INSL4 Pseudogenes Help Define the Relaxin Family Repertoire in the Common Ancestor of Placental Mammals

The relaxin/insulin-like (RLN/INSL) gene family comprises a group of signaling molecules that perform physiological roles related mostly to reproduction and neuroendocrine regulation. They are found on three different locations in the mammalian genome, which have been called relaxin family locus (RFL) A, B, and C. Early in placental mammalian evolution, the ancestral proto-RLN gene at the RFLB locus underwent successive rounds of small-scale duplications resulting in variable number of paralogous genes in different placental lineages. Most placental mammals harbor copies of the RLN2 and INSL6 paralogs in the RFLB. However, the origin of an additional paralog, INSL4 (also known as placentin), has been controversial as its phyletic distribution does not converge with its phylogenetic position. In principle, by searching for INSL4 genes in representative species of all major groups of mammals we can gain insights into when the gene originated and better reconstruct its evolutionary history. Here we identified INSL4 pseudogenes in two laurasiatherian, (alpaca and dolphin) and one xenarthran (armadillo) species. Phylogenetic and synteny analyses confirmed that the identified pseudogenes are orthologs of INSL4. According to these results, the proto-RLN gene in the RFLB underwent two successive tandem duplications which gave rise the INSL6 and INSL4 paralogs in the last common ancestor of placental mammals. The INSL4 gene was subsequently inactivated or lost from the genome in all placentals other than catarrhine primates, where its product became functionally relevant. Our results highlight the contribution of relatively old gene duplicates to the gene complement of extant species.     

Genome sequences of Lactococcus garvieae TB25, isolated from Italian cheese, and Lactococcus garvieae LG9, isolated from Italian rainbow trout

Lactococcus garvieae is a fish pathogen and an emerging zoonotic opportunistic pathogen as well as a component of natural microbiota in dairy products. Here, we present the first report of a genome sequence of L. garvieae TB25, isolated from a dairy source, and that of L. garvieae LG9, isolated from rainbow trout.     

Mechanisms of monomeric and dimeric glycogenin autoglucosylation

Initiation of glucose polymerization by glycogenin autoglucosylation at Tyr-194 is required to prime de novo biosynthesis of glycogen. It has been proposed that the synthesis of the primer proceeds by intersubunit glucosylation of dimeric glycogenin, even though it has not been demonstrated that this mechanism is responsible for the described polymerization extent of 12 glucoses produced by the dimer. We reported previously the intramonomer glucosylation capability of glycogenin without determining the extent of autoglucopolymerization. Here, we show that the maximum specific autoglucosylation extent (MSAE) produced by the non-glucosylated glycogenin monomer is 13.3 ± 1.9 glucose units, similar to the 12.5 ± 1.4 glucose units measured for the dimer. The mechanism and capacity of the dimeric enzyme to carry out full glucopolymerization were also evaluated by construction of heterodimers able to glucosylate exclusively by intrasubunit or intersubunit reaction mechanisms. The MSAE of non-glucosylated glycogenin produced by dimer intrasubunit glucosylation was 16% of that produced by the monomer. However, partially glucosylated glycogenin was able to almost complete its autoglucosylation by the dimer intrasubunit mechanism. The MSAE produced by heterodimer intersubunit glucosylation was 60% of that produced by the wild-type dimer. We conclude that both intrasubunit and intersubunit reaction mechanisms are necessary for the dimeric enzyme to acquire maximum autoglucosylation. The full glucopolymerization capacity of monomeric glycogenin indicates that the enzyme is able to synthesize the glycogen primer without the need for prior dimerization.     

