Role of T-loop phosphorylation in PDK1 activation, stability, and substrate binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates the T-loop of several AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family protein kinases, resulting in their activation. Previous structural studies have revealed that the alpha C-helix, located in the small lobe of the kinase domain of PDK1, is a key regulatory element, as it links a substrate interacting site termed the hydrophobic motif (HM) pocket with the phosphorylated Ser-241 in the T-loop. In this study we have demonstrated by mutational analysis that interactions between the phosphorylated Ser-241 and the alpha C-helix are not required for PDK1 activity or substrate binding through the HM-pocket but are necessary for PDK1 to be activated or stabilized by a peptide that binds to this site. The structure of an inactive T-loop mutant of PDK1, in which Ser-241 is changed to Ala, was also determined. This structure, together with surface plasmon resonance binding studies, demonstrates that the PDK1(S241A)-inactive mutant possesses an intact HM-pocket as well as an ordered alpha C-helix. These findings reveal that the integrity of the alpha C-helix and HM-pocket in PDK1 is not regulated by T-loop phosphorylation.     

Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7

We generated and characterized novel antibody-cytokine fusion proteins (“immunocytokines”) based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed “F8-mIL7”) of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed “F8-mIL7-F8”), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio = 16:1, 24 h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.     

Curcumin derivatives as new ligands of Aβ peptides

Curcumin derivatives with high chemical stability, improved solubility and carrying a functionalized appendage for the linkage to other entities, have been synthesized in a straightforward manner. All compounds retained Curcumin ability to bind Aβ peptide oligomers without inducing their aggregation. Moreover all Curcumin derivatives were able to stain very efficiently Aβ deposits.     

High‐Performance and Stable All‐Polymer Solar Cells Using Donor and Acceptor Polymers with Complementary Absorption

To explore the advantages of emerging all‐polymer solar cells (all‐PSCs), growing efforts have been devoted to developing matched donor and acceptor polymers to outperform fullerene‐based PSCs. In this work, a detailed characterization and comparison of all‐PSCs using a set of donor and acceptor polymers with both conventional and inverted device structures is performed. A simple method to quantify the actual composition and light harvesting contributions from the individual donor and acceptor is described. Detailed study on the exciton dissociation and charge recombination is carried out by a set of measurements to understand the photocurrent loss. It is unraveled that fine‐tuned crystallinity of the acceptor, matched donor and acceptor with complementary absorption and desired energy levels, and device architecture engineering can synergistically boost the performance of all‐PSCs. As expected, the PBDTTS‐FTAZ:PNDI‐T10 all‐PSC attains a high and stable power conversion efficiency of 6.9% without obvious efficiency decay in 60 d. This work demonstrates that PNDI‐T10 can be a potential alternative acceptor polymer to the widely used acceptor N2200 for high‐performance and stable all‐PSCs.     

Guiding the Selection of Processing Additives for Increasing the Efficiency of Bulk Heterojunction Polymeric Solar Cells

In bulk heterojunction (BHJ) polymeric organic solar cells (OSCs), the use of processing additives in the material formulation has emerged as a promising, cost‐effective, and widely applicable method for optimizing the phase separation between the donor (D) and acceptor (A) materials, thus increasing their efficiency. So far, however, there has been no systematic approach for identifying suitable processing additives for a given D:A system. A method based on the Hansen solubility parameters (HSPs) is proposed for guiding the selection of processing additives for a given D:A combination. The method is applied to the archetypical poly(3‐hexylthiophene) (P3HT) and [6,6]‐phenyl‐C₆₁‐butyric acid methyl ester (PCBM) system. The HSPs of these materials are determined and used to define a set of numerical criteria that need to be satisfied by a processing additive in order for it to be effective in realizing a higher efficiency OSC. Applying the selection criteria results in the identification of three novel processing additives. OSCs made of these formulations demonstrate an increase in their short‐circuit current density (J_SC) and power conversion efficiency (PCE). These results demonstrate the efficiency of these novel processing additives and show that the HSPs represent a useful tool to determine and explore new types of processing additives for BHJ‐OSCs.     

