Academic literature on the topic 'Astrophysical Interest -Weakly Bound Molecules'

At the low temperatures of interstellar dust grains, it is well established that surface chemistry proceeds via diffusive mechanisms of H atoms weakly bound (physisorbed) to the surface. Until recently, however, it was unknown whether atoms heavier than hydrogen could diffuse rapidly enough on interstellar grains to react with other accreted species. In addition, models still require simple reduction as well as oxidation reactions to occur on grains to explain the abundances of various molecules. In this paper we investigate O-atom diffusion and reactivity on a variety of astrophysically relevant surfaces (water ice of three different morphologies, silicate, and graphite) in the 6.5–25 K temperature range. Experimental values were used to derive a diffusion law that emphasizes that O atoms diffuse by quantum mechanical tunnelling at temperatures as low as 6.5 K. The rates of diffusion on each surface, based on modelling results, were calculated and an empirical law is given as a function of the surface temperature. The relative diffusion rates are k_H2Oice > k_sil > k_graph ≫ k_expected. The implications of efficient O-atom diffusion over astrophysically relevant time-scales are discussed. Our findings show that O atoms can scan any available reaction partners (e.g., either another H atom, if available, or a surface radical like O or OH) at a faster rate than that of accretion. Also, as dense clouds mature, H₂ becomes far more abundant than H and the O : H ratio grows, and the reactivity of O atoms on grains is such that O becomes one of the dominant reactive partners together with H.

TCR (T-cell receptor) recognition of antigenic peptides bound and presented by MHC (major histocompatibility complex) molecules forms the basis of the cellular immune response to pathogens and cancer. TCRs bind peptide–MHC complexes weakly and with fast kinetics, features which have hindered detailed biophysical studies of these interactions. Modified peptides resulting in enhanced TCR binding could help overcome these challenges. Furthermore, there is considerable interest in using modified peptides with enhanced TCR binding as the basis for clinical vaccines. In the present study, we examined how fluorine substitutions in an antigenic peptide can selectively impact TCR recognition. Using a structure-guided design approach, we found that fluorination of the Tax peptide [HTLV (human T-cell lymphotropic virus)-1 Tax11-19] enhanced binding by the Tax-specific TCR A6, yet weakened binding by the Tax-specific TCR B7. The changes in affinity were consistent with crystallographic structures and fluorine chemistry, and with the A6 TCR independent of other substitutions in the interface. Peptide fluorination thus provides a means to selectively modulate TCR binding affinity without significantly perturbing peptide composition or structure. Lastly, we probed the mechanism of fluorine's effect on TCR binding and we conclude that our results were most consistent with a ‘polar hydrophobicity’ mechanism, rather than a purely hydrophobic- or electrostatic-based mechanism. This finding should have an impact on other attempts to alter molecular recognition with fluorine.

Abstract Among new treatment options for mantle cell lymphoma (MCL), the targeted drug lenalidomide appears as one of the most efficient molecules. Lenalidomide has multiple modes of action targeting the tumor cell and its environment including the immune system. It is widely reported that cancer patients are deficient in vitamin D3 (1,25-dihydroxyvitamin D3, VD3) and recent studies have shown in non Hodgkin lymphomas (NHL) that VD3 levels have a prognostic value on survival (Drake, J Clin Oncol. 2010;28:4191). While the relations between VD3 and cancer incidence remain unresolved, it has been shown that VD3 displays anti-tumoral properties via its anti-proliferative, pro-differentiation, anti-inflammatory and anti-angiogenic properties. We assessed the efficacy of VD3 to potentiate cell death induced by lenalidomide in MCL cell lines and patients’ samples and explored the mechanisms of cell death in this context. Experiments were conducted on a panel of 6 MCL cell lines (JEKO-1, MINO, GRANTA-519, UPN-1, REC-1 and Z138) and 8 primary peripheral blood samples. After 6 days of treatment, MCL cells were weakly sensitive to low doses of lenalidomide (1µM and 10µM for cell lines and samples, respectively). Addition of physiological doses of VD3 (100nM) significantly and synergistically increased cell death in 67% of cell lines (Z138, JEKO-1, MINO, REC-1) and in 63% of primary samples (p<0.05). However resistance