STAT-Related Profiles Are Associated with Patient Response to Targeted Treatments in Locally Advanced SCCHN

The anti-epidermal growth factor receptor antibody cetuximab (Erbitux, CTX) is currently used for the treatment of locally advanced squamous cell carcinoma of the head and neck (LA-SCCHN), as yet with modest effectiveness, prompting for the identification of response predictors to this treatment and for the targeting of additional pathways implicated in this disease. Within this scope, we investigated the effect of SRC/STAT pathway components on LA-SCCHN patient outcome. SRC, STAT1, STAT3, STAT5A, STAT5B, ANXA1, CAV1, IGFBP2, EPHA2, EPHB2, and MSN relative gene expression, as well as Stat protein activation, were assessed on LA-SCCHN tumor tissues from 35 patients treated with combined radiotherapy (RT) and CTX-based regimens. Stat1, Stat3, and Stat5 proteins were usually found activated in neoplastic nuclei (70.4%, 85.7%, and 70.8%, respectively). Activated Stat3 and Stat5 were associated with each other (P = .017) and with a CAV1(high)/MSN(high)/IGFBP2(low) profile. All patients with tumors expressing high STAT5A/EPHA2 experienced a complete response on RT-CTX-based treatments (12/15 complete responders, P < .0001) and a longer progression-free survival (P = .024). Few tumors expressed high ANXA1/CAV1/EPHA2 and low IGFBP2, a putative dasatinib response-related profile, whereas high ANXA1 was associated with shorter overall survival (P = .003). In conclusion, Stat activation is common in LA-SCCHN, where overexpression of STAT5A and EPHA2 may predict for response to RT-CTX treatments. The STAT5A/EPHA2 profile seems of particular interest for validation in larger cohorts and in multiple tumor types because markers for the positive selection of patients to benefit from CTX-containing treatments are currently lacking.     

Synthetic shRNAs as potent RNAi triggers

Designing potent silencing triggers is key to the successful application of RNA interference (RNAi) in mammals. Recent studies suggest that the assembly of RNAi effector complexes is coupled to Dicer cleavage. Here we examine whether transfection of optimized Dicer substrates results in an improved RNAi response. Dicer cleavage of chemically synthesized short hairpin RNAs (shRNAs) with 29-base-pair stems and 2-nucleotide 3' overhangs produced predictable homogeneous small RNAs comprising the 22 bases at the 3' end of the stem. Consequently, direct comparisons of synthetic small interfering RNAs and shRNAs that yield the same small RNA became possible. We found synthetic 29-mer shRNAs to be more potent inducers of RNAi than small interfering RNAs. Maximal inhibition of target genes was achieved at lower concentrations and silencing at 24 h was often greater. These studies provide the basis for an improved approach to triggering experimental silencing via the RNAi pathway.     

Leishmania histone H1 overexpression delays parasite cell-cycle progression, parasite differentiation and reduces Leishmania infectivity in vivo

Episomal expression of Leishmania histone H1 sense mRNAs in Leishmania major promastigotes was found previously to result in overexpression of this molecule and to reduce parasite infectivity in vitro. Herein, we evaluated the in vivo infectivity of these transfectants, in BALB/c mice, and showed that it is dramatically reduced. No lesions were observed in this group of mice and this was associated with an extremely low number of parasites both in the footpad and in the draining lymph nodes. Interestingly, the transfectants-reduced infectivity was associated with a delay in their cell-cycle progression and differentiation to axenic amastigotes, assessed in vitro. Therefore, the dramatic reduction in their infectivity may be attributed to the above-mentioned phenotypic modifications. As the metazoan linker histone H1(0) homologue is known to delay cell-cycle progression in mammalian cells we investigated whether its Leishmania counterpart, which possesses homology to its C-terminal region, when expressed in mammalian cells may also affect their cell-cycle progression. It was thus shown that Leishmania histone H1 expressed in COS7 and NIH 3T3 cells, delays cell-cycle progression in these cells too. The latter strengthens the phenotype observed in Leishmania and provides evidence that critical functions of histone H1 molecules are conserved throughout evolution.     

