Biochemical and structural characterization of BLM Helicase

Les Hélicases sont un sous-ensemble d'enzymes qui couplent l'énergie provenant de l'hydrolyse des nucléosides triphosphates (NTP) à la séparation des duplex d'acides nucléiques. Elles sont essentielles au métabolisme de l'ADN et de l'ARN (réparation, réplication, recombinaison et transcription). Les hélicases de la famille de RecQ sont essentielles et ont été très conservées lors de l'évolution. Chez l'humain, trois hélicases de type RecQ sont liées à de graves désordres autosomiques. Ils s'agissent du syndrome de Bloom (BS), du syndrome de Werner (WS) et du syndrome de Rothmund-Thomson (RTS) liés respectivement aux hélicases BLM, WRN et RecQ4. Cette thèse se focalise sur l'analyse des relations structurales et fonctionnelles de BLM. La formation d'érythèmes après exposition au soleil et le retard de croissance sont les principaux phénotypes du BS. La stérilité (ou l'infertilité), l'immunodéficience, et la prédisposition à un large spectre de cancers sont également les symptômes cliniques des patients BS. Les brèches et cassures de chromatides, les réarrangements structuraux de chromosomes, la configuration quadriradiale symétrique, tout comme les associations télomériques ou la quantité excessive de ponts d'anaphase représentent autant d'autres caractéristiques des patients BS. Mais, la haute fréquence d'échanges entre chromatides sœurs (SCE) détectable sous le microscope dans les lymphocytes du sang reste à l'heure actuelle le moyen le plus efficace de diagnostiquer la maladie. Cette thèse présente des résultats nouveaux, publiés dans trois articles scientifiques dan des journaux internationau x, sur les relations entre structure et fonctions de l'hélicase BLM qui peuvent être utiles dans l'identification de nouvelles stratégies thérapeutiques du syndrome de BLOOM.Bloom's syndrome (BS) is an autosomal recessive disorder, showing high frequency of sister chromatid exchange in lymphocyte of the patients. Since BS is preposition of a wide spectrum of cancer, the syndrome has been studied for understanding of the mechanism of cancer extensively. ln the first part, we proved the existence of a zinc-binding domain in which a zinc ion is coordinated by four cysteines residues in RecQ-Ct domain of BLM. This conclusion is drawn from our biophysical and biochemical studies. We modeled the 3D structure of BLM protein based on that of E.coli RecQ helicase, which revealed a similar structural domain in both helicases that coordinate zinc. The results from experiments with three mutants showed that their enzymatic activities were severely reduced or abrogated. The demetalization of zinc from BLM had no influence on the activities of BLM, but it would decrease the themostability of the protein. ln conclusion, BLM contains a zinc binding domain with one zinc ion in each protein. The second part of our studies includes the work for understanding of causative molecular mechanism of missense mutations which happened in helicase domain of BLM found in BS patients. On the basis of the work inthe fist part, we further modeled the 3D structure of BLM in complex with A TPyS and DNA. With the model, we deduced the possible causative mechanism of mutants. We produced mutant proteins and purified them to homogeneity. The A TPase activity, A TP binding activity, DNA binding activity and helicase activity ofthe mutants were ail checked. ln conclusion 1 showed that: 1. BLM642-129o possibly employ an "inchworm" model mechanism; 2. Amino acid residues from 861 to 901 are imprtant for DNA binding; 3. DNA binding ofBLM is mainly controlled by lobe2 and lobe3, lobel contribute to a transient ssDNA binding; 4.The annealing activity of RecQ helicase suggests a weak DNA binding activity.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Neutral Lipid Content in Lipid Droplets: Potential Biomarker of Cordycepin Accumulation in Cordycepin-Producing Fungi

To clarify the relationship between neutral lipid content and cordycepin accumulation in Cordyceps militaris, mutants were generated from mixed spores of two C. militaris strains with varying cordycepin-producing capacities. Fifteen stable mutants producing from 0.001 to 2.363 mg/mL cordycepin were finally selected. The relative fluorescence intensities of the 15 mutants, two C. militaris strains and an Aspergillus nidulans strain at different concentrations of lyophilized mycelium powder were then investigated using the Nile red method. The mutant CM1-1-1 with the highest relative fluorescence intensity among the eighteen strains was selected for optimizing the Nile red method. Relative fluorescence intensity was linearly correlated with cordycepin concentration in liquid broth (R2 = 0.9514) and in lyophilized mycelium powder (R2 = 0.9378) for the 18 cordycepin-producing strains under identical culture conditions and with cordycepin concentration in liquid broth (R2 = 0.9727) and in lyophilized mycelium powder (R2 = 0.9613) for CM1-1-1 under eight different sets of conditions. In addition, the cordycepin content in lyophilized mycelium powder measured by the Nile red method was linearly correlated with that determined by an HPLC method (R2 = 0.9627). In conclusion, neutral lipids in lipid droplets are required during cordycepin accumulation; these neutral lipids are potential biomarkers of cordycepin biosynthesis

