Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6

Here we investigate the role of rab5 and its cognate exchange factors rabex-5 and hRME-6 in the regulation of AP2 uncoating from endocytic clathrin-coated vesicles (CCVs). In vitro, we show that the rate of AP2 uncoating from CCVs is dependent on the level of functional rab5. In vivo, overexpression of dominant-negative rab5^S34N, or small interfering RNA (siRNA)–mediated depletion of hRME-6, but not rabex-5, resulted in increased steady-state levels of AP2 associated with endocytic vesicles, which is consistent with reduced uncoating efficiency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to α-adaptin ear, which displaces the ear-associated μ2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-μ2 levels, and expression of a phosphomimetic μ2 mutant increases levels of endocytic vesicle-associated AP2. Depletion of hRME-6 or rab5^S35N expression also increases the levels of phosphoinositide 4,5-bisphosphate (PtdIns(4,5)P₂) associated with endocytic vesicles. These data are consistent with a model in which hRME-6 and rab5 regulate AP2 uncoating in vivo by coordinately regulating μ2 dephosphorylation and PtdIns(4,5)P₂ levels in CCVs.     

Biological versus geochemical control and environmental change drivers of the base metal budgets of a tropical montane forest in Ecuador during 15 years

To assess the susceptibility of the base metal budget of a remote tropical montane forest in Ecuador to environmental change, we determined the extent of biological control of base metal fluxes and explored the impact of atmospheric inputs and precipitation, considered as potential drivers of ecosystem change, on the base metal fluxes. We quantified all major base metal fluxes in a ca. 9.1 ha forested catchment from 1998 to 2013. Mean (±s.d.) annual flux to the soil via throughfall + stemflow + litterfall was 13800 ± 1500 mg m^?2 Ca, 19000 ± 1510 mg m^?2 K, 4690 ± 619 mg m^?2 Mg and 846 ± 592 mg m^?2 Na of which 22 ± 6, 45 ± 16, 39 ± 10 and 84 ± 33%, respectively, were leached to below the organic layer. The mineral soil retained 79–94% of this Ca, K and Mg, while Na was released. Weathering rates estimated with three different approaches ranged from not detected (ND) to 504 mg m^?2 year^?1 Ca, ND-1770 mg m^?2 year^?1 K, 287–597 mg m^?2 year^?1 Mg and 403–540 mg m^?2 year^?1 Na. The size of mainly biologically controlled aboveground fluxes of Ca, K and Mg was 1–2 orders of magnitude larger than that of mainly geochemically controlled fluxes (sorption to soil and weathering). The elemental catchment budgets (total deposition ? streamflow) were positive for Ca (574 ± 893 mg m^?2) and K (1330 ± 773 mg m^?2), negative for Na (?370 ± 1300 mg m^?2) and neutral for Mg (1.89 ± 304 mg m^?2). Our results demonstrate that biological processes controlled element retention for Ca, K and Mg in the biological part of the ecosystem. This was different for Na, which was mainly released by weathering from the study catchment, while the biological part of the ecosystem was Na-poor. The deposition of base metals was the strongest driver of their budgets suggesting that the base metal cycling of the study ecosystem is susceptible to changing deposition.     

Oxidation promotes insertion of the CLIC1 chloride intracellular channel into the membrane

Members of the chloride intracellular channel (CLIC) family exist primarily as soluble proteins but can also auto-insert into cellular membranes to form ion channels. While little is known about the process of CLIC membrane insertion, a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural metamorphosis between a monomeric glutathione-S-transferase homolog and an all-helical dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis facilitates CLIC1 membrane insertion is unclear. In this work, we have sought to characterise the role of oxidation in the process of CLIC1 membrane insertion. We examined how redox conditions modify the ability of CLIC1 to associate with and insert into the membrane using fluorescence quenching studies and a sucrose-loaded vesicle sedimentation assay to measure membrane binding. Our results suggest that oxidation of monomeric CLIC1, in the presence of membranes, promotes insertion into the bilayer more effectively than the oxidised CLIC1 dimer.     

Broadly directed SARS-CoV-2-specific CD4+ T cell response includes frequently detected peptide specificities within the membrane and nucleoprotein in patients with acute and resolved COVID-19

The aim of this study was to define the breadth and specificity of dominant SARS-CoV-2-specific T cell epitopes using a comprehensive set of 135 overlapping 15-mer peptides covering the SARS-CoV-2 envelope (E), membrane (M) and nucleoprotein (N) in a cohort of 34 individuals with acute (n = 10) and resolved (n = 24) COVID-19. Following short-term virus-specific in vitro cultivation, the single peptide-specific CD4+ T cell response of each patient was screened using enzyme linked immuno spot assay (ELISpot) and confirmed by single-peptide intracellular cytokine staining (ICS) for interferon-γ (IFN-γ) production. 97% (n = 33) of patients elicited one or more N, M or E-specific CD4+ T cell responses and each patient targeted on average 21.7 (range 0–79) peptide specificities. Overall, we identified 10 N, M or E-specific peptides that showed a response frequency of more than 36% and five of them showed high binding affinity to multiple HLA class II binders in subsequent in vitro HLA binding assays. Three peptides elicited CD4+ T cell responses in more than 55% of all patients, namely Mem_P30 (aa146-160), Mem_P36 (aa176-190), both located within the M protein, and Ncl_P18 (aa86-100) located within the N protein. These peptides were further defined in terms of length and HLA restriction. Based on this epitope and restriction data we developed a novel DRB*11 tetramer (Mem_aa145-164) and examined the ex vivo phenotype of SARS-CoV-2-specific CD4+ T cells in one patient. This detailed characterization of single T cell peptide responses demonstrates that SARS-CoV-2 infection universally primes a broad T cell response directed against multiple specificities located within the N, M and E structural protein.     