Synthesis, conformational characteristics and anti-influenza virus A activity of some 2-adamantylsubstituted azacycles

The broad-spectrum antiviral activity of 2-(2-adamantyl)piperidines 11, 13a,b, and 15, 3-(2-adamantyl)pyrrolidines 27, 21a-g and 2-(2-adamantylmethyl)piperidines 30, 32a-c, and 35a-d was examined. Several compounds in the new series were potent against influenza A H(3)N(2) virus. When 1-aminoethyl pharmacophore group of 2-rimantadine 4 (2-isomer of rimantadine) is included into a saturated nitrogen heterocycle, see compound 11, potency was retained. The diamine derivatives 21e-g and particularly 35a-c possessing three pharmocophoric groups, that is, the adamantyl and the two amine groups, exhibited high potency. The new compounds did not afford specific activity at non-toxic concentrations against any of the other viruses tested. According to NMR spectroscopy and molecular mechanics calculations it is striking that the parent structures 11 and 27 adopt a fixed trans conformation around C2-C2' bond. In the parent amines, which proved to be active compounds, the distance between nitrogen and adamantyl pharmacophoric groups was different; N-C2' distance is 3.7, 3.8 A for 27, 30 and 2.5 A for 11 suggesting that M2 receptor site can accommodate different in size and orientation lipophilic cages.     

Organizing cell renewal in the intestine: stem cells, signals and combinatorial control

The lining of the intestine is renewed at an extraordinary rate, outpacing all other tissues in the vertebrate body. The renewal process is neatly organized in space, so that the whole production line, from the ever-youthful stem cells to their dying, terminally differentiated progeny, is laid out to view in histological sections. A flurry of recent papers has clarified the key regulatory signals and brought us to the point where we can begin to give a coherent account, for at least one tissue, of how these signals collaborate to organize the architecture and behaviour of a stem-cell system.     

Silencing Relaxin-3 in Nucleus Incertus of Adult Rodents: A Viral Vector-based Approach to Investigate Neuropeptide Function

Relaxin-3, the most recently identified member of the relaxin peptide family, is produced by GABAergic projection neurons in the nucleus incertus (NI), in the pontine periventricular gray. Previous studies suggest relaxin-3 is a modulator of stress responses, metabolism, arousal and behavioural activation. Knockout mice and peptide infusions in vivo have significantly contributed to understanding the function of this conserved neuropeptide. Yet, a definitive role remains elusive due to discrepancies between models and a propensity to investigate pharmacological effects over endogenous function. To investigate the endogenous function of relaxin-3, we generated a recombinant adeno-associated viral (rAAV) vector expressing microRNA against relaxin-3 and validated its use to knock down relaxin-3 in adult rats. Bilateral stereotaxic infusion of rAAV1/2 EmGFP miR499 into the NI resulted in significant reductions in relaxin-3 expression as demonstrated by ablation of relaxin-3-like immunoreactivity at 3, 6 and 9 weeks and by qRT-PCR at 12 weeks. Neuronal health was unaffected as transduced neurons in all groups retained expression of NeuN and stained for Nissl bodies. Importantly, qRT-PCR confirmed that relaxin-3 receptor expression levels were not altered to compensate for reduced relaxin-3. Behavioural experiments confirmed no detrimental effects on general health or well-being and therefore several behavioural modalities previously associated with relaxin-3 function were investigated. The validation of this viral vector-based model provides a valuable alternative to existing in vivo approaches and promotes a shift towards more physiologically relevant investigations of endogenous neuropeptide function.     

Flowering phenology of Campanula on Mt Olynipos, Greece

We studied the fowering phenology of the genus Campanula (represented by nine species) along the elevation gradient of Mt Olympos (Greece) in order to assess whether there are elevation patterns at the genus level and whether these relate to patterns previously observed along such gradients at the community level. The traits examined were time and duration of the flowering period, flower life span, and duration of male and female flower phases. Floral attributes, such as number of flowers per plant, flower biomass, flower size, were also studied in order to examine whether they change with elevation or influence flower phenology. Flowering of Campanula species started in mid-May and ended in late September. The average duration of flowering of the genus was ca 27 d and the average floral longevity 4 d. In all but one species, the female phase lasted longer than the male. Campanula versicolor differed remarkably from all others in flower phenology and other floral trails. Nearly all Campanula populations studied had right or positively skewed flowering distributions indicating that flowering begins more abruptly than it ends. At the genus level, the time of flowering increased with elevation by 2 3d for every 100 m. Floral longevity also increased with elevation, by 0,2 d for every 100 m. Neither duration of flowering nor duration of the flower phases showed any consistent change with elevation. The same is true of the non–phenological floral traits examined. No trade–off between duration of flowering and flower life span or between structure and maintenance of flowers was apparent. The pattern of increasing floral longevity along the elevation gradient suggests a mechanism of compensation for reduecd pollinator availability.