to lenalidomide alone was not reversed by VD3 since both GRANTA-519 and UPN-1 remained unsensitive. Apoptosis, characterized by Annexin V staining, appearance of a subG1 peak and caspase 9 activation, was dependent on Bax expression, since transient extinction of BAX by siRNA in JEKO-1 cells inhibited cell death (mean of inhibition 30%±5%, p=0.03). The combination of lenalidomide and VD3 dramatically increased expression of the BH3-only Bik (Bcl2-Interacting Killer) protein in sensitive (Z138, JEKO-1, MINO, REC-1) but not resistant (GRANTA-519, UPN-1) cell lines, without affecting the expression of other molecules of the Bcl2 family. By immunoprecipitation assays, we showed that induced-Bik was not bound to the anti-apoptotic molecules Bcl2, BclxL or Mcl1 in treated cells but was free to activate such pro-apoptotic molecules as Bax. Moreover, siBIK RNA significantly decreased the proportion of Annexin V+ cells observed after treatment with lenalidomide and VD3, respectively by 36%±9% (p=0.04) and 28%±4% (p=0.04) in JEKO-1 and MINO cells. This confirmed the involvement of Bik in the cell death induced by this synergistic combination. Q-RT-PCR assays disclosed that Bik accumulation was related to an increase in BIK mRNA expression. BIK expression is controlled by the transcription factor TEF and is regulated by epigenetic modifications, its expression being silenced by methylation in many cancer cells. We showed that Bik accumulation induced by lenalidomide and VD3 was not related to an increase in TEF expression. To determine whether Bik expression could be induced or increased upon demethylation in MCL, we treated cell lines for 3 days with 1µM 5-azadecytidine (5-aza). Indeed, higher expression of Bik was observed after this treatment in the four cell lines sensitive to lenalidomide. Of note, cell death induced by 5-aza correlated linearly to that induced by lenalidomide and VD3 (p<0.001, r=0.95, n=6), suggesting that BIK demethylation could be a key point in the response to this combination. To directly assess the level of BIK methylation in MCL cell lines, we then performed a DNA methylation specific PCR assay on bisulfite-treated DNA, which targets the CpG rich region located within intron 1, as previously described by Hatzimichael et al (Leuk Lymphoma. 2012;53:1709). Indeed, we showed that lenalidomide and VD3 increased the proportion of unmethylated over methylated BIKDNA CpG islands in sensitive (2 to 5-fold increase) but not in resistant cell lines. These data show that the association of lenalidomide and VD3, by increasing BIK expression through DNA demethylation, is an efficient combination to induce the apoptosis of MCL cells. They also underline the interest of measuring the level of VD3 in MCL patients especially those receiving lenalidomide, since supplementation in deprived patients might improve the effect of therapy. Disclosures: No relevant conflicts of interest to declare.

Clinical efficacy of targeted signaling inhibitors for hematologic malignancies is limited bynoutgrowth of subpopulations with alternative pathways independent of the drug target. The eIF4F complex responsible for translation initiation is a convergence point for cancer-promoting signaling pathways and its inhibition leads to decreased expression of key oncoproteins and apoptosis. Lymphomas and leukemias show particular dependence on constitutive eIF4F activation. Indeed, natural compounds targeting the eIF4F enzymatic component, eIF4A1, demonstrate activities in vitro and in vivo against lymphoma and leukemia model systems, among other tumor types. The natural compound silvestrol is a potent inhibitor of eIF4A1, results in cancer cell cytotoxicity, and has an established therapeutic window in vivo. Silvestrol shows potent antitumor activity against 924 pan-cancer tumor cell lines with 830/924 (90%) sensitive at IC50 <100nM with lymphoma and leukemia cell lines being particularly sensitive. Silvestrol and other natural compounds, however, lack core drug-like properties and synthetic tractability. To discover new, specific and tractable inhibitors of eIF4A1 that are more drug-like, we have constructed several molecular models that we used to virtually screen more than 20 million compounds. eIF4A1 is the founding member of the DEAD-box RNA helicases, which include its paralogs eIF4A2 (91% amino-acid identity with eIF4A1) and eIF4A3 (60% identity). All DEAD-box helicases contain two RecA-like domains separated by a flexible linker. The cleft between these domains is lined with helicase motifs that mediate nucleotide binding and hydrolysis. In an absence of RNA or nucleotide, eIF4A proteins adopt diffuse open conformations; binding