The Relationship Between Foraging,Learning Abilities and Neophobia in Two Species of Darwin’s Finches

The ability to unlearn a previously established association is an important component of behavioural flexibility and may vary according to species ecology. Previously, two closely related sympatric Darwin’s finches were found to differ in their learning abilities. Small tree finches (Camarhynchus parvulus) outperformed woodpecker finches (Cactospiza pallida) in reversal learning but performed worse in an operant task. We attributed this difference to the habit of woodpecker finches to engage in long bouts of energetic pecking during extractive foraging. Persistently repeating one action without reward could favour performance in operant tasks but also limit behavioural flexibility. Here, we tested whether perseverance is the reason for woodpecker finches’ depressed reversal learning performance. Two new reversal conditions allowed the disentanglement of two sources of error in reversal learning: perseverant choice of the previously rewarded stimulus and failure to respond to the previously non‐rewarded stimulus. For the within‐species comparison, we predicted that woodpecker finches should find it more difficult to learn to avoid the previously rewarded stimulus than learning to choose the previously non‐rewarded stimulus. For the species comparison, we predicted the woodpecker finches should make more errors of perseverance than small tree finches. As performance could also be influenced by reaction to novelty, we compared neophobic responses between species and related them to reversal learning proficiency. We found no significant difference in reversal learning in the predicted direction, but found a negative correlation between neophobia and reversal learning at the inter‐ and the intraspecific level, which points towards a general relationship between reaction to novelty and flexibility.     

Network-Based Data Integration for Selecting Candidate Virulence Associated Proteins in the Cereal Infecting Fungus Fusarium graminearum

The identification of virulence genes in plant pathogenic fungi is important for understanding the infection process, host range and for developing control strategies. The analysis of already verified virulence genes in phytopathogenic fungi in the context of integrated functional networks can give clues about the underlying mechanisms and pathways directly or indirectly linked to fungal pathogenicity and can suggest new candidates for further experimental investigation, using a ‘guilt by association’ approach. Here we study 133 genes in the globally important Ascomycete fungus Fusarium graminearum that have been experimentally tested for their involvement in virulence. An integrated network that combines information from gene co-expression, predicted protein-protein interactions and sequence similarity was employed and, using 100 genes known to be required for virulence, we found a total of 215 new proteins potentially associated with virulence of which 29 are annotated as hypothetical proteins. The majority of these potential virulence genes are located in chromosomal regions known to have a low recombination frequency. We have also explored the taxonomic diversity of these candidates and found 25 sequences, which are likely to be fungal specific. We discuss the biological relevance of a few of the potentially novel virulence associated genes in detail. The analysis of already verified virulence genes in phytopathogenic fungi in the context of integrated functional networks can give clues about the underlying mechanisms and pathways directly or indirectly linked to fungal pathogenicity and can suggest new candidates for further experimental investigation, using a ‘guilt by association’ approach.     

Microalgal assemblages in a poikilohaline pond

Microalgal strains for algal biofuels production in outdoor ponds will need to have high net growth rates under diverse environmental conditions. A small, variable salinity pond in the San Elijo Lagoon estuary in southern California was chosen to serve as a model pond due to its routinely high chlorophyll content. Profiles of microalgal assemblages from water samples collected from April 2011 to January 2012 were obtained by constructing 18S rDNA environmental clone libraries. Pond assemblages were found to be dominated by green algae Picochlorum sp. and Picocystis sp. throughout the year. Pigment analysis suggested that the two species contributed most of the chlorophyll a of the pond, which ranged from 21.9 to 664.3 μg · L^?1 with the Picocystis contribution increasing at higher salinities. However, changes of temperature, salinity or irradiance may have enabled a bloom of the diatom Chaetoceros sp. in June 2011. Isolates of these microalgae were obtained and their growth rates characterized as a function of temperature and salinity. Chaetoceros sp. had the highest growth rate over the temperature test range while it showed the most sensitivity to high salinity. All three strains showed the presence of lipid bodies during nitrogen starvation, suggesting they have potential as future biofuels strains.     

A new method for reconstitution of highly fusogenic sendai virus envelopes

A new way for reconstituting highly fusogenic Sendai virus envelopes is described. As opposed to previously described methods, in the present one the detergent (Triton X-100) is removed by direct addition of SM-2 Bio-beads to the detergent solubilized mixture of the viral phospholipids and glycoproteins, thus avoiding the long dialysis step. The vesicles obtained in the present work resemble, in their composition, size and features, envelopes of intact Sendai virus particles. The present method allows the enclosure of low and high molecular weight material within the reconstituted viral envelopes.