Recent publications have argued that there are potentially serious consequences for researchers in recognising distinct genera in the terminal fusarioid clade of the family Nectriaceae . Thus, an alternate hypothesis, namely a very broad concept of the genus Fusarium was proposed. In doing so, however, a significant body of data that supports distinct genera in Nectriaceae based on morphology, biology, and phylogeny is disregarded. A DNA phylogeny based on 19 orthologous protein-coding genes was presented to support a very broad concept of Fusarium at the F1 node in Nectriaceae . Here, we demonstrate that re-analyses of this dataset show that all 19 genes support the F3 node that represents Fusarium sensu stricto as defined by F. sambucinum (sexual morph synonym Gibberella pulicaris ). The backbone of the phylogeny is resolved by the concatenated alignment, but only six of the 19 genes fully support the F1 node, representing the broad circumscription of Fusarium. Furthermore, a re-analysis of the concatenated dataset revealed alternate topologies in different phylogenetic algorithms, highlighting the deep divergence and unresolved placement of various Nectriaceae lineages proposed as members of Fusarium . Species of Fusarium s. str. are characterised by Gibberella sexual morphs, asexual morphs with thin- or thick-walled macroconidia that have variously shaped apical and basal cells, and trichothecene mycotoxin production, which separates them from other fusarioid genera. Here we show that the Wollenweber concept of Fusarium presently accounts for 20 segregate genera with clear-cut synapomorphic traits, and that fusarioid macroconidia represent a character that has been gained or lost multiple times throughout Nectriaceae . Thus, the very broad circumscription of Fusarium is blurry and without apparent synapomorphies, and does not include all genera with fusarium-like macroconidia, which are spread throughout Nectriaceae ( e.g. , Cosmosporella , Macroconia , Microcera ). In this study four new genera are introduced, along with 18 new species and 16 new combinations. These names convey information about relationships, morphology, and ecological preference that would otherwise be lost in a broader definition of Fusarium . To assist users to correctly identify fusarioid genera and species, we introduce a new online identification database, Fusarioid-ID, accessible at www.fusarium.org . The database comprises partial sequences from multiple genes commonly used to identify fusarioid taxa ( ...

The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

The Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is primarily expressed by neural crest cells during embryogenesis. Following a complete downregulation after birth, ROR1 was shown to re-express in various types of cancers. Little is known about ROR1 expression and function in melanoma. Here we show that ROR1 is aberrantly expressed in both melanoma cell lines and tumors and that its expression associates with poor Post-Recurrence Survival of melanoma. Using gain- and loss-of-function approaches we found that ROR1 enhances both anchorage-dependent and -independent growth of melanoma cells. In addition, ROR1 decreases cell adhesion and increases cell motility and migration. Mechanistically, ROR1 was found to induce upregulation of Akt and the mesenquimal markers N-cadherin and vimentin. The regulation of N-cadherin by ROR1 relies on both Akt dependent and independent mechanisms. ROR1 does not affect Wnt canonical pathway but was found to be engaged in a positive feedback loop with Wnt5a. In summary, we show that ROR1 contributes to melanoma progression and is a candidate biomarker of poor prognosis. Although further studies are needed to confirm this possibility, the present work indicates that ROR1 is a good prospective target for melanoma cancer therapy. © 2015 Wiley Periodicals, Inc.

The idea of G protein-coupled receptors (GPCRs) coupling to G protein solely in their active form was abolished when it was found that certain ligands induce a G protein-coupled but inactive receptor form. This receptor form interferes with signaling of other receptors by sequestering G protein. However, the spontaneous existence of this receptor species has never been established. The aim of the present work was to evaluate the existence of the spontaneous conformation of the receptor inactively coupled to G protein able to interfere with the response of other GPCRs. According to the law of mass action, receptor overexpression should lead to increased amounts of all spontaneously occurring species. Based on this, we generated Chinese hamster ovary (CHO-K1)-derived cell lines expressing various amounts of the human histamine H2 receptor. In these systems, the signaling of other endogenously and transiently expressed GPCRs was attenuated proportionally to human H2 receptor expression levels. G protein transfection specifically reverted this attenuation, strongly suggesting hijacking of the G protein from a common pool. Similar attenuation effects were observed when the ␤ 2 -adrenergic receptor was overexpressed, suggesting that this is a more general phenomenon. Moreover, in human mammary MDA-MB-231 cells, a consistent increase in the response of other GPCRs was observed when endogenous expression of ␤ 2 -adrenergic receptor was knocked down using specific small interfering RNAs. Our findings show that GPCRs may interact with the signaling of other receptors by modulating the availability of the G protein and suggest the existence of GPCR spontaneous coupling to G proteins in an inactive form. G protein-coupled receptors (GPCRs)2 form a large and functionally diverse superfamily of proteins that transduce signals across cell membranes. Although much is known about structural features of GPCRs involved in ligand recognition and G protein binding, the actual mechanism underlying GPCR signaling remains unclear.Traditionally, agonist occupancy of GPCRs is believed to result in a conformational change in the receptor, leading to activation of G proteins (1). However, in genetically engineered systems where receptors can be expressed at high densities, Costa and Herz (2) noted that high levels of receptor expression uncovered the existence of a population of spontaneously (unliganded) active receptors, resulting in an elevated basal response in the system.The histamine H2 receptor (H2R) is an extensively characterized member of the GPCR family, which in most systems couples to G s proteins to activate adenylyl cyclase (3-6). Compared with other GPCRs, the H2R is unique in that the wildtype receptor possesses a remarkably high degree of constitutive activity. With a receptor density of 300 fmol/mg protein, constitutive H2 receptor activity could be detected in Chinese hamster ovary (CHO-K1) cells (7).The notion that GPCRs also signal without an external chemical trigger, i.e. in a constitutive or spontaneous manner, res...