Return to the RNAi world: rethinking gene expression and evolution (Nobel Lecture)

It's wonderful to be here today, I would like to start with the most important part, by saying thank you. First of all, I want to thank Andy Fire for being such a tremendous colleague, friend, and collaborator going back over the years. Without Andy I definitely wouldn't be here today. I need to thank the University of Massachusetts for providing for my laboratory, for believing in me, and for giving me not only a place and money, but great colleagues with whom to pursue my research. Without UMass and the great environment provided for me there, I probably would not be here today. And, of course my family; I'm not going to spend time now thanking them individually, but they know how important they are.     

Hibernating Bears,Antibiotics, and the Evolving Ribosome (Nobel Lecture)

High‐resolution structures of ribosomes, the cellular machines that translate the genetic code into proteins, revealed the decoding mechanism, detected the mRNA path, identified the sites of the tRNA molecules in the ribosome, elucidated the position and the nature of the nascent proteins exit tunnel, illuminated the interactions of the ribosome with non‐ribosomal factors, such as the initiation, release and recycling factors. Notably, these structures proved that the ribosome is a ribozyme whose active site, namely where the peptide bonds are being formed, is situated within a universal symmetrical region that is embedded in the otherwise asymmetric ribosome structure. As this symmetrical region is highly conserved and provides the machinery required for peptide bond formation and for ribosome polymerase activity, it may be the remnant of the proto‐ribosome, a dimeric prebiotic machine that formed peptide bonds and non‐coded polypeptide chains. Structures of complexes of ribosomes with antibiotics targeting them revealed the principles allowing for their clinical use, identified resistance mechanisms and showed the structural bases for discriminating pathogenic bacteria from hosts, hence providing valuable structural information for antibiotics improvement and for the design of novel compounds that can serve as antibiotics.     

Mass spectrometrical analysis of bilin-binding protein from the wing of Hebomoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)

Bilin-binding protein (BBP) is a member of the lipocalin superfamily and a pigment protein in Lepidoptera. It is binding to a series of lipidic compounds but its functions remain to be elucidated. Working on wing proteins in Hebomoia glaucippe, we observed this protein on gels and decided to characterize BBP. A gel-based mass spectrometrical method using two-dimensional gel electrophoresis followed by in-gel digestion of protein spots followed by nano-LC-ESI-MS/MS (ion trap, HCT) identification and characterization of proteins was applied. An antibody was generated against the protein and immunoblotting in the butterfly and mouse brain was carried out. Two spots were identified from the butterfly wing as BBP (P09464) with high sequence coverage. Nitrotyrosination (Y163; as aminotyrosine) was observed and nitration was verified using immunoblotting. Additional posttranslational modifications (PTMs) as hypusine, carboxylation, kynurenine, aminoadipic acid, were proposed. The presence of BBP-immunoreactive protein was also observed in mouse brain. The characterization of BBP showed high sequence similarity with mouse apolipoprotein D and the findings suggest a tentative function of BBP comparable to apolipoproteins. The role of the PTMs remains elusive but nitration, in analogy to nitration effects reported in literature, proposes a role for mechanoelastic proteins and protein-protein interactions.     

Primary structure of p-momorcharin, a ribosome-inactivating protein from the seeds of Momordica Charantia Linn (Cucurbitaceae)

The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S. aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 5 1 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.     

Primary structure of β-momorcharin,a ribosome-inactivating protein from the seeds of Momordica Charantia Linn (Cucurbitaceae)

The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S.aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 51 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.     

The human keratinocyte two-dimensional gel protein database (update 1992): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.

The master two-dimensional gel database of human keratinocytes currently lists 2980 cellular proteins (2098 isoelectric focusing, IEF; and 882 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to posttranslational modifications. About 20% of all recorded proteins have been identified (protein name, organelle components, etc.) and they are listed in alphabetical order together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Also, we have listed 145 microsequenced proteins that are recorded in this database. As an aid in localizing the polypeptides we have included blow-ups of the master images (IEF, NEPHGE) displaying all the protein numbers. In the long run, the master keratinocyte database is expected to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease.     

pH-induced changes in the fabrication of multilayers of poly(acrylic acid) and chitosan: fabrication, properties, and tests as a drug storage and delivery system

