The unique Phe–His dyad of 2-ketopropyl coenzyme M oxidoreductase/carboxylase selectively promotes carboxylation and S–C bond cleavage

The 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) enzyme is the only member of the disulfide oxidoreductase (DSOR) family of enzymes, which are important for reductively cleaving S–S bonds, to have carboxylation activity. 2-KPCC catalyzes the conversion of 2-ketopropyl-coenzyme M to acetoacetate, which is used as a carbon source, in a controlled reaction to exclude protons. A conserved His–Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC, the dyad is substituted by Phe–His. Here, we propose that this difference is important for coupling carboxylation with C–S bond cleavage. We substituted the Phe–His dyad in 2-KPCC to be more DSOR like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E), and solved crystal structures of F501H and the double variant F501H_H506E. We found that F501 protects the enolacetone intermediate from protons and that the F501H variant strongly promotes protonation. We also provided evidence for the involvement of the H506 residue in stabilizing the developing charge during the formation of acetoacetate, which acts as a product inhibitor in the WT but not the H506E variant enzymes. Finally, we determined that the F501H substitution promotes a DSOR-like charge transfer interaction with flavin adenine dinucleotide, eliminating the need for cysteine as an internal base. Taken together, these results indicate that the 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.     

The decline of Afro‐Palaearctic migrants and an assessment of potential causes

There is compelling evidence that Afro‐Palaearctic (A‐P) migrant bird populations have declined in Europe in recent decades, often to a greater degree than resident or short‐distance migrants. There appear to have been two phases of decline. The first in the 1960s–1970s, and in some cases into the early 1980s, largely affected species wintering predominantly in the arid Sahelian zone, and the second since the 1980s has mostly affected species wintering in the humid tropics and Guinea forest zone. Potential drivers of these declines are diverse and are spread across and interact within the migratory cycle. Our knowledge of declining species is generally better for the breeding than the non‐breeding parts of their life cycles, but there are significant gaps in both for many species. On the breeding grounds, degradation of breeding habitats is the factor affecting the demography of the largest number of species, particularly within agricultural systems and woodland and forests. In the non‐breeding areas, the interacting factors of anthropogenic habitat degradation and climatic conditions, particularly drought in the Sahel zone, appear to be the most important factors. Based on our synthesis of existing information, we suggest four priorities for further research: (1) use of new and emerging tracking technologies to identify migratory pathways and strategies, understand migratory connectivity and enable field research to be targeted more effectively; (2) undertake detailed field studies in sub‐Saharan Africa and at staging sites, where we understand little about distribution patterns, habitat use and foraging ecology; (3) make better use of the wealth of data from the European breeding grounds to explore spatial and temporal patterns in demographic parameters and relate these to migratory pathways and large‐scale patterns of habitat change and climatic factors; and (4) make better use of remote sensing to improve our understanding of how and where land cover is changing across these extensive areas and how this impacts A‐P migrants. This research needs to inform and underpin a flyway approach to conservation, evaluating a suite of drivers across the migratory cycle and combining this with an understanding of land management practices that integrate the needs of birds and people in these areas.     

Impaired glomerulogenesis and endothelial cell migration in Pkd1-deficient renal organ cultures

The PKD1 gene is essential for a number of biological functions, and its loss-of-function causes autosomal dominant polycystic kidney disease (ADPKD). The gene is developmentally regulated and believed to play an essential role in renal development. Previous studies have shown that manipulating murine renal organ cultures with dominant-negative forms of the Pkd1 gene impaired ureteric bud (UB) branching. In the current study, we analyzed different stages of renal development in two distinct mouse models carrying either a null mutation or inactivation of the last two exons of Pkd1. Surprisingly, metanephric explants from Pkd1-deleted kidneys harvested at day E11.5 did not show defects of UB branching and elongation, estimated by cytokeratin staining on fixed tissues or by Hoxb7-GFP time-lapse imaging. However, renal explants from Pkd1-mutants isolated at day E14.5 showed impaired nephrogenesis. Notably, we observed cell migratory defects in the developing endothelial compartment. Previous studies had implicated the Pkd1 gene in controlling cell migration and collagen deposition through PI3 kinases. In line with these studies, our results show that wild-type explants treated with PI3-kinase inhibitors recapitulate the endothelial defects observed in Pkd1 mutants, whereas treatment with VEGF only partially rescued the defects. Our data are consistent with a role for the Pkd1 gene in the endothelium that may be required for proper nephrogenesis.     