Differential susceptibility in a specialised aphidophagous ladybird,Platynaspis luteorubra (Coleoptera: Coccinellidae), facing intraguild predation by exotic and native generalist predators

The establishment of exotic generalist predators raises concern about possible domination of the invaded community and displacement of native species. In this respect, recent studies suggest that specialists are likely to be more at risk of niche displacement than generalists, thereby compromising biological control in agroecosystems. Under laboratory conditions, we examined the susceptibility of a specialised myrmecophilous ladybird, Platynaspis luteorubra Goeze, as intraguild (IG) prey of generalist native (Oenopia conglobata L. and Adalia bipunctata L.) and exotic (Harmonia axyridis Pallas) coccinellid species. We described parameters of predatory behaviour (for example, attack rate and effectiveness of antipredator defence) and their variation among predators. Furthermore, we estimated the effect of these parameters on the time to predation using a Cox proportional hazards model. We found that A. bipunctata was the most effective IG predator of P. luteorubra, with the highest rate of successful attacks; however, P. luteorubra was not very susceptibile to attack by O. conglobata or H. axyridis, and these species exhibited lower rates of successful attack and predation. Our results, together with field observations, suggest that IG predation is a factor that fosters niche specialization in P. luteorubra and decreases the realised niche of this species in central Italy. Furthermore the lower susceptibility of P. luteorubra to H. axyridis may be due to a lack of historical co-occurrence between preimmaginal stages, possibly arising from differental selection of oviposition sites.     

Discovery of the first small molecule inhibitor of human DDX3 specifically designed to target the RNA binding site: towards the next generation HIV-1 inhibitors

Efficacy of currently approved anti-HIV drugs is hampered by mutations of the viral enzymes, leading invariably to drug resistance and chemotherapy failure. Recent data suggest that cellular co-factors also represent useful targets for anti-HIV therapy. Here we describe the identification of the first small molecules specifically designed to inhibit the HIV-1 replication by targeting the RNA binding site of the human DEAD-Box RNA helicase DDX3. Optimization of a easily synthetically accessible hit (1) identified by application of a high-throughput docking approach afforded the promising compounds 6 and 8 which proved to inhibit both the helicase and ATPase activity of DDX3 and to reduce the viral load of peripheral blood mononuclear cells (PBMC) infected with HIV-1.     

Synthesis and biological evaluation of novel small non-peptidic HIV-1 PIs: the benzothiophene ring as an effective moiety

Synthesis and biological evaluation of a new series of potential HIV-1 protease inhibitors incorporating different heterocycles are described. The variation of heteroatom in such molecules has displayed totally different biological activities and a benzothiophene containing inhibitor has shown high potency against wild type HIV-1 protease with IC(50)=60 nM, thanks to the lower desolvation penalty to be payed by such hydrophobic moiety.     

Ferrocene labelings as inhibitors and dual electrochemical sensors of human glutathione S-transferase P1-1

The inhibitory and sensor properties of two ferrocene conjugates, in which the ferrocene and glutathione are linked through a spacer arm of different length and chemical structure, on human Pi glutathione S-transferase, were examined by activity assays, ITC, fluorescence spectroscopy and voltammetry. Such ferrocene conjugates are strong competitive inhibitors of this enzyme with an enhanced binding affinity, the one bearing the longest spacer arm being the most potent inhibitor. Voltammetric measurements showed a strong decrease of the peak current intensity and an increase of the oxidation potential upon binding of ferrocene–glutathione conjugates to GST P1-1 showing that both conjugates can be used as dual electrochemical sensors for GST P1-1.     

Efficient synthesis and biological evaluation of proximicins A, B and C

A quick and efficient synthesis and the biological evaluation of promising antitumor-antibiotics proximicins A, B and C are reported. The characteristic repetitive unit of these molecules, the methyl 4-Boc-aminofuran-2-carboxylate 15, was prepared in three synthetic steps in good yield using an optimised copper-catalysed amidation method. The proximicins were evaluated for their antitumor activity using cellular methods. Proximicin B induced apoptosis in both Hodgkin's lymphoma and T-cell leukemia cell lines and proximicin C exhibited significantly high cytotoxicity against glioblastoma and breast carcinoma cells. The proximicins were also screened against Escherichia coli, Enterococcus faecalis and several strains of methicillin-and multidrug-resistant Staphylococcus aureus. Proximicin B showed noteworthy activity against antibiotic-resistant Gram-positive cocci.     

Protein Secondary Structure Determination by Constrained Single-Particle Cryo-Electron Tomography

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Highlights? Efficient extraction of high-resolution information from cryo-EM tilt series ? Successful CTF correction strategy despite low-contrast and quality of tilted images ? Use of geometric constraints in refinement improves orientational accuracy of images ? Enforcement of tomographic constraints reduces model bias and overfitting artifacts