Aging exacerbates negative remodeling and impairs endothelial regeneration after balloon injury

Many older patients, because of their high prevalence of coronary artery disease, are candidates for percutaneous coronary interventions (PCI), but the effects of vascular aging on restenosis after PCI are not yet well understood. Balloon injury to the right carotid artery was performed in adult and old rats. Vascular smooth muscle cell (VSMC) proliferation, apoptotic cell death, together with Akt induction, telomerase activity, p27kip1, and endothelial nitric oxide synthase (eNOS) expression was assessed in isolated arteries. Neointima hyperplasia and vascular remodeling along with endothelial cell regeneration were also measured after balloon injury. Arteries isolated from old rats exhibited a significant reduction of VSMC proliferation and an increase in apoptotic death after balloon injury when compared with adult rats. In the vascular wall of adult rats, balloon dilation induced Akt phosphorylation, and this was barely present in old rats. In arteries from old rats, Akt-modulated cell cycle check points like telomerase activity and p27kip1 expression were decreased and increased, respectively, compared with adults. After balloon injury, old rats showed a significant reduction of neointima formation and an increased vascular negative remodeling compared with adults. These results were coupled by a marked delay in endothelial regeneration in aged rats, partially mediated by a decreased eNOS expression and phosphorylation. Interestingly, chronic administration of L-arginine prevented negative remodeling and improved reendothelialization after balloon injury in aged animals. A decreased neointimal proliferation, an impaired endothelial regeneration, and an increase in vascular remodeling after balloon injury were observed in aged animals. The molecular mechanisms underlying these responses seem to be a reduced Akt and eNOS activity.     

Dioxygen affinity in heme proteins investigated by computer simulation

We present an investigation of the molecular basis of the modulation of oxygen affinity in heme proteins using computer simulation. QM-MM calculations are applied to explore distal and proximal effects on O(2) binding to the heme, while classical molecular dynamics simulations are employed to investigate ligand migration across the polypeptide to the active site. Trends in binding energies and in the kinetic constants are illustrated through a number of selected examples highlighting the virtues and the limitations of the applied methodologies. These examples cover a wide range of O(2)-affinities, and include: the truncated-N and truncated-O hemoglobins from Mycobacterium tuberculosis, the mammalian muscular O(2) storage protein: myoglobin, the hemoglobin from the parasitic nematode Ascaris lumbricoides, the oxygen transporter in the root of leguminous plants: leghemoglobin, the Cerebratulus lacteus nerve tissue hemoglobin, and the Alcaligenes xyloxidans cytochrome c'.     

Characterization of reproduction-specific genes in a marine bivalve mollusc: influence of maturation stage and sex on mRNA expression

Numerous studies have investigated the reproduction mechanisms in mollusc species at a biochemical and physiological level; few have described these mechanisms at a molecular level, despite great commercial interest in several mollusc species. We investigated genes involved in gonad maturation of the marine scallop Argopecten purpuratus. A cDNA library was made from gonad tissue. After sequence analysis, 418 unique genes were characterized, of these, about 80% were of unknown function. Among the identified sequences, we analyzed the mRNA expression by real-time PCR of 7 genes involved in reproduction mechanisms, either directly: testis-specific serine/threonine-protein kinase (TSSK), vitellogenin (Vg), and spermatogenesis and centriole associated 1 (SCA) or indirectly: calcineurin A (CNA), centrin, RNA-specific adenosine deaminase (ADAR), and cytidine deaminase (CDA). The real-time PCR analyses were conducted on different tissues of mature and immature scallops (testis, ovary, immature gonad, gill, digestive gland and mantle). The genes studied, presented (1) a strong tissue-dependent expression pattern (higher expression in gonad tissues than in all other tissues) and (2) a sex- and maturation-specific expression pattern (except centrin). This is the first time that the expression of specific genes involved in reproduction mechanisms in a marine mollusc has been described at the molecular level.