of RNA and ATP triggers transition to a more stable closed state. Modeling small-molecule interactions in the nucleotide cleft of eIF4A1 therefore assesses ability of molecules to lock eIF4A1 in a conformation unable to cycle through ATPase and helicase activities. A new crystal structure of eIF4A1 has become available (2019) with a resolution of 2 angstroms. The protein is co-crystallized with ANP in the nucleotide binding site at the interface of the N and C-terminal domains and with known inhibitor, Rocaglamide, bound to the interface of the eIF4A1and a polypurine RNA. We used this high-resolution crystal structure to build models predicting interactions of small molecules in the interdomain nucleotide-binding cleft. We then performed all-atom explicit-water molecular dynamics (MD) simulations for 500-700 ns to study conformational dynamics and atomic interactions of ATP-bound and ATP-unbound states. Extended molecular dynamic simulations confirm the hypothesis that rocaglamide stabilizes the interaction between the helicase and a polypurine sequence on RNA, thus preventing further ATPase activity and RNA unwinding. Pooling these results, we constructed two homology models of human eIF4A1 with both open and closed conformations as structural templates. Over 50 compounds identified as hits in silicowere ordered and tested thus far in our biochemical and cell-based validation platform. Using our machine learning and virtual screening approach targeted to the ATP binding site of eIF4A1, we identified a promising piperazine-amide fragment scaffold (UM107; ~300 MW) with similar electronics to nucleotide triphosphates. UM107 caused cellular toxicities with an LD50 of 50 uM and was weakly active in the biochemical screen against eIF4A1 with an IC50 of 250 uM. We will increase molecular weight by adding more groups to maximize hydrogen bond interactions in the active site. These analogs will be synthesized and screened virtually building on the core using established medicinal chemistry optimization tools followed by biochemical and cellular validation. We therefore have developed an accurate and novel in silico models of eIF4A1 highly useful in assessing interactions of small-molecule ATPase inhibitors, with focus on the ATP-binding cleft. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction The fate of organic micro-pollutants in soil largely depends on their interactions with soil organic matter (SOM). Indeed, the intensity and nature of such interactions are major factors in the control of micro-pollutant bioavailability, and hence degradation and micro-pollutant mobility in the environment [Khan and Hamilton, 1980 ; Calderbank, 1989 ; Dec et al., 1990, 1997 ; Barriuso and Koshinen, 1996]. Residual micro-pollutants, i.e. the remaining micro-pollutants and their various transformation products (metabolites), occur in soil both in the water phase and associated with the solid phase. A part of these associated molecules, termed bound residues (BRs), cannot be released via extraction with water or organic solvents like methanol. SOM plays a major role in BR formation [e.g. Loiseau et al., 2000] and different types of interaction (like covalent bonds and trapping in organo-mineral aggregates) can be implied [Hsu and Bartha, 1976 ; Schiavon et al., 1977 ; Bollag et al., 1992 ; Dec and Bollag, 1997 ; Steinberg et al., 1987; Providenti et al., 1993]. The present study is concerned with the fate of atrazine in three silty loamy soils from the Paris Basin. Atrazine is largely used (ca. 5000 T/year in France) as herbicide for maize cropping and may cause important pollution problems through transfer to water resources. Experimental Incubations were performed for 60 days, using 14C labelled atrazine, with the selected soils. These incubations were carried out with 50 g of soil in closed vessels at room temperature in darkness. The production of 14C CO2 was regularly measured so as to determine the rate of atrazine mineralisation in the three incubated soils. These three soils exhibit large differences in pH and atrazine use (tabl. I). Following incubations, radioactivity measurements were performed on the different fractions isolated : aqueous extract, methanol extract, BRs in various size fractions (&lt; 20 μm, 20–50 μm, &gt; 50 μm). In addition, BR distribution was determined in the fulvic and humic acids and humin isolated from the &lt; 20 μm fraction. In a second set of incubation experiments, the distribution of residual atrazine was examined in size fractions of organo-mineral aggregates separated by sieving in water (&lt; 50 μm, 50–200 μm, 200 μm-1 mm, 1–2 mm, &gt; 2 mm) and in free plant debris separated by