Semipreparative enantioseparation of Tröger base derivatives by HPLC

We describe the enantioseparation of functionalized derivatives of the Tr?ger base by HPLC on commercially available chiral stationary phases. Cellulose-derived Chiralcel OJ and brush-type Whelk O1 are demonstrated to be complementary to each other in their scope. On the basis of the results obtained, the separation of selected compounds was successfully transferred onto semipreparative columns. We believe that the availability of enantiopure functionalized derivatives of the Tr?ger base will stimulate the further use of this interesting molecular scaffold in molecular recognition, asymmetric catalysis, and related areas of research and technology.     

Tactile perception and manual dexterity in computer users

Recent studies suggest that sensory input generated during highly repetitive tasks can degrade the sensory representation of the hand and eventually lead to sensory and motor problems. In this study, we investigated whether early changes in tactile perception and manual dexterity could be detected in persons exposed to computer tasks. Performance in tests designed to assess tactile perception (grating orientation task for spatial acuity and roughness discrimination) and manual dexterity (grooved pegboard test) was compared between two groups of healthy individuals, matched for age, gender, and experience, who differed in terms of computer habits. One group consisted of frequent users (FU, > 2 h/day, n = 36) and the other of non or occasional users (OU, < 2 h/day, n = 28). Comparison of performance between groups with subjects sorted by gender revealed significant differences ( t -test, p < 0.05) in female, but not male, participants. Grating resolution thresholds at the tip on the second and fifth digits were, on average, 40% higher in female FU ( n = 13) than in female OU ( n = 10) and performance scores on the dexterity test were significantly higher for the left hand. The results of this study indicate that early signs of deterioration in hand function can be present in persons constantly exposed to computer tasks and that these signs are more readily apparent in women than in men. The loss of tactile spatial acuity found in female FU possibly reflect an early consequence of the degraded sensory representation of the hand resulting from constant repetitions of fine motor tasks.     

Syntheses and properties of trimethylaminophenoxy-substituted Zn((II))-phthalocyanines

The syntheses, photophysical properties and in vitro biological behavior of a series of nine Zn((II))-phthalocyanines (ZnPcs) bearing one to eight positively-charged trimethylaminophenoxy groups are reported. All ZnPcs are highly soluble in polar organic solvents, and show fluorescence and singlet oxygen quantum yields in the ranges 0.11-0.21 and 0.16-0.47, respectively. The cytotoxicity of the ZnPcs depends on both the number of charges and their site of substitution (α vs. β) on the Pc isoindole units; the most promising for PDT application are the α-substituted di-cationic ZnPcs 6a and 17a.     

An accurate definition of the status of inactive hepatitis B virus carrier by a combination of biomarkers (FibroTest-ActiTest) and viral load

Background

The combination of transaminases (ALT), biopsy, HBeAg and viral load have classically defined the inactive status of carriers of chronic hepatitis B. The use of FibroTest (FT) and ActiTest (AT), biomarkers of fibrosis and necroinflammatory activity, has been previously validated as alternatives to biopsy. We compared the 4-year prognostic value of combining FT-AT and viral load for a better definition of the inactive carrier status.

Methods and Findings

1,300 consecutive CHB patients who had been prospectively followed since 2001 were pre-included. The main endpoint was the absence of liver-related complications, transplantation or death. We used the manufacturers'' definitions of normal FT (< = 0.27), normal AT (< = 0.29) and 3 standard classes for viral load. The adjustment factors were age, sex, HBeAg, ethnic origin, alcohol consumption, HIV-Delta-HCV co-infections and treatment.

Results

1,074 patients with baseline FT-AT and viral load were included: 41 years old, 47% African, 27% Asian, 26% Caucasian. At 4 years follow-up, 50 complications occurred (survival without complications 93.4%), 36 deaths occurred (survival 95.0%), including 27 related to HBV (survival 96.1%). The prognostic value of FT was higher than those of viral load or ALT when compared using area under the ROC curves [0.89 (95%CI 0.84–0.93) vs 0.64 (0.55–0.71) vs 0.53 (0.46–0.60) all P<0.001], survival curves and multivariate Cox model [regression coefficient 5.2 (3.5–6.9; P<0.001) vs 0.53 (0.15–0.92; P = 0.007) vs −0.001 (−0.003−0.000;P = 0.052)] respectively. A new definition of inactive carriers was proposed with an algorithm combining “zero” scores for FT-AT (F0 and A0) and viral load classes. This new algorithm provides a 100% negative predictive value for the prediction of liver related complications or death. Among the 275 patients with the classic definition of inactive carrier, 62 (23%) had fibrosis presumed with FT, and 3 died or had complications at 4 year.