Phenomenological partial-specific volumes for G-quadruplex DNAs

Accurate partial-specific volume ([`(v)] \overline{v} ) values are required for sedimentation velocity and sedimentation equilibrium analyses. For nucleic acids, the estimation of these values is complicated by the fact that [`(v)] \overline{v} depends on base composition, secondary structure, solvation and the concentrations and identities of ions in the surrounding buffer. Here we describe sedimentation equilibrium measurements of the apparent isopotential partial-specific volume ϕ′ for two G-quadruplex DNAs and a single-stranded DNA of similar molecular weight and base composition. The G-quadruplex DNAs are a 22 nucleotide fragment of the human telomere consensus sequence and a 27 nucleotide fragment from the human c-myc promoter. The single-stranded DNA is 26 nucleotides long and is designed to have low propensity to form secondary structures. Parallel measurements were made in buffers containing NaCl and in buffers containing KCl, spanning the range 0.09 M ≤ [salt] ≤ 2.3 M. Limiting values of ϕ′, extrapolated to [salt] = 0 M, were: 22-mer (NaCl-form), 0.525 ± 0.004 mL/g; 22-mer (KCl-form), 0.531 ± 0.006 mL/g; 27-mer (NaCl-form), 0.548 ± 0.005 mL/g; 27-mer (KCl-form), 0.557 ± 0.006 mL/g; 26-mer (NaCl-form), 0.555 ± 0.004 mL/g; 26-mer (KCl-form), 0.564 ± 0.006 mL/g. Small changes in ϕ′ with [salt] suggest that large changes in counterion association or hydration are unlikely to take place over these concentration ranges.     

Amphibian chytrid fungus and ranaviruses in the Northwest Territories, Canada

Pathogens can cause serious declines in host species, and knowing where pathogens associated with host declines occur facilitates understanding host-pathogen ecology. Suspected drivers of global amphibian declines include infectious diseases, with 2 pathogens in particular, Batrachochytrium dendrobatidis (Bd) and ranaviruses, causing concern. We explored the host range and geographic distribution of Bd and ranaviruses in the Taiga Plains ecoregion of the Northwest Territories, Canada, in 2007 and 2008. Both pathogens were detected, greatly extending their known geographic distributions. Ranaviruses were widespread geographically, but found only in wood frogs. In contrast, Bd was found at a single site, but was detected in all 3 species of amphibians in the survey area (wood frogs, boreal chorus frogs, western toads). The presence of Bd in the Northwest Territories is not congruent with predicted distributions based on niche models, even though findings from other studies at northern latitudes are consistent with those same models. Unexpectedly, we also found evidence that swabs routinely used to collect samples for Bd screening detected fewer infections than toe clips. Our use and handling of the swabs was consistent with other studies, and the cause of the apparent lack of integrity of swabs is unknown. The ranaviruses detected in our study were confirmed to be Frog Virus 3 by sequence analysis of a diagnostic 500 bp region of the major capsid protein gene. It is unknown whether Bd or ranaviruses are recent arrivals to the Canadian north. However, the genetic analyses required to answer that question can inform larger debates about the origin of Bd in North America as well as the potential effects of climate change and industrial development on the distributions of these important amphibian pathogens.     

Ric-8A and Giα Recruit LGN,NuMA, and Dynein to the Cell Cortex To Help Orient the Mitotic Spindle

In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein α (Gα) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and Gα_i function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to Gα_i, thus preventing its GEF activity for Gα_i. Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased Gα_i expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.The cortical capture of astral microtubules is essential to generate the forces needed for mitotic spindle positioning for both symmetric and asymmetric cell divisions (23, 29). Failure to either capture astral microtubules or the inappropriate application of pulling forces adversely affects mitotic spindle orientation, and can impede embryogenesis and alter cell fate decisions. Studies examining mitotic spindle orientation in Drosophila embryonic and larval neuroblasts have identified two critical pathways, the Gα/Pins/Mud pathway and the Pins/Dlg/Khc73 pathway (29). The heterotrimeric G-protein α subunit (Gα), Pins (Partner-of-Inscuteable), and Mud (Mushroom body defect) are members of an evolutionarily conserved noncanonical G-protein signaling pathway, which form a tripartite protein complex linked to the apical Par complex by the adapter protein Inscuteable (29, 37). Reducing the level of Gα_i, Pins, or Mud prevents neuroblast mitotic spindle alignment. A second spindle orientation pathway involves Pins, the tumor suppressor Discs large (Dlg) and the microtubule plus-end-directed kinesin heavy chain 73 (Khc73). Khc73 binds Dlg and coimmunoprecipitates with Pins. Khc73 localized to astral microtubules can induce Pins-Dlg cortical polarity (27).In canonical G-protein signaling pathways, the binding of ligand to a seven-transmembrane receptor triggers a heterotrimeric G-protein α subunit (Gα) to exchange GTP for GDP, resulting in the dissociation of the Gα subunit from its associated Gβγ heterodimer (12, 20). This exposes interactive sites in the Gα and Gβγ subunits, allowing their binding to and activation of downstream effectors. Since Gα subunits possess an intrinsic GTPase activity, GTP hydrolysis leads to the reassembly of heterotrimeric G protein causing signaling to cease. In noncanonical G-protein signaling the seven-transmembrane receptor is replaced by an intracellular guanine nucleotide exchange factor, such as Ric-8 (37). In studies in Drosophila and Caenorhabditis elegans Ric-8 has been shown to positively regulate Gα_i activity and is essential for asymmetric cell divisions (1, 2, 5, 8, 11, 36). Although initially characterized as a guanine nucleotide exchange factor (GEF) for isolated G_αsubunits, more recent biochemical studies have shown that Ric-8A (the mammalian equivalent of Ric-8) also acts on a complex of GDP-Gα_i, the mammalian Pins homolog LGN, and NuMA (nuclear mitotic apparatus protein; the mammalian equivalent of Mud) catalytically releasing GTP-Gα_i and causing liberation of NuMA from LGN (30, 31). Ric-8A can also catalyze guanine nucleotide exchange on Gα_i1 bound to the GPR/GoLoco exchange inhibitor AGS3, a paralog of LGN (33). During mitosis the N-terminal portion of LGN binds NuMA and the C-terminal domain binds GDP-Gα_i and the trimolecular complex localizes to the cell cortex, where the dynamic release of NuMA from LGN may regulate aster microtubule pulling during cell division (3, 9, 10, 22).In the present study we examined the role of Ric-8A in mitotic spindle orientation in adherent cells and in polarized MDCK cells. In nonpolarized adherent cells cell such as HeLa, integrin mediated cell-substrate adhesion orients the mitotic spindle parallel to the substratum, and thereby both daughter cells remain attached. This requires the actin cytoskeleton, astral microtubules, the microtubule plus end tracking protein EB1, myosin X, cdc42, LIM kinase 1, and phosphatidylinositol(3,4,5)-triphosphate (PIP₃) (13, 18, 32, 34, 35). PIP₃ may direct dynein/dynactin-dependent pulling forces on the spindle midcortex to orient the mitotic spindle (34). In polarized cells such as Madin-Darby canine kidney (MDCK) cells, the mitotic spindle is constrained by the topology of the cell and cortical cues provided by adherens junctions (24). In contrast to HeLa cells these cues are insensitive to phosphatidylinositol 3-kinase (PI3K) inhibition, which blocks the generation of PIP₃ (34). We found that inhibiting either Ric-8A or Gα_i expression impairs the orientation of the metaphase mitotic spindle in HeLa cells and pertussis toxin, which blocks Ric-8A triggered nucleotide exchange, disrupts the normal mitotic spindle alignment of both HeLa and MDCK cells. Impairment of Ric-8A expression or function inhibits the localization of Gα_i1, LGN, NuMA, and dynein to the metaphase cortex opposite the spindle poles.     