floating. The GB1 soil was also incubated for 60 days using 13C labelled atrazine. Curie point flash pyrolysis combined both with isotopic measurements on individual compounds (Py-GC-C-irMS) and with gas chromatography/mass spectrometry (GC-MS) analyses was used to examine atrazine BRs following the latter incubation. Results and discussion Atrazine mineralisation The extent of atrazine mineralisation during incubation is sharply different for the three soils (fig. 1). The GM1 soil shows an intense mineralisation and ca. 85 % of the applied atrazine is transformed into CO2. Moreover, this degradation is fast and mostly takes place during the first week. In contrast, much lower levels of mineralisation are observed for the other two soils, especially for VMSA (only ca. 8 % after 60 days). For the GB1 soil, weak mineralisation is noted, during the first week of incubation. However, following this lag phase, substantial mineralisation occurs and a value of ca. 20 % is obtained at the end of the experiment. It was previously observed [Houot et al., 1998] that soil microflora can adapt to atrazine mineralisation following regular inputs of atrazine. Accordingly, the pronouced differences observed in the present study reflect the presence of a microflora adapted to atrazine degradation in the GM1 soil, due to yearly application of atrazine over 25 years whereas such a microflora is absent in the GB1 and VMSA soils, which had never been treated with atrazine before incubation. The substantial difference in mineralisation intensity observed between the latter two soils is probably related to the relatively low pH of the VMSA soil since such a feature is known to limit atrazine mineralisation [Houot et al., 1998 ; Castéraz, 1998]. BRs and distribution of residual atrazine The distribution of residual atrazine between the different fractions controls its biodisponibility and fate. Water-soluble molecules are considered as directly bioavailable, whereas the degree of availability is much lower for the bound compounds and, to a lesser extent, for the methanol-soluble ones. It appears (fig. 2) that, as expected, the relative abundance of water– and methanol-soluble residual atrazine is inversely correlated to mineralisation intensity. As a result, the contribution of BRs to total residual atrazine is much higher for GM1 (68 %) where soluble residues were strongly affected by mineralisation when compared to GB1 (36 %) and VMSA (51 %). The higher percentage observed for VMSA relative to GB1 suggests that BR formation is favored by a low pH. BR distribution between the different size fractions (fig. 2) shows that the largest absolute amount of BRs is found for the three soils in the &lt; 20 μm fraction. This fraction is both the most humified and the most abundant (as wt %). However, if BR concentration relative to OM is considered (tabl. II), it appears that the highest concentrations in BRs tend to occurs in the fractions which contain non- (or weakly-) humified organic material. Atrazine BRs thus show a higher affinity with the latter material, as also previously observed for various soils [Barriuso et al., 1991 ; Barriuso and Koshinen, 1996 ; Loiseau et al., 2000]. BR distribution between the fulvic and humic acids and humin was examined for the &lt; 20 μm fraction (fig. 3). For the three soils, the largest amount of BRs is associated with humin, especially in the case of GB1 and GM1. Substantial amounts of BRs are also found with the fulvic acid fractions. Moreover, when BR concentration is considered (tabl. III), a higher affinity is noted for fulvic acids. The latter features should have important environmental consequences since fulvic acids are rather readily transfered from soil to surface and ground water. The high affinity of atrazine BRs for fulvic acids is probably related to interactions with the polar groups that abundantly occur in the latter fraction [Khan and Hamilton, 1980 ; Bertin and Schiavon, 1989 ; Capriel and Haisch, 1983 ; Capriel et al., 1985]. Comparison of the three soils therefore shows pronounced differences in mineralisation levels, related to microflora adaptation and pH. Large differences are also observed in the distribution of residual atrazine between the BRs and the soluble fractions. In contrast, the three soils exhibit similar features for BR distribution (absolute amounts and concentrations) between the three size fractions and also between the fulvic acids, humic acids and humin of the &lt; 20 μm fraction. Concentration values show higher affinity of atrazine BRs for weakly humified OM and for fulvic acids. Atrazine distribution in organo-mineral aggregates and fresh debris of plants This study was