氟中毒及补钙干预大鼠骨骼差异蛋白的分离与鉴定

为从蛋白水平探索钙缓解氟中毒的作用机制,本试验建立氟中毒和补钙干预动物模型进行骨骼差异蛋白的分离鉴定。通过双向凝胶电泳技术(Two-dimensional electrophoresis,2-DE)和基质辅助质量飞行时间质谱技术(MALDI-TOF MS)鉴定骨骼中的差异蛋白质,并通过gene ontology (GO)注释、pathway富集和互作网络对差异蛋白进行分析。结果表明,与对照组(C)相比,在氟中毒组(F)和氟中毒补钙组(F+Ca)中有17种差异性的蛋白,包括Ⅰ型胶原蛋白(Col1a1)、肌动蛋白(Actb)、蛋白质谷氨酰胺转移酶2 (Tgm2)等。这些差异蛋白富集到黏着斑(Focal adhesion)、PI3K-Akt信号通路、AMPK信号通路等38条骨代谢通路。并且这些蛋白的功能主要与细胞骨架、能量代谢、物质转运、离子通道、细胞凋亡有关。因此推测,钙可能通过调控黏着斑、PI3K-Akt、AMPK等信号通路进而缓解氟对骨骼带来的损伤,具体作用机制还需要进一步验证。     

MALDI-TOF Mass Spectrometry: A Powerful Tool for Clinical Microbiology at H?pital Principal de Dakar,Senegal (West Africa)

Our team in Europe has developed the routine clinical laboratory identification of microorganisms by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). To evaluate the utility of MALDI-TOF MS in tropical Africa in collaboration with local teams, we installed an apparatus in the Hôpital Principal de Dakar (Senegal), performed routine identification of isolates, and confirmed or completed their identification in France. In the case of discordance or a lack of identification, molecular biology was performed. Overall, 153/191 (80.1%) and 174/191 (91.1%) isolates yielded an accurate and concordant identification for the species and genus, respectively, with the 2 different MALDI-TOF MSs in Dakar and Marseille. The 10 most common bacteria, representing 94.2% of all bacteria routinely identified in the laboratory in Dakar (Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, Enterobacter cloacae, Enterococcus faecalis, and Staphylococcus epidermidis) were accurately identified with the MALDI-TOF MS in Dakar. The most frequent misidentification in Dakar was at the species level for Achromobacter xylosoxidans, which was inaccurately identified as Achromobacter denitrificans, and the bacteria absent from the database, such as Exiguobacterium aurientacum or Kytococcus schroeteri, could not be identified. A few difficulties were observed with MALDI-TOF MS for Bacillus sp. or oral streptococci. 16S rRNA sequencing identified a novel bacterium, “Necropsobacter massiliensis.” The robust identification of microorganisms by MALDI-TOF MS in Dakar and Marseille demonstrates that MALDI-TOF MS can be used as a first-line tool in clinical microbiology laboratories in tropical countries.     

Peroxidase Activity and Involvement in the Oxidative Stress Response of Roseobacter denitrificans Truncated Hemoglobin

Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm^-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (k_cat/K_m) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pK_a value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry.     

Onchocerciasis: Target product profiles of in vitro diagnostics to support onchocerciasis elimination mapping and mass drug administration stopping decisions

In June 2021, the World Health Organization (WHO), recognizing the need for new diagnostics to support the control and elimination of onchocerciasis, published the target product profiles (TPPs) of new tests that would support the two most immediate needs: (a) mapping onchocerciasis in areas of low prevalence and (b) deciding when to stop mass drug administration programs. In both instances, the test should ideally detect an antigen specific for live, adult O. volvulus female worms. The preferred format is a field-deployable rapid test. For mapping, the test needs to be ≥ 60% sensitive and ≥ 99.8% specific, while to support stopping decisions, the test must be ≥ 89% sensitive and ≥ 99.8% specific. The requirement for extremely high specificity is dictated by the need to detect with sufficient statistical confidence the low seroprevalence threshold set by WHO. Surveys designed to detect a 1–2% prevalence of a given biomarker, as is the case here, cannot tolerate more than 0.2% of false-positives. Otherwise, the background noise would drown out the signal. It is recognized that reaching and demonstrating such a stringent specificity criterion will be challenging, but test developers can expect to be assisted by national governments and implementing partners for adequately powered field validation.     

Mechanistic study of HCV polymerase inhibitors at individual steps of the polymerization reaction

Little is known about the mechanism of HCV polymerase-catalyzed nucleotide incorporation and the individual steps employed by this enzyme during a catalytic cycle. In this paper, we applied various biochemical tools and examined the mechanism of polymerase catalysis. We found that formation of a productive RNA-enzyme complex is the slowest step followed by RNA dissociation and initiation of primer strand synthesis. Various groups have reported several classes of small molecule inhibitors of hepatitis C virus NS5B polymerase; however, the mechanism of inhibition for many of these inhibitors is not clear. We undertook a series of detailed mechanistic studies to characterize the mechanisms of inhibition for these HCV polymerase inhibitors. We found that the diketoacid derivatives competitively bind to the elongation NTP pocket in the active site and inhibit both the initiation and elongation steps of polymerization. While both benzimidazoles and benzothiadiazines are noncompetitive with respect to the active site elongation NTP pocket, benzothiadiazine compounds competitively bind to the initiation pocket in the active site and inhibit only the initiation step of de novo RNA polymerization. The benzimidazoles bind to the thumb allosteric pocket and inhibit the conformational changes during RNA synthesis. We also observed a cross interaction between the thumb allosteric pocket and the initiation pocket using inhibitor-inhibitor cross competition studies. This information will be very important in designing combination therapies using two small molecule drugs to treat hepatitis C virus.     

