Stabilities of nanohydrated thymine radical cations: insights from multiphoton ionization experiments and ab initio calculations

Multi-photon ionization experiments have been carried out on thymine-water clusters in the gas phase. Metastable H2O loss from T+(H2O)n was observed at n ≥ 3 only. Ab initio quantum-chemical calculations of a large range of optimized T+(H2O)n conformers have been performed up to n = 4, enabling binding energies of water to be derived. These decrease smoothly with n, consistent with the general trend of increasing metastable H2O loss in the experimental data. The lowest-energy conformers of T+(H2O)3 and T+(H2O)4 feature intermolecular bonding via charge-dipole interactions, in contrast with the purely hydrogen-bonded neutrals. We found no evidence for a closed hydration shell at n = 4, also contrasting with studies of neutral clusters

A threshold level of NFATc1 activity facilitates thymocyte differentiation and opposes notch-driven leukaemia development.

International audienceNFATc1 plays a critical role in double-negative thymocyte survival and differentiation. However, the signals that regulate Nfatc1 expression are incompletely characterized. Here we show a developmental stage-specific differential expression pattern of Nfatc1 driven by the distal (P1) or proximal (P2) promoters in thymocytes. Whereas, preTCR-negative thymocytes exhibit only P2 promoter-derived Nfatc1beta expression, preTCR-positive thymocytes express both Nfatc1beta and P1 promoter-derived Nfatc1alpha transcripts. Inducing NFATc1alpha activity from P1 promoter in preTCR-negative thymocytes, in addition to the NFATc1beta from P2 promoter impairs thymocyte development resulting in severe T-cell lymphopenia. In addition, we show that NFATc1 activity suppresses the B-lineage potential of immature thymocytes, and consolidates their differentiation to T cells. Further, in the pTCR-positive DN3 cells, a threshold level of NFATc1 activity is vital in facilitating T-cell differentiation and to prevent Notch3-induced T-acute lymphoblastic leukaemia. Altogether, our results show NFATc1 activity is crucial in determining the T-cell fate of thymocytes

Glycogen synthase kinase 3 (GSK-3) inactivation compensates for the lack of CD28 in the priming of CD8+ cytotoxic T-cells: implications for anti-PD-1 immunotherapy

The rescue of exhausted CD8+ cytolytic T-cells (CTLs) by anti-PD-1 blockade has been found to require CD28 expression. At the same time, we have shown that the inactivation of the serine/threonine kinase GSK‐3α/β with small interfering RNAs (siRNAs) and small molecule inhibitors (SMIs) specifically down-regulate PD-1 expression for enhanced CD8+ CTL function and clearance of tumours and viral infections.  Despite this, it has been unclear whether the GSK‐3α/β pathway accounts for CD28 co‐stimulation of CD8+ CTL function.  In this paper, we show that inactivation of GSK‐3α/β through siRNA or by SMIs during priming can substitute CD28 stimulation in the potentiation of cytotoxic CD8+ CTL function. This increased response was observed in the blockade of CD28 co-receptor by CTLA-4-IgG in OT-1 T-cells responding to OVA peptide as presented by the lymphoma cell line EL4. The effect was seen using several GSK-3 SMIs, and was accompanied by an increase in Lamp-1 and GZMB expression. Conversely, CD28 crosslinking obviated the need for GSK‐3α/β inhibition in its enhancement of CTL function.  Our findings support a model where GSK‐3 is the central co-signal for CD28 priming of CD8+ CTLs in anti-PD-1 immunotherapy

A family of unsymmetrical hydroxyl-substituted BEDT-TTF donors: syntheses, structures and preliminary thin film studies

Three new unsymmetrical hydroxyl-functionalized donors H1–H3 closely related to hydroxymethyl-BEDT-TTF have been synthesised and characterised. Cyclic voltammetry studies showed that the compounds exhibit reversible two one-electron redox processes typical for BEDT-TTF derivatives. X-ray diffraction studies of H1 and H2 reveal π-stacking interactions between pairs of donors that are organized into distinct H-bonded square motifs and DFT calculations indicate that the HOMO is located on the central 1,3-dithiole rings. Protection of the hydroxyl group with acetyl in 13 eliminates co-facial S...S interactions between the dimers to accommodate the bulkier side chains, but short edge-to-edge S...S contacts offer an alternative pathway for electron mobility. Chemical oxidation of H1 and HMET 2 with I2 afforded single crystals of two 1 : 1 charge transfer salts, 18 and 19. The molecules pack as dimers with close π-stacking interactions between pairs of radical cations whose crystal structures are further stabilized via an interplay of S...S and S...I contacts. Iodine-doped surface conducting polystyrene blend films of H3 deposited on a silica substrate exhibit quasiconducting properties, but afford no OFET response when fabricated into devices. Visible-NIR studies of a doped polystyrene blend film of H3 cast on a glass substrate show absorption bands at λ =9 50 and 3000 nm, consistent with mixed valence states due to the presence of charge-transfer species on the surface of the films