performed on the GM1 and VMSA soils. Measurements were performed (i) at time zero (just one hour after the addition of 14C atrazine to soils) i.e. when no significant atrazine mineralisation and transformation had occurred and (ii) at the end of the experiment after 60 days of incubations. Distribution at time 0 is similar for the two soils (fig. 4a) and the bulk of the atrazine is mostly associated with the 0.2–1 mm fraction followed by the 1–2 mm fraction. When content is considered (fig. 4b), the highest value is observed, by far, for the free plant debris. Such a feature reflects, as observed in previous studies [Barriuso et al., 1994 ; Puget et al., 2000], the high affinity of atrazine for fresh or weakly humified material. These results are therefore consistent with the preferential association of atrazine and of its metabolites with weakly humified OM observed in the BRs in the first set of incubations. After 60 days, the amount of residual atrazine observed in a given fraction shall both reflect the extent of mineralisation in this fraction and possible transfer from other fractions. As already stressed, only weak mineralisation occurs in the VMSA soil and similar atrazine distributions are noted for both soils at time 0. Thus, the results obtained after incubation shall mostly reflect the transfer processes for the VMSA soil and a combination of such processes and of biodegradation for GM1. Accordingly, the relative changes observed, between the two soils, for label concentration in the different fractions (fig. 4c) reflect the relative efficiency of the stabilisation of residual atrazine. Large (ca. 90 %) and similar decreases are observed for all the fractions of organo-mineral aggregates whereas only a limited decrease of ca. 15 % occurs for the free debris. Therefore residual atrazine stabilisation is more important in the latter fraction than in the organo-mineral aggregates and no preferential stabilisation takes place in some size fractions of these aggregates. Pyrolytic studies of atrazine BRs Various types of interactions between bound residual atrazine and SOM, ranging from relatively weak linkages like hydrogen bonds to much stronger linkages like covalent bonds, can occur in soil. The nature of such interactions should have major consequences for the stabilisation and fate of residual atrazine. Information on this nature could be derived from successive pyrolyses at increasing temperatures, as recently shown for pyrene retention in sediments [Guthrie et al., 1999] and 2-aminobenzothiazole interactions with humic acids [Schulze et al., 1997]. This type of pyrolytic study, combined with GC/MS identifications, should thus be useful to decipher (i) the structure of the different metabolites in bound residual atrazine, (ii) their relative abundance and (iii) their modes of linkage with SOM. However, this is made difficult by the extremely high dilution of bound residual atrazine both by soil minerals and SOM and it seems that no such study has been reported so far. The above drawbacks could be overcome via incubation with labelled atrazine followed by elimination of the bulk of soil minerals. To test the suitability of this approach, the GB1 soil was incubated for 60 days with 13C atrazine. After incubation, soluble compounds were eliminated by water and methanol extractions and minerals via a mild treatment with 2 % HF which should not largely affect organic components in soil [Skjemstad et al., 1994]. Atrazine BRs were then examined by pyrolysis combined both with GC-C-irMS and GC-MS. The pyrolysis products derived from bound residual atrazine can be located by isotopic analysis owing to their very high enrichment in 13C (in the 100-1 000 ‰ range) (fig. 5a). As expected, when the GC-MS trace is considered (fig. 5b), the latter products are overwhelmed by the pyrolysis products from the natural OM of the soil. However, they can be identified and analysed owing to the above isotopic localisation and to the presence of typical fragments of the triazine ring (such as m/z 68) in their mass spectra. The retention times and mass spectra of the five compounds derived from bound residual atrazine thus identified indicate that they do not correspond either to atrazine itself or to the metabolites, like hydroxy– and dealkylatrazines, classically observed in the soluble phase. Residual atrazine occurring as BRs and as soluble components in the incubated soil thus appears to exhibit different chemical composition. These preliminary results confirm the suitability and interest of such a combined pyrolytic approach, applied for the first time to the best of our knowledge, for the study of BRs of micro-pollutants.

