Comparative cytogenetic analysis of the “Sylvaemus” group of Apodemus (Rodentia, Muridae): A. sylvaticus from Sicily and A. flavicollis from the central Apennines

Chromosome mapping of three common markers, ie major and 5S rRNA genes (rDNA) and telomeric repeats, and conventional chromosome bandings were applied to two sibling species, Apodemus sylvaticus Linnaeus, 1758 and A. flavicollis Melchior, 1834, to further investigate intra- and interspecific karyological differentiation in the genus Apodemus. A slight variation of the rDNA-patterns was detected between the two Apodemus species. In both of them, the major NORs were located on autosome pairs 8, 11, 12 and 22, while the two other rDNA sites detected on chromosomes 7 and 21 were variable in, respectively, A. sylvaticus and A. flavicollis. Several tiny rDNA sites were present on the sex chromosomes in both species, but their incidence was lower in A. flavicollis. Single 5S rDNA chromosomal sites were conserved on chromosome pair 20. No interstitial sites of telomeric repeats were present in either species. In the Sicilian population of A. sylvaticus, the constitutive heterochromatin pattern corresponded to the “sylvaticus-E1” cytotype, while A. flavicollis had a species-specific pattern restricted to centromeres of all chromosomes. The results are discussed in relation to cytogenetic data available for the genus, with emphasis on the Sylvaemus group/subgenus.     

The 5-hydroxytryptamine(1A) receptor is stably palmitoylated, and acylation is critical for communication of receptor with Gi protein

In the present study, we verified that the mouse 5-hydroxytryptamine(1A) (5-HT(1A)) receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT(1A) receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal C-terminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT(1A) receptor acylation, we have analyzed the ability of acylation-deficient mutants to interact with heterotrimeric G(i) protein and to modulate downstream effectors. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with G(i) protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G alpha(i) subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT(1A) receptor is critical for the enabling of G(i) protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G beta gamma-mediated pathway, in addition to the G alpha(i)-mediated signaling.     

Photoacoustically‐guided photothermal killing of mosquitoes targeted by nanoparticles

In biomedical applications, nanoparticles have demonstrated the potential to eradicate abnormal cells in small localized pathological zones associated with cancer or infections. Here, we introduce a method for nanotechnology‐based photothermal (PT) killing of whole organisms considered harmful to humans or the environment. We demonstrate that laser‐induced thermal, and accompanying nano‐ and microbubble phenomena, can injure or kill C. elegans and mosquitoes fed carbon nanotubes, gold nanospheres, gold nanoshells, or magnetic nanoparticles at laser energies that are safe for humans. In addition, a photoacoustic (PA) effect was used to control nanoparticle delivery. Through the integration of this technique with molecular targeting, nanoparticle clustering, magnetic capturing and spectral sharpening of PA and PT plasmonic resonances, our laser‐based PA‐PT nano‐theranostic platform can be applied to detection and the physical destruction of small organisms and carriers of pathogens, such as malaria vectors, spiders, bed bugs, fleas, ants, locusts, grasshoppers, phytophagous mites, or other arthropod pests, irrespective of their resistance to conventional treatments. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The influence of water on the nanomechanical behavior of the plant biopolyester cutin as studied by AFM and solid-state NMR

Atomic force microscopy and solid-state nuclear magnetic resonance have been used to investigate the effect of water absorption on the nanoscale elastic properties of the biopolyester, cutin, isolated from tomato fruit cuticle. Changes in the humidity and temperature at which fruits are grown or stored can affect the plant surface (cuticle) and modify its susceptibility to pathogenic attack by altering the cuticle's rheological properties. In this work, atomic force microscopy measurements of the surface mechanical properties of isolated plant cutin have been made as a first step to probing the impact of water uptake from the environment on surface flexibility. A dramatic decrease in surface elastic modulus (from approximately 32 to approximately 6 MPa) accompanies increases in water content as small as 2 wt %. Complementary solid-state nuclear magnetic resonance measurements reveal enhanced local mobility of the acyl chain segments with increasing water content, even at molecular sites remote from the covalent cross-links that are likely to play a crucial role in cutin's elastic properties.     

Virtual screening,selection and development of a benzindolone structural scaffold for inhibition of lumazine synthase

Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a K_i of 70 μM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (K_i 38 μM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve π–π stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.     

Developmental regulation of human cytomegalovirus receptors in cytotrophoblasts correlates with distinct replication sites in the placenta

Cytomegalovirus (CMV), the major viral cause of congenital disease, infects the uterus and developing placenta and spreads to the fetus throughout gestation. Virus replicates in invasive cytotrophoblasts in the decidua, and maternal immunoglobulin G (IgG)-CMV virion complexes, which are transcytosed by the neonatal Fc receptor across syncytiotrophoblasts, infect underlying cytotrophoblasts in chorionic villi. Immunity is central to protection of the placenta-fetal unit: infection can occur when IgG has a low neutralizing titer. Here we used immunohistochemical and function-blocking methods to correlate infection in the placenta with expression of potential CMV receptors in situ and in vitro. In placental villi, syncytiotrophoblasts express the virion receptor epidermal growth factor receptor (EGFR) but lack integrin coreceptors, and virion uptake occurs without replication. Focal infection can occur when transcytosed virions reach EGFR-expressing cytotrophoblasts that selectively initiate expression of alphaV integrin. In cell columns, proximal cytotrophoblasts lack receptors and distal cells express integrins alpha1beta1 and alphaVbeta3, enabling virion attachment. In the decidua, invasive cytotrophoblasts expressing coreceptors upregulate EGFR, thereby dramatically increasing susceptibility to infection. Our findings indicate that virion interactions with cytotrophoblasts expressing receptors in the placenta (i) change as the cells differentiate and (ii) correlate with spatially distinct sites of CMV replication in maternal and fetal compartments.