脊椎动物的大脑皮层

脊椎动物的大脑皮层历经由无到有,由简单到复杂的进化过程。简要概述了脊椎动物的大脑皮层的发生,以及在各类群的差异和演化情况。     

Hepatitis C NS5B polymerase inhibitors: 4,4-Dialkyl-1-hydroxy-3-oxo-3,4-dihydronaphthalene-3-yl benzothiadiazine derivatives

4,4-Dialkyl-1-hydroxy-3-oxo-3.4-dihydronaphthalene-3-yl benzothiadiazine derivatives were synthesized and evaluated as inhibitors of genotypes 1a and 1b HCV NS5B polymerase. A number of these compounds exhibited potent activity against genotypes 1a and 1b HCV polymerase in both enzymatic and cell culture activities. A representative compound also showed favorable pharmacokinetics in the rat.     

Filarial infection suppresses malaria-specific multifunctional Th1 and Th17 responses in malaria and filarial coinfections

The mechanisms underlying the modulation of both the malaria-specific immune response and the course of clinical malaria in the context of concomitant helminth infection are poorly understood. We used multiparameter flow cytometry to characterize the quality and the magnitude of malaria-specific T cell responses in filaria-infected and -uninfected individuals with concomitant asymptomatic Plasmodium falciparum malaria in Mali. In comparison with filarial-uninfected subjects, filarial infection was associated with higher ex vivo frequencies of CD4(+) cells producing IL-4, IL-10, and IL-17A (p = 0.01, p = 0.001, and p = 0.03, respectively). In response to malaria Ag stimulation, however, filarial infection was associated with lower frequencies of CD4(+) T cells producing IFN-γ, TNF-α, and IL-17A (p < 0.001, p = 0.04, and p = 0.04, respectively) and with higher frequencies of CD4(+)IL10(+)T cells (p = 0.0005). Importantly, filarial infection was associated with markedly lower frequencies of malaria Ag-specific Th1 (p < 0.0001), Th17 (p = 0.012), and "TNF-α" (p = 0.0008) cells, and a complete absence of malaria-specific multifunctional Th1 cells. Filarial infection was also associated with a marked increase in the frequency of malaria-specific adaptive regulatory T/Tr1 cells (p = 0.024), and the addition of neutralizing anti-IL-10 Ab augmented the amount of Th1-associated cytokine produced per cell. Thus, among malaria-infected individuals, concomitant filarial infection diminishes dramatically the frequencies of malaria-specific Th1 and Th17 T cells, and alters the quality and magnitude of malaria-specific T cell responses.     

Impact of esterified bacteriochlorophylls on the biogenesis of chlorosomes in Chloroflexus aurantiacus

A chlorosome is an antenna complex located on the cytoplasmic side of the inner membrane in green photosynthetic bacteria that contains tens of thousands of self-assembled bacteriochlorophylls (BChls). Green bacteria are known to incorporate various esterifying alcohols at the C-17 propionate position of BChls in the chlorosome. The effect of these functional substitutions on the biogenesis of the chlorosome has not yet been fully explored. In this report, we address this question by investigating various esterified bacteriochlorophyll c (BChl c) homologs in the thermophilic green non-sulfur bacterium Chloroflexus aurantiacus. Cultures were supplemented with exogenous long-chain alcohols at 52 °C (an optimal growth temperature) and 44 °C (a suboptimal growth temperature), and the morphology, optical properties and exciton transfer characteristics of chlorosomes were investigated. Our studies indicate that at 44 °C Cfl. aurantiacus synthesizes more carotenoids, incorporates more BChl c homologs with unsaturated and rigid polyisoprenoid esterifying alcohols and produces more heterogeneous BChl c homologs in chlorosomes. Substitution of phytol for stearyl alcohol of BChl c maintains similar morphology of the intact chlorosome and enhances energy transfer from the chlorosome to the membrane-bound photosynthetic apparatus. Different morphologies of the intact chlorosome versus in vitro BChl aggregates are suggested by small-angle neutron scattering. Additionally, phytol cultures and 44 °C cultures exhibit slow assembly of the chlorosome. These results suggest that the esterifying alcohol of BChl c contributes to long-range organization of BChls, and that interactions between BChls with other components are important to the assembly of the chlorosome. Possible mechanisms for how esterifying alcohols affect the biogenesis of the chlorosome are discussed.