Abstract Several extensions to the Standard Model of particle physics, including light dark matter candidates and unification theories predict deviations from Newton’s law of gravitation. For macroscopic distances, the inverse-square law of gravitation is well confirmed by astrophysical observations and laboratory experiments. At micrometer and shorter length scales, however, even the state-of-the-art constraints on deviations from gravitational interaction, whether provided by neutron scattering or precise measurements of forces between macroscopic bodies, are currently many orders of magnitude larger than gravity itself. Here we show that precision spectroscopy of weakly bound molecules can be used to constrain non-Newtonian interactions between atoms. A proof-of-principle demonstration using recent data from photoassociation spectroscopy of weakly bound Yb2 molecules yields constraints on these new interactions that are already close to state-of-the-art neutron scattering experiments. At the same time, with the development of the recently proposed optical molecular clocks, the neutron scattering constraints could be surpassed by at least two orders of magnitude.

AbstractPII proteins constitute a widespread signal transduction superfamily in the prokaryotic world. The canonical PII signal proteins sense metabolic state of the cells by binding the metabolite molecules ATP, ADP and 2-oxoglutarate. Depending on bound effector molecule, PII proteins interact with and modulate the activity of multiple target proteins. To investigate the complexity of interactions of PII with target proteins, analytical methods that do not disrupt the native cellular context are required. To this purpose, split luciferase proteins have been used to develop a novel complementation reporter called NanoLuc Binary Technology (NanoBiT). The luciferase NanoLuc is divided in two subunits: a 18 kDa polypeptide termed “Large BiT” and a 1.3 kDa peptide termed “Small BiT”, which only weakly associate. When fused to proteins of interest, they reconstitute an active luciferase when the proteins of interest interact. Therefore, we set out to develop a new NanoBiT sensor based on the interaction of PII protein from Synechocystis sp. PCC6803 with PII-interacting protein X (PipX) and N-acetyl-L-glutamate kinase (NAGK). The novel NanoBiT sensor showed unprecedented sensitivity, which made it possible to detect even weak and transient interactions between PII variants and their interacting partners, thereby shedding new light in PII signalling processes.

Abstract Immunopeptidomics is the survey of all peptides displayed on a cell or tissue when bound to human leukocyte antigen (HLA) molecules using tandem mass spectrometry. When attempting to determine the targets of tumour-specific CD8+ T cells, a survey of the potential ligands in tumour tissues is invaluable, and, in comparison with in-silico predictions, provides greater certainty of the existence of individual epitopes, as immunopeptidomics-confirmed CD8+ T-cell epitopes are known to be immunogenic, and direct observation should avoid the risk of autoreactivity which could arise following immunisation with structural homologues. The canonical sources of CD8+ T-cell tumour specific epitopes, such as tumour associated antigens, may be well conserved between patients and tumour types, but are often only weakly immunogenic. Direct observation of tumour-specific neoantigens by immunopeptidomics is rare, although valuable. Thus, there has been increasing interest in the non-canonical origins of tumour-reactive CD8+ T-cell epitopes, such as those arising from proteasomal splicing events, translational/turnover defects and alternative open reading frame reads. Such epitopes can be identified in silico, although validation is more challenging. Non-self CD8+ T-cell epitopes such as viral epitopes may be useful in certain cancer types with known viral origins, however these have been relatively unexplored with immunopeptidomics to date, possibly due to the paucity of source viral proteins in tumour tissues. This review examines the latest evidence for canonical, non-canonical and non-human CD8+ T-cell epitopes identified by immunopeptidomics, and concludes that the relative contribution for each of these sources to anti-tumour CD8+ T-cell reactivity is currently uncertain.

There has been great interest in studying optical and transport properties of multiple quantum well (MQW) structures. In these “artificial molecules”, energy quantization and the wave nature of the carriers have been used to design new devices, e.g., intersubband lasers. The understanding of carrier transport in MQWs is important for the design of lasers with high modulation speed. In this talk, we report on a new observation of a quantum interference effect in the photocurrent spectrum of weakly coupled bound-to-continuum MQWs. Using this effect, we analyze the electric field domain (EFD) formation in the superlattice [1].