High-speed liquid chromatographic method for the monitoring of carbamazepine and its active metabolite, carbamazepine-10,11-epoxide, in human plasma

The assays of antiepileptic drugs, which are performed by central laboratories in Phase II and III clinical trials, require both a very fast turn-around time and a suitable specificity. In order to decrease the run time and to keep the powerful specificity of the liquid chromatography (HPLC), the use of a reversed-phase 1.5 μm monosized non-porous silicon dioxide microspheres column instead of regular columns containing spherical porous C₁₈ material was studied. The determination of carbamazepine (CBZ) and its active metabolite, carbamazepine-10,11-epoxide (CBZ-E), in human plasma or serum was chosen to demonstrate the utility of these columns. As a prerequisite of this work, no modification of a regular HPLC system was allowed. The samples were prepared in autosampler vials by protein precipitation with acetonitrile, followed by a quick centrifugation. Without any change to a conventional HPLC system, CBZ and CBZ-E are well separated in less than 2.5 min using a Kovasil MS C₁₄ column. No interference was observed with endogenous compounds and with nine antiepileptic drugs commonly prescribed as co-medication, and their metabolites. Due to the very low specific surface area of the packing, the required organic modifier volume per chromatographic run was decreased by a factor of 25. The method was validated. The developed method is well suited for the determination of CBZ and CBZ-E in clinical trials. It can be easily adapted to the monitoring of other antiepileptic drugs. No modification of a regular HPLC system was required.     

Spécificité des interactions DNA-peptides et DNA-histones en spectroscopie de résonance Raman dans l'ultraviolet

Resonance Raman spectra of complexes between DNA and peptides on one hand, DNA and histones on the other, were obtained.Our work has clearly shown a DNA-base recognition by various protides. The basic residues selectively modify the environment of DNA bases : G-C bases for the lysine residues, A-T bases for the arginine residues, wether the residues are alone or included in a protein. This selectivity of interaction has allowed us to confirm the particular role played by H4 in the structure of nucleosomes and specially of the arginine residues.     

Cutting Edge: Influenza A virus activates TLR3-dependent inflammatory and RIG-I-dependent antiviral responses in human lung epithelial cells

Influenza A virus (IAV) triggers a contagious acute respiratory disease that causes considerable mortality annually. Recently, we established a role for the pattern-recognition TLR3 in the response of lung epithelial cells to IAV-derived dsRNA. However, additional nucleic acid-recognition proteins have lately been implicated as key viral sensors, including the RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene (MDA)-5. In this study, we investigated the respective role of TLR3 vs RIG-I/MDA-5 signaling in human respiratory epithelial cells infected by IAV using BEAS-2B cells transfected with vectors encoding either a dominant-negative form of TLR3 or of mitochondrial antiviral signaling protein (MAVS; a signaling intermediate of RIG-I and MDA-5), or with plasmids overexpressing functional RIG-I or MDA-5. We demonstrate that the sensing of IAV by TLR3 primarily regulates a proinflammatory response, whereas RIG-I (but not MDA-5) mediates both a type I IFN-dependent antiviral signaling and a proinflammatory response.     

Leprosy and cutaneous leishmaniasis affecting the same individuals: A retrospective cohort analysis in a hyperendemic area in Brazil

BackgroundLeprosy and cutaneous leishmaniasis (CL) are neglected tropical diseases (NTDs) affecting the skin. Their control is challenging but the integration of skin NTDs control programs is recommended to improve timely detection and treatment. However, little is known about the occurrence of leprosy and CL in the same individuals, and what are the characteristics of such patients. This study aimed to identify and characterize patients diagnosed with both leprosy and CL (i.e., outcome) in the hyperendemic state of Mato Grosso, Brazil. Also, we investigated the demographic risk factors associated with the period between the diagnosis of both diseases.Methodology/principal findingsA retrospective cohort study was conducted with patients diagnosed between 2008 and 2017. From the leprosy (n = 28,204) and CL (n = 24,771) databases of the national reporting system, 414 (0.8%; 414/52,561) patients presenting both diseases were identified through a probabilistic linkage procedure. This observed number was much higher than the number of patients that would be expected by chance alone (n = 22). The spatial distribution of patients presenting the outcome was concentrated in the North and Northeast mesoregions of the state. Through survival analysis, we detected that the probability of a patient developing both diseases increased over time from 0.2% in the first year to 1.0% within seven years. Further, using a Cox model we identified male sex (HR: 2.3; 95% CI: 1.7–2.9) and low schooling level (HR: 1.5; 95% CI: 1.2–1.9) as positively associated with the outcome. Furthermore, the hazard of developing the outcome was higher among individuals aged 40–55 years.Conclusions/significanceLeprosy and CL are affecting the same individuals in the area. Integration of control policies for both diseases will help to efficiently cover such patients. Measures should be focused on timely diagnosis by following-up patients diagnosed with CL, active case detection, and training of health professionals.     

Molecular architecture and oligomerization of Candida glabrata Cdc13 underpin its telomeric DNA-binding and unfolding activity

The CST complex is a key player in telomere replication and stability, which in yeast comprises Cdc13, Stn1 and Ten1. While Stn1 and Ten1 are very well conserved across species, Cdc13 does not resemble its mammalian counterpart CTC1 either in sequence or domain organization, and Cdc13 but not CTC1 displays functions independently of the rest of CST. Whereas the structures of human CTC1 and CST have been determined, the molecular organization of Cdc13 remains poorly understood. Here, we dissect the molecular architecture of Candida glabrata Cdc13 and show how it regulates binding to telomeric sequences. Cdc13 forms dimers through the interaction between OB-fold 2 (OB2) domains. Dimerization stimulates binding of OB3 to telomeric sequences, resulting in the unfolding of ssDNA secondary structure. Once bound to DNA, Cdc13 prevents the refolding of ssDNA by mechanisms involving all domains. OB1 also oligomerizes, inducing higher-order complexes of Cdc13 in vitro. OB1 truncation disrupts these complexes, affects ssDNA unfolding and reduces telomere length in C. glabrata. Together, our results reveal the molecular organization of C. glabrata Cdc13 and how this regulates the binding and the structure of DNA, and suggest that yeast species evolved distinct architectures of Cdc13 that share some common principles.