Rule-based simulation of temperate bacteriophage infection: Restriction–modification as a limiter to infection in bacterial populations

An individual-based model (IbM) for bacterial adaptation and evolution, COSMIC-Rules, has been employed to simulate interactions of virtual temperate bacteriophages (phages) and their bacterial hosts. Outcomes of infection mimic those of a phage such as lambda, which can enter either the lytic or lysogenic cycle, depending on the nutritional status of the host. Infection of different hosts possessing differing restriction and modification systems is also simulated. Phages restricted upon infection of one restricting host can be adapted (by host-controlled modification of the phage genome) and subsequently propagate with full efficiency on this host. However, such ability is lost if the progeny phages are passaged through a new host with a different restriction and modification system before attempted re-infection of the original restrictive host. The simulations show that adaptation and re-adaptation to a particular host-controlled restriction and modification system result in lower efficiency and delayed lysis of bacterial cells compared with infection of non-restricting host bacteria.     

Protein kinase D as a potential new target for cancer therapy

Protein kinase D is a novel family of serine/threonine kinases and diacylglycerol receptors that belongs to the calcium/calmodulin-dependent kinase superfamily. Evidence has established that specific PKD isoforms are dysregulated in several cancer types, and PKD involvement has been documented in a variety of cellular processes important to cancer development, including cell growth, apoptosis, motility, and angiogenesis. In light of this, there has been a recent surge in the development of novel chemical inhibitors of PKD. This review focuses on the potential of PKD as a chemotherapeutic target in cancer treatment and highlights important recent advances in the development of PKD inhibitors.     

Internalization of Flax Rust Avirulence Proteins into Flax and Tobacco Cells Can Occur in the Absence of the Pathogen

Translocation of pathogen effector proteins into the host cell cytoplasm is a key determinant for the pathogenicity of many bacterial and oomycete plant pathogens. A number of secreted fungal avirulence (Avr) proteins are also inferred to be delivered into host cells, based on their intracellular recognition by host resistance proteins, including those of flax rust (Melampsora lini). Here, we show by immunolocalization that the flax rust AvrM protein is secreted from haustoria during infection and accumulates in the haustorial wall. Five days after inoculation, the AvrM protein was also detected within the cytoplasm of a proportion of plant cells containing haustoria, confirming its delivery into host cells during infection. Transient expression of secreted AvrL567 and AvrM proteins fused to cerulean fluorescent protein in tobacco (Nicotiana tabacum) and flax cells resulted in intracellular accumulation of the fusion proteins. The rust Avr protein signal peptides were functional in plants and efficiently directed fused cerulean into the secretory pathway. Thus, these secreted effectors are internalized into the plant cell cytosol in the absence of the pathogen, suggesting that they do not require a pathogen-encoded transport mechanism. Uptake of these proteins is dependent on signals in their N-terminal regions, but the primary sequence features of these uptake regions are not conserved between different rust effectors.