2,2′-Diazinodimethylidyne)di-o-phenyl­ene) dibenzoate

The title compound, C28H20N2O4, was synthesized by the reaction of 2-(hydrazonometh­yl)phenyl benzoate with iodine. The mol­ecule possesses a crystallographically imposed center of symmetry at the mid-point of the hydrazine N—N bond. The substituents at the ends of the C=N bonds adopt an E,E configuration. Inter­molecular C—H⋯π(arene) hydrogen bonds and aromatic π–π stacking inter­actions [centroid–centroid distance 3.900 (1) Å] link the mol­ecules into (100) sheets. In addition, there is an inter­molecular C—H⋯O hydrogen-bond inter­action

Triethyl­ammonium 4-nitro­benzene­sulfonate

In the anion of the title molecular salt, C6H16N+·C6H4O5S−, the nitro group is twisted slightly from the benzene ring, making a dihedral angle of 3.16 (10)°. In the crystal structure, the cations and anions are linked into a two-dimensional network parallel to the ab plane by C—H⋯O and N—H⋯O hydrogen bonds

TGN1412: From Discovery to Disaster

After a drug is confirmed as safe and efficacious in preclinical studies, it is tested in healthy human volunteers for first in man trials. In 2006, a phase I clinical study was conducted for a CD28 superagonist antibody TGN1412 in six human volunteers. After very first infusion of a dose 500 times smaller than that found safe in animal studies, all six human volunteers faced life-threatening conditions involving multiorgan failure for which they were moved to intensive care unit. After this particular incident, a lot was changed over how first in man trials are approved by regulatory authorities and the way clinical trials are conducted. This review primarily deals with preclinical studies conducted by TeGenero, results of which encouraged them to test the antibody on human subjects, reasons why this drug failed in human trial and aftermath of this drug trial. In addition, another drug—Fialuridine which failed in phase 2 clinical trial leading to death of five human subjects is briefly reviewed

Deletion of IL-4Rα on CD4 T Cells Renders BALB/c Mice Resistant to Leishmania major Infection

Effector responses induced by polarized CD4(+) T helper 2 (Th2) cells drive nonhealing responses in BALB/c mice infected with Leishmania major. Th2 cytokines IL-4 and IL-13 are known susceptibility factors for L. major infection in BALB/c mice and induce their biological functions through a common receptor, the IL-4 receptor α chain (IL-4Rα). IL-4Rα–deficient BALB/c mice, however, remain susceptible to L. major infection, indicating that IL-4/IL-13 may induce protective responses. Therefore, the roles of polarized Th2 CD4(+) T cells and IL-4/IL-13 responsiveness of non-CD4(+) T cells in inducing nonhealer or healer responses have yet to be elucidated. CD4(+) T cell–specific IL-4Rα (Lck(cre)IL-4Rα(−/lox)) deficient BALB/c mice were generated and characterized to elucidate the importance of IL-4Rα signaling during cutaneous leishmaniasis in the absence of IL-4–responsive CD4(+) T cells. Efficient deletion was confirmed by loss of IL-4Rα expression on CD4(+) T cells and impaired IL-4–induced CD4(+) T cell proliferation and Th2 differentiation. CD8(+), γδ(+), and NK–T cells expressed residual IL-4Rα, and representative non–T cell populations maintained IL-4/IL-13 responsiveness. In contrast to IL-4Rα(−/lox) BALB/c mice, which developed ulcerating lesions following infection with L. major, Lck(cre)IL-4Rα(−/lox) mice were resistant and showed protection to rechallenge, similar to healer C57BL/6 mice. Resistance to L. major in Lck(cre)IL-4Rα(−/lox) mice correlated with reduced numbers of IL-10–secreting cells and early IL-12p35 mRNA induction, leading to increased delayed type hypersensitivity responses, interferon-γ production, and elevated ratios of inducible nitric oxide synthase mRNA/parasite, similar to C57BL/6 mice. These data demonstrate that abrogation of IL-4 signaling in CD4(+) T cells is required to transform nonhealer BALB/c mice to a healer phenotype. Furthermore, a beneficial role for IL-4Rα signaling in L. major infection is revealed in which IL-4/IL-13–responsive non-CD4(+) T cells induce protective responses

Signaling Signatures and Functional Properties of Anti-Human CD28 Superagonistic Antibodies

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4+ T cells but not in cynomolgus T lymphocytes. The sustained Ca++-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-γ and TNF-α. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-γ and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells