Crystal structure of HydF scaffold protein provides insights into [FeFe]-hydrogenase maturation

[FeFe]-hydrogenases catalyze the reversible production of H₂ in some bacteria and unicellular eukaryotes. These enzymes require ancillary proteins to assemble the unique active site H-cluster, a complex structure composed of a 2Fe center bridged to a [4Fe-4S] cubane. The first crystal structure of a key factor in the maturation process, HydF, has been determined at 3 Å resolution. The protein monomer present in the asymmetric unit of the crystal comprises three domains: a GTP-binding domain, a dimerization domain, and a metal cluster-binding domain, all characterized by similar folding motifs. Two monomers dimerize, giving rise to a stable dimer, held together mainly by the formation of a continuous β-sheet comprising eight β-strands from two monomers. Moreover, in the structure presented, two dimers aggregate to form a supramolecular organization that represents an inactivated form of the HydF maturase. The crystal structure of the latter furnishes several clues about the events necessary for cluster generation/transfer and provides an excellent model to begin elucidating the structure/function of HydF in [FeFe]-hydrogenase maturation.     

Isospora canaria Box, 1975 (Apicomplexa: Eimeriidae) from canaries Serinus canaria Linnaeus (Passeriformes: Fringillidae) in Brazil

Isospora canaria Box, 1975 (Protozoa, Apicomplexa, Eimeriidae) is reported and described from canaries Serinus canaria (Linnaeus) in southeast Brazil. Its oöcysts are subspheroidal to ellipsoidal, 24.4 × 22.2 μm, with smooth, bilayered wall, ~1.2 μm. The micropyle and the oöcyst residuum are absent, but a polar granule is present. The sporocysts are lemon-shaped, 17.6 × 10.6 μm. The Stieda body is nipple-like, and substieda body is prominent and homogeneous. The sporocyst residuum is composed of scattered granules. The sporozoite has anterior and posterior refractile bodies and a nucleus. The report of this coccidium recovered from exotic canaries in South America is relevant to native passerines, mainly to Carduelis yarrellii, which are listed as vulnerable species by IUCN (International Union for Conservation of Nature and Natural Resources).     

Disentangling the molecular determinants for Cenp‐F localization to nuclear pores and kinetochores

Cenp‐F is a multifaceted protein implicated in cancer and developmental pathologies. The Cenp‐F C‐terminal region contains overlapping binding sites for numerous proteins that contribute to its functions throughout the cell cycle. Here, we focus on the nuclear pore protein Nup133 that interacts with Cenp‐F both at nuclear pores in prophase and at kinetochores in mitosis, and on the kinase Bub1, known to contribute to Cenp‐F targeting to kinetochores. By combining in silico structural modeling and yeast two‐hybrid assays, we generate an interaction model between a conserved helix within the Nup133 β‐propeller and a short leucine zipper‐containing dimeric segment of Cenp‐F. We thereby create mutants affecting the Nup133/Cenp‐F interface and show that they prevent Cenp‐F localization to the nuclear envelope, but not to kinetochores. Conversely, a point mutation within an adjacent leucine zipper affecting the kinetochore targeting of Cenp‐F KT‐core domain impairs its interaction with Bub1, but not with Nup133, identifying Bub1 as the direct KT‐core binding partner of Cenp‐F. Finally, we show that Cenp‐E redundantly contributes together with Bub1 to the recruitment of Cenp‐F to kinetochores.     

Isospora leptopogoni n. sp. (Apicomplexa: Eimeriidae) from the sepia-capped flycatcher Leptopogon amaurocephalus Tschudi, 1846 (Passeriformes: Rhynchocyclidae) in South America

Coccidian protozoan species recorded from flycatchers are few, but they have been described with a certain frequency in recent years. In this context, the present study describes a new Isospora sp. from sepia-capped flycatchers Leptopogon amaurocephalus Tschudi, 1846 captured in the Itatiaia National Park and in a reforestation area which is about 60 km away from the park boundaries, in addition to providing a molecular identification via sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene. Isospora leptopogoni n. sp. has oöcysts that are subspheroidal to ovoidal, measuring on average 22.0 × 19.7 μm, with a smooth, bi-layered wall, c.1.7 μm thick. The micropyle is delicate or inconspicuous. Oöcyst residuum is absent, but one to three polar granules are present. Sporocysts are lemon-shaped, measuring on average 14.7 × 9.3 μm, with a knob-like Stieda body and a rectangular to rounded sub-Stieda body. Sporocyst residuum is present, consisting of compactly bounded granules. Sporozoites are vermiform, with refractile bodies and nucleus. Isospora leptopogoni is different from other Isospora spp. mainly due to its lemon-shaped sporocysts, the presence of micropyle and details of Stieda and sub-Stieda bodies. Phylogenetic analysis placed I. leptopogoni close to other Isospora spp. recorded from phylogenetically related hosts and from the same biogeographic region. Finally, the recurrent finding of this coccidian species in the same L. amaurocephalus specimen in a specific locality in the Itatiaia National Park suggests that the dispersion of I. leptopogoni needs continuous transmissions between susceptible passerines as the area of movement of each L. amaurocephalus specimen appears to be quite small.

     

Secundum atrial septal defect in adults: a practical review and recent developments

Secundum atrial septal defect (ASDII) is a common congenital heart defect that causes shunting of blood between the systemic and pulmonary circulations. Patients with an isolated ASDII often remain asymptomatic during childhood and adolescence. If the defect remains untreated, however, the rates of exercise intolerance, supraventricular arrhythmias, right ventricular dysfunction and pulmonary arterial hypertension (PAH) increase with patient age, and life expectancy is reduced. Transcatheter and surgical techniques both provide valid options for ASDII closure, the former being the preferred method. With the exception of those with severe and irreversible PAH, closure is beneficial to, and thus indicated in all patients with significant shunts, regardless of age and symptoms. The symptomatic and survival benefits conferred by defect closure are inversely related to patient age and the presence of PAH, supporting timely closure after diagnosis. In this paper we review the management of adult patients with an isolated ASDII, with a focus on aspects of importance to the decision regarding defect closure and medical follow-up.     

Re-challenge with pemetrexed in advanced mesothelioma: a multi-institutional experience

ABSTRACT: BACKGROUND: Although first-line therapy for patients affected by advanced mesothelioma is well established, there is a lack of data regarding the impact of second-line treatment. METHODS: We retrospectively collected data of patients affected by advanced mesothelioma, already treated with first-line therapy based on pemetrexed and platin, with a response (partial response or stable disease) lasting at least 6 months, and re-treated with a pemetrexed-based therapy at progression. The primary objective was to describe time to progression and overall survival after re-treatment. RESULTS: Overall across several Italian oncological Institutions we found 30 patients affected by advanced mesothelioma, in progression after a 6-month lasting clinical benefit following a first-line treatment with cisplatin and pemetrexed, and re-challenged with a pemetrexed-based therapy. In these patients we found a disease control rate of 66%, with reduction of pain in 43% of patients. Overall time to progression and survival were promising for a second-line setting of patients with advanced mesothelioma, being 5.1 and 13.6 months, respectively. CONCLUSIONS: In our opinion, when a patient experience a long-lasting benefit from previous treatment with pemetrexed combined with a platin compound, the same treatment should be offered at progression.     

Biochemical Analysis of the Interactions between the Proteins Involved in the [FeFe]-Hydrogenase Maturation Process

[FeFe]-hydrogenases are iron-sulfur proteins characterized by a complex active site, the H-cluster, whose assembly requires three conserved maturases. HydE and HydG are radical S-adenosylmethionine enzymes that chemically modify a H-cluster precursor on HydF, a GTPase with a dual role of scaffold on which this precursor is synthesized, and carrier to transfer it to the hydrogenase. Coordinate structural and functional relationships between HydF and the two other maturases are crucial for the H-cluster assembly. However, to date only qualitative analysis of this protein network have been provided. In this work we showed that the interactions of HydE and HydG with HydF are distinct events, likely occurring in a precise functional order driven by different kinetic properties, independently of the HydF GTPase activity, which is instead involved in the dissociation of the maturases from the scaffold. We also found that HydF is able to interact with the hydrogenase only when co-expressed with the two other maturases, indicating that under these conditions it harbors per se all the structural elements needed to transfer the H-cluster precursor, thus completing the maturation process. These results open new working perspectives aimed at improving the knowledge of how these complex metalloenzymes are biosynthesized.     

The DCR Protein TTC3 Affects Differentiation and Golgi Compactness in Neurons through Specific Actin-Regulating Pathways

In neuronal cells, actin remodeling plays a well known role in neurite extension but is also deeply involved in the organization of intracellular structures, such as the Golgi apparatus. However, it is still not very clear which mechanisms may regulate actin dynamics at the different sites. In this report we show that high levels of the TTC3 protein, encoded by one of the genes of the Down Syndrome Critical Region (DCR), prevent neurite extension and disrupt Golgi compactness in differentiating primary neurons. These effects largely depend on the capability of TTC3 to promote actin polymerization through signaling pathways involving RhoA, ROCK, CIT-N and PIIa. However, the functional relationships between these molecules differ significantly if considering the TTC3 activity on neurite extension or on Golgi organization. Finally, our results reveal an unexpected stage-dependent requirement for F-actin in Golgi organization at different stages of neuronal differentiation.