ripening of this cheese. Several approaches were combined to determine the quantitative changes in the different bacterial populations during 20 months of ripening of Parmigiano Reggiano cheeses from the same cheesemaking. Total and viable cells were enumerated after fluorescent labeling. Culturable bacteria were enumerated on different plate count agar media, including original media prepared from curd and ripened cheese. Six peptidase activities were quantified in curd and cheese samples free from cells. While the total bacterial cultivable population remained high and similar for the first six months, a decrease in viable starter lactic acid bacteria was observed during the first 48 h. The non-starter lactic acid bacteria populations, initially present in low numbers, began to grow after the brining and remained at high levels (about 10$^{7 }$ CFU$\cdot$g$^{-1})$ for at least 10 months. During ripening, a strong decrease in the total bacterial population and a marked increase in 4 out of 6 peptidase activities were observed. In the external and internal zones of Parmigiano Reggiano cheese different trends in microbial growth, cell autolysis and peptidase activity were observed. This study gives for the first time a global view of the possible contribution of total, viable, cultivable and lysed bacterial cells throughout the ripening of Parmigiano Reggiano cheese.Le Parmigiano Reggiano est un fromage d'Appellation d'Origine Protégée à affinage long, fabriqué à partir de lait cru supplémenté d'un levain naturel issu du lactosérum. Ce fromage contient, en conséquence, une large biodiversité microbienne. L'objectif de cette étude était de comprendre la dynamique de population de la microflore lactique pendant la fabrication et l'affinage du Parmigiano Reggiano. Plusieurs approches ont été combinées pour déterminer l'évolution des différentes populations bactériennes dans ce fromage au cours de 20 mois d'affinage de fromages issus de la même série de fabrications. Le nombre de bactéries totales et viables a été mesuré après marquage fluorescent. Les bactéries cultivables ont été déterminées en utilisant différents milieux gélosés, incluant des milieux originaux préparés à partir de caillé et de fromage affiné. Six activités peptidasiques ont été quantifiées dans des échantillons de caillé et de fromages exempts de cellules bactériennes. La population de bactéries viables diminuait dans le caillé pendant les 48 premières heures, alors que la population totale de bactéries cultivables restait élevée et constante sur les six premiers mois d'affinage. Les populations de bactéries lactiques non levains, présentes initialement en faible nombre, commençaient à se développer dès le saumurage puis restaient à un niveau élevé (environ 10$^{7 }$ UFC$\cdot$g$^{-1})$ pendant plus de 10 mois. Une forte chute de la population bactérienne totale était observée peandant l'affinage, accompagnée d'une hausse marquée de 4 activités peptidasiques. Dans les parties externes et internes du Parmigiano Reggiano, différentes tendances de croissance, d'autolyse et d'activité peptidasique étaient observées. Cette étude donne pour la première fois un aperçu global de la contribution possible des populations totales, viables, cultivables et lysées tout au long de l'affinage du Parmigiano Reggiano