Integrated transcriptome and microbiome analyses of residual feed intake in ducks during high production period

ABSTRACT: Residual feed intake (RFI) is a crucial parameter for assessing the feeding efficiency of poultry. Minimizing RFI can enhance feed utilization and reduce costs. In this study, 315 healthy female ducks were individually housed in cages. Growth performance was monitored during the high laying period, from 290 to 325 d of age. The cecal transcriptome and microbiome of 12 ducks with high RFI and 12 with low residual feed intake (LRFI) were analyzed. Regarding growth performance, the LRFI group exhibited significantly lower RFI, feed conversion ratio (FCR), and feed intake (Fi) compared to the HRFI group (p 0.05). Microbiome analysis demonstrated that RFI impacted gut microbial abundance, particularly affecting metabolism and disease-related microorganisms such as Romboutsia, Enterococcus, and Megamonas funiformis. Transcriptome analysis revealed that varying RFI changed the expression of genes related to glucose metabolism and lipid metabolism, including APOA1, G6PC1, PCK1, and PLIN1. The integrated analysis indicated that host genes were closely linked to the microbiota and primarily function in lipid metabolism, which may enhance feeding efficiency by influencing metabolism and maintaining gut homeostasis

Comparative analysis of the hypothalamus transcriptome of laying ducks with different residual feeding intake

ABSTRACT: Feed costs account for approximately 60 to 70% of the cost of poultry farming, and feed utilization is closely related to the profitability of the poultry industry. To understand the causes of the differences in feeding in Shan Partridge ducks, we compared the hypothalamus transcriptome profiles of 2 groups of ducks using RNA-seq. The 2 groups were: 1) low-residual feed intake (LRFI) group with low feed intake but high feed efficiency, and 2) high-residual feed intake (HRFI) group with high feed intake but low feed efficiency. We found 78 DEGs were enriched in 9 differential Kyoto Encyclopedia of Genes and Genome (KEGG) pathways, including neuroactive ligand-receptor interaction, GABAergic synapse, nitrogen metabolism, cAMP signaling pathway, calcium signaling pathway, nitrogen metabolism, tyrosine metabolism, ovarian steroidogenesis, and gluconeogenesis. To further identify core genes among the 78 DEGs, we performed protein-protein interaction and coexpression network analyses. After comprehensive analysis and experimental validation, 4 core genes, namely, glucagon (GCG), cholecystokinin (CCK), gamma-aminobutyric acid type A receptor subunit delta (GABRD), and gamma-aminobutyric acid type A receptor subunit beta1 (GABRB1), were identified as potential core genes responsible for the difference in residual feeding intake between the 2 breeds. We also investigated the level of cholecystokinin (CCK), neuropeptide Y (NPY), peptide YY (PYY), ghrelin, and glucagon-like peptide1 (GLP-1) hormones in the sera of Shan Partridge ducks at different feeding levels and found that there was a difference between the 2 groups with respect to GLP-1 and NPY levels. The findings will serve as a reference for future research on the feeding efficiency of Shan Partridge ducks and assist in promoting their genetic breeding

The impact of global dimming on crop yields is determined by the source–sink imbalance of carbon during grain filling

Global dimming reduces incident global radiation but increases the fraction of diffuse radiation, and thus affects crop yields; however, the underlying mechanisms of such an effect have not been revealed. We hypothesized that crop source–sink imbalance of either carbon (C) or nitrogen (N) during grain filling is a key factor underlying the effect of global dimming on yields. We presented a practical framework to assess both C and N source–sink relationships, using data of biomass and N accumulation from periodical sampling conducted in field experiments for wheat and rice from 2013 to 2016. We found a fertilization effect of the increased diffuse radiation fraction under global dimming, which alleviated the negative impact of decreased global radiation on source supply and sink growth, but the source supply and sink growth were still decreased by dimming, for both C and N. In wheat, the C source supply decreased more than the C sink demand, and as a result, crops remobilized more pre-heading C reserves, in response to dimming. However, these responses were converse in rice, which presumably stemmed from the more increment in radiation use efficiency and the more limited sink size in rice than wheat. The global dimming affected source supply and sink growth of C more significantly than that of N. Therefore, yields in both crops were dependent more on the source–sink imbalance of C than that of N during grain filling. Our revealed source–sink relationships, and their differences and similarities between wheat and rice, provide a basis for designing strategies to alleviate the impact of global dimming on crop productivity.</p