RSF Governs Silent Chromatin Formation via Histone H2Av Replacement

Human remodeling and spacing factor (RSF) consists of a heterodimer of Rsf-1 and hSNF2H, a counterpart of Drosophila ISWI. RSF possesses not only chromatin remodeling activity but also chromatin assembly activity in vitro. While no other single factor can execute the same activities as RSF, the biological significance of RSF remained unknown. To investigate the in vivo function of RSF, we generated a mutant allele of Drosophila Rsf-1 (dRsf-1). The dRsf-1 mutant behaved as a dominant suppressor of position effect variegation. In dRsf-1 mutant, the levels of histone H3K9 dimethylation and histone H2A variant H2Av were significantly reduced in an euchromatic region juxtaposed with heterochromatin. Furthermore, using both genetic and biochemical approaches, we demonstrate that dRsf-1 interacts with H2Av and the H2Av-exchanging machinery Tip60 complex. These results suggest that RSF contributes to histone H2Av replacement in the pathway of silent chromatin formation

Specific Nutritional Requirements of Coconut Calli (Cocos nucifera L.) during Somatic Embryogenesis Induction

Summary Coconut calli were cultivated on two somatic embryogenesis induction media (SEIMs), differing in their 2,4-D content. Gain in dry matter weight, composition of soluble sugars within calli, but also pH and contents of glucose and macroelements in media were analysed at O, 15, 28, and 60 days of culture. Relationships between contents of endogenous sugars, on the one hand, and between contents of media macroelements, on the other hand, were analysed. Comparison was made with calli maintained on a control multiplication medium. Traits could be classified into 3 types of response with regard to condition of somatic embryogenesis induction (SEI condition). The first correspond to traits that were modified by the SEI condition and varying over time. Two phases were determined. During the first phase (TO-T15), soluble sugar contents within calli decreased over time. The higher the 2,4-D content in SEIMs, the higher the sugar contents. Consumption of glucose and macroelements in media was negligible. However, strong relationships in the contents of chloride, nitrate, phosphate, and sulfate were modified in the SEI condition. During the second phase (T15-T60, growth became lower in the SEI condition. Requirements for glucose, nitrate and phosphate and acidification .of media were higher. The relationship, determined by changes in nitrate and phosphate (R>0.98), was modified by the SEI conditions, showing a preferential consumption for nitrate in this case. Endogenous sucrose content decreased to become lower in the SEI condition. The higher the 2,4-D content in SEIMs, the higher the requirements for media compounds, the higher the contents of sugars within calli, but the lower the growth. The second type of response corresponded to traits modified by the SEI condition, but constant over time. It concerned relationships between contents of some cations in the media. The third type of response corresponded to traits unchanges by the SEI condition and over time. It concerned the high relationship contents of endogenous glucose and fructose (R = 0.88), and between contents of chloride, ammonium, calcium, magnesium, and potassium. Abbreviations: ANOVA = analysis of variance; ANCOVA = analysis of covariance; BAP = 6-benzylaminopurine; D M W = dry matter weight; FMW = fresh matter weight; % WC = percentage of water content; SEIM = somatic embryogenesis induction medium; TFMW = total fresh matter weight

Development and validation of a multiplex UHPLC-MS/MS method for the determination of the investigational antibiotic against multi-resistant tuberculosis macozinone (PBTZ169) and five active metabolites in human plasma.

The emergence of Mycobacterium tuberculosis strains resistant to current first-line antibiotic regimens constitutes a major global health threat. New treatments against multidrug-resistant tuberculosis (MDR-TB) are thus eagerly needed in particular in countries with a high MDR-TB prevalence. In this context, macozinone (PBTZ169), a promising drug candidate with an unique mode of action and highly potent in vitro tuberculocidal properties against MDR Mycobacterium strains, has now reached the clinical phase and has been notably tested in healthy male volunteers in Switzerland. To that endeavor, a multiplex UHPLC-MS/MS method has been developed for the sensitive and accurate human plasma levels determination of PBTZ169 along with five metabolites retaining in vitro anti-TB activity. Plasma protein precipitation with methanol was carried out as a simplified sample clean-up procedure followed by direct injection of the undiluted supernatant for the bioanalysis of the six analytes within 5 min, using 1.8 μm reversed-phase chromatography coupled to triple quadrupole mass spectrometry employing electrospray ionization in the positive mode. Stable isotopically-labelled PBTZ169 was used as internal standard (ISTD), while metabolites could be reliably quantified using two unlabeled chemical analogues selected as ISTD from a large in-house analogous compounds library. The overall methodology was fully validated according to current recommendations (FDA, EMEA) for bioanalytical methods, which include selectivity, carryover, qualitative and quantitative matrix effect, extraction recovery, process efficiency, trueness, precision, accuracy profiles, method and instrument detection limits, integrity to dilution, anticoagulant comparison and short- and long-term stabilities. Stability studies on the reduced metabolite H2-PBTZ169 have shown no significant impact on the actual PBTZ169 concentrations determined with the proposed assay. This simplified, rapid, sensitive and robust methodology has been applied to the bioanalysis of human plasma samples collected within the frame of a phase I clinical study in healthy volunteers receiving PBTZ169