Multilayers of poly(acrylic acid), PAA, and chitosan, CHI, have been built by the layer-by-layer (LbL) method from aqueous solutions at different pH values and analyzed by the dissipative quartz crystal microbalance (D-QCM) and ellipsometry. The results showed that under all of the assembly conditions considered the growth of the films is nonlinear. The thickness of the PAA layers increases as the pH of the assembling solutions decreases, whereas the adsorption of CHI is almost unaffected by the pH conditions. The comparison of the thickness obtained by D-QCM and by ellipsometry has allowed us to calculate the water content of the films, showing that the multilayers are highly hydrated, with an average water content higher than 20%. The analysis of D-QCM data has provided high-frequency values of the complex shear modulus that are in the megapascal range and shows a transition from mainly viscous to mainly elastic behavior for the added PAA layers, depending on the pH. The monomer surface density in each layer (obtained from the combination of ellipsometry and differential refractive index measurements) indicated that the monomer density depends on the assembly conditions. It was found that the adsorption kinetics is a bimodal process, with characteristic times that depend on the number and nature of the layers. Finally, the possibility of using of these multilayers as a drug storage and delivery system has been evaluated.     

Facile and efficient synthesis of polyfunctionalized benzofurans: three-component coupling reactions from an alkynylsilane, an o-hydroxybenzaldehyde derivative, and a secondary amine by a Cu(I)-Cu(II) cooperative catalytic system

The combination of 5 mol % Cu(OTf)₂ and CuCl in the presence of DMAP effectively catalyzed a three-component coupling reaction involving an alkynylsilane, an o-hydroxybenzaldehyde derivative, and a secondary amine. The reaction proceeded via intramolecular 5-exo-dig cyclization, resulting in direct synthesis of the corresponding benzofuran derivatives in moderate to excellent yields.     

Investigation of the reduced high-potential iron-sulfur protein from chromatium vinosum and relevant model compounds: a unified picture of the electronic structure of [Fe(4)S(4)](2+) systems through magnetic and optical studies

Magnetization measurements and variable temperature optical spectroscopy have been used to investigate, within the 4-300 K temperature range, the electronic structure of the reduced high-potential iron protein (HiPIP) from Chromatium vinosum and the model compounds (Cat)(2)[Fe(4)S(4)(SR)(4)], where RS(-) = 2,4,6-triisopropylphenylthiolate (1), 2,6-diphenylphenylthiolate (2), diphenylmethylthiolate (3), 2,4,6-triisopropylbenzylthiolate (4, 4'), 2,4,6-triphenylbenzylthiolate (5, 5'), 2,4,6-tri-tert-butylbenzylthiolate (6), and Cat(+) = (+)NEt(4) (1, 2, 3, 4', 5', 6), (+)PPh(4) (4, 5). The newly synthesized 2(2)(-), 3(2)(-), 5(2)(-), and 6(2)(-) complexes are, as 1(2)(-) and 4(2)(-), excellent models of the reduced HiPIPs: they exhibit the [Fe(4)S(4)](3+/2+) redox couple, because of the presence of bulky ligands which stabilize the [Fe(4)S(4)](3+) oxidized core. Moreover, the presence of SCH(2) groups in 4(2)(-), 5(2)(-), and 6(2)(-), as in the [Fe(4)S(4)] protein cores, makes them good biomimetic models of the HiPIPs. The X-ray structure of 2 is reported: it crystallizes in the orthorhombic space group Pcca with no imposed symmetry and a D(2)(d)()-distorted geometry of the [Fe(4)S(4)](2+) core. Fit of the magnetization data of the reduced HiPIP and of the 1, 2, 3, 4, 5, and 6 compounds within the exchange and double exchange theoretical framework leads to exchange coupling parameters J = 261-397 cm(-)(1). A firm determination of the double exchange parameters B or, equivalently, the transfer integrals beta = 5B could not be achieved that way. The obtained |B| values remain however high, attesting thus to the strength of the spin-dependent electronic delocalization which is responsible for lowest lying electronic states being characterized by delocalized mixed-valence pairs of maximum spin (9)/(2). Electronic properties of these systems are then accounted for by the population of a diamagnetic ground level and excited paramagnetic triplet and quintet levels, which are respectively J and 3J above the ground level. Optical studies of 1, 2, 4', 5', and 6 but also of (NEt(4))(2)[Fe(4)S(4)(SCH(2)C(6)H(5))(4)] and the isomorph (NEt(4))(2)[Fe(4)S(4)(S-t-Bu)(4)] and (NEt(4))(2)[Fe(4)Se(4)(S-t-Bu)(4)] compounds reveal two absorption bands in the near infrared region, at 705-760 nm and 1270-1430 nm, which appear to be characteristic of valence-delocalized and ferromagnetically coupled [Fe(2)X(2)](+) (X = S, Se) units. The |B| and |beta| values can be directly determined from the location at 10|B| of the low-energy band, and are respectively of 699-787 and 3497-3937 cm(-)(1). Both absorption bands are also present in the 77 K spectrum of the reduced HiPIP, at 700 and 1040 nm (Cerdonio, M.; Wang, R.-H.; Rawlings, J.; Gray, H. B. J. Am. Chem. Soc. 1974, 96, 6534-6535). The blue shift of the low-energy band is attributed to the inequivalent environments of the Fe sites in the protein, rather than to an increase of |beta| when going from the models to the HiPIP. The small differences observed in known geometries of [Fe(4)S(4)](2+) clusters, especially in the Fe-Fe distances, cannot probably lead to drastic changes in the direct Fe-Fe interactions (parameter beta) responsible for the delocalization phenomenon. These differences are however magnetostructurally significant as shown by the 261-397 cm(-)(1) range spanned by J. The cluster's geometry, hence the efficiency of the Femicro(3)-S-Fe superexchange pathways, is proposed to be controlled by the more or less tight fit of the cluster within the cavity provided by its environment.     