Conclusion

In patients with chronic hepatitis B, a combination of FibroTest-ActiTest and viral load testing accurately defined the prognosis and the inactive carrier status.     

Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.     

Reduced fertility of female mice lacking CD81

In somatic cells, the tetraspanins CD81 and CD9 associate with each other, with additional tetraspanins and with non-tetraspanin molecules to form proteolipidic complexes. Here we show that CD81 is expressed on the surface of oocytes where it associates with tetraspanin-enriched membrane structures. A major CD9 and CD81 partner, CD9P-1, is also expressed by oocytes. Deletion of CD81 gene in mice results in a 40% reduction of female fertility. In vitro insemination indicated that this infertility is due to a deficiency of oocytes to fuse with sperm. While the fertility of CD9-/- mice is severely but not completely impaired, double knock-out CD9-/- CD81-/- mice were completely infertile indicating that CD9 and CD81 play complementary roles in sperm-egg fusion. Finally, a fraction of CD9 was transferred from CD81-/- oocytes to sperm present in the perivitelline space indicating that the defect of fusion of CD81-/- oocytes does not result from an impaired initial gamete interaction.     

Effects of chemical modifications of crotoxin B, the phospholipase A(2) subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activities.

Crotoxin B, the basic Asp49-PLA(2) subunit from crotoxin, the main component of Crotalus durissus terrificus venom, displays myotoxic, edema-inducing, bactericidal (upon Escherichia coli), liposomal-disrupting and anticoagulant activities. Chemical modifications of His (with 4-bromophenacyl bromide, BPB), Tyr (with 2-nitrobenzenesulphonyl fluoride, NBSF), Trp (with o-nitrophenylsulphenyl chloride, NPSC) and Lys (with acetic anhydride) residues of this protein, in addition to cleavage with cyanogen bromide (CNBr) and inhibition with ethylenediaminetetraacetic acid (EDTA), were carried out in order to study their effects on enzymatic and pharmacological activities. Lethality was reduced after modification of His or Lys residues, as well as after cleavage with CNBr, while enzymatic activity was completely abolished after modification of His or incubation with EDTA. Modification of Lys or Tyr, or cleavage with CNBr, partially reduced enzymatic activity. Anticoagulant activity was modified similarly to enzymatic activity, evidencing the dependency of this pharmacological effect on catalytic activity. Myotoxicity was reduced after modification of His or Lys, as well as after cleavage with CNBr, whereas EDTA reduced this effect to a lesser extent. Bactericidal effect was significantly reduced only after modification of Lys and after cleavage with CNBr. Edema-inducing activity was partially inhibited after treatment with EDTA and strongly reduced after acetylation of Lys residues and cleavage with CNBr, being only partially reduced after His alkylation. On the other hand, liposome disrupting activity was only partially reduced after modification of His and Tyr or after cleavage with CNBr. Modification of Trp residue partially reduced lethality and myotoxicity but did not affect enzymatic or anticoagulant activities. These data indicate that enzymatic activity is relevant for some pharmacological effects induced by crotoxin B (mainly lethal, myotoxic and anticoagulant activities), and also evidence that this subunit of crotoxin displays regions different from the active catalytic site which are involved in some of the toxic and pharmacological effects induced by this phospholipase A(2).     

Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.     

Dihydroxynonene mercapturic acid, a urinary metabolite of 4-hydroxynonenal, as a biomarker of lipid peroxidation

The objective of our study was to compare the information obtained through the use of three different urinary biomarkers of lipoperoxidation during the time course of a bromotrichloromethane (BrCCl3) induced oxidative stress in rats. These biomarkers were malondialdehyde (MDA) measured by LC/MS after derivatization, the isoprostane 8-iso-PGF2alpha measured by enzyme immunoassay and 1,4-dihydroxynonene mercapturic acid (DHN-MA), the major 4-hydroxynonenal urinary metabolite [1], measured by LC-MS. Male Wistar rats received a single dose of 100 microL/kg BrCCl3 per os and lipid peroxidation was estimated every day for a 4-day-period after treatment. MDA, 8-iso-PGF2alpha and DHN-MA significantly increased in response to BrCCl3 treatment for this period of time, and DHN-MA showed the main increase during the 24-48 h period after treatment.