Expanding the Chemical Space of Tetracyanobuta-1,3-diene (TCBD) through a Cyano-Diels-Alder Reaction: Synthesis,Structure, and Physicochemical Properties of an Anthryl-fused-TCBD Derivative

Tetracyanobuta-1,3-diene (TCBD) is a powerful and versatile electron-acceptor moiety widely used for the preparation of electroactive conjugates. While many reports addressing its electron-accepting capability have appeared in the literature, significantly scarcer are those dealing with its chemical modification, a relevant topic which allows to broaden the chemical space of this interesting functional unit. Here, we report on the first example of a high-yielding cyano-Diels-Alder (CDA) reaction between TCBD, that is, where a nitrile group acts as a dienophile, and an anthryl moiety, that is, acting as a diene. The resulting anthryl-fused-TCBD derivative, which structure was unambiguously identified by X-ray diffraction, shows high thermal stability, remarkable electron-accepting capability, and interesting electronic ground- and excited-state features, as characterized by a thorough theoretical, electrochemical, and photophysical investigation. Moreover, a detailed kinetic analysis of the intramolecular CDA reaction transforming the anthryl-TCBD-based reactant into the anthryl-fused-TCBD product was carried out at different temperatures.     

Novel chromium imprinted polymer: synthesis,characterization and analytical applicability for the selective remediation of Cr(VI) from an aqueous system

In this study, we report the synthesis of chromium imprinted polymer (Cr-IP) by a simple and facile one-pot precipitation polymerization technique by using 1-vinyl imidazole as ligand and methacrylic acid as functional monomer and their application for the specific removal of toxic hexavalent chromium from aqueous media. To attain maximum adsorption capacity various components i.e. functional monomer, cross-linker, and the molar ratio of ligand, monomer, and cross-linker were evaluate. Furthermore, several parameters i.e. pH, adsorbent dose, agitation time and shaking speed were also optimized to obtained maximum adsorption efficiency. The synthesized Cr-IP was characterized by SEM, EDX and FT-IR. The kinetic data were well described by pseudo-second-order while Langmuir model is best fitted to isotherm data. The maximum adsorption capacity of Cr-IP was 76.92mg/g. The relative standard deviation, limit of detection and quantification of this method was found to be 4.7%, 0.71µg/L and 2.36µg/L. The relative selective coefficient of Cr(VI) ions and competitive ions is greater than one due to the imprinting effect. This Cr-IP polymer also possesses good reproducibility and stability. The synthesized imprinted polymer has successfully applied for elimination of toxic Cr(VI) from real water samples.     

Methanesulfenyl fluoride, CH3SF, a missing link in the family of sulfenyl halides: formation and characterization through the matrix photochemistry of methyl thiofluoroformate, FC(O)SCH3

Matrix-isolation experiments have afforded the means of preparing the hitherto unknown sulfur(II) fluoride, methanesulfenyl fluoride, CH3SF. Broadband UV-visible irradiation of methyl thiofluoroformate, FC(O)SCH3, isolated in a solid Ar matrix results, first, in photoisomerization of the syn into the anti form of the molecule, and, subsequently, in the elimination of CO with the concomitant formation of CH3SF. Continued irradiation brings about tautomerization of this product with the detachment and migration of a hydrogen from the methyl group to give the molecular complex H2C==S...HF. The changes have been monitored and the photoproducts detected and identified by the IR spectra of the matrices, and the conclusions confirmed: 1) with reference to the corresponding behavior of the perdeuterated molecule FC(O)SCD3; 2) by analogy with the properties of related molecules, for example, ClC(O)SCH3, CH3SCl, and H2C==S...HCl, and; 3) by comparison with the vibrational properties simulated for the different molecules by ab initio and density functional theory methods.