The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment

Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges’ role in Giardia biology. Live imaging revealed that the flange grows to around 1 μm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia’s unconventional actin cytoskeleton has an important role in supporting parasite attachment.     

Plants,microorganisms, and soil temperatures contribute to a decrease in methane fluxes on a drained Arctic floodplain

As surface temperatures are expected to rise in the future, ice‐rich permafrost may thaw, altering soil topography and hydrology and creating a mosaic of wet and dry soil surfaces in the Arctic. Arctic wetlands are large sources of CH₄, and investigating effects of soil hydrology on CH₄ fluxes is of great importance for predicting ecosystem feedback in response to climate change. In this study, we investigate how a decade‐long drying manipulation on an Arctic floodplain influences CH₄‐associated microorganisms, soil thermal regimes, and plant communities. Moreover, we examine how these drainage‐induced changes may then modify CH₄ fluxes in the growing and nongrowing seasons. This study shows that drainage substantially lowered the abundance of methanogens along with methanotrophic bacteria, which may have reduced CH₄ cycling. Soil temperatures of the drained areas were lower in deep, anoxic soil layers (below 30 cm), but higher in oxic topsoil layers (0–15 cm) compared to the control wet areas. This pattern of soil temperatures may have reduced the rates of methanogenesis while elevating those of CH₄ oxidation, thereby decreasing net CH₄ fluxes. The abundance of Eriophorum angustifolium, an aerenchymatous plant species, diminished significantly in the drained areas. Due to this decrease, a higher fraction of CH₄ was alternatively emitted to the atmosphere by diffusion, possibly increasing the potential for CH₄ oxidation and leading to a decrease in net CH₄ fluxes compared to a control site. Drainage lowered CH₄ fluxes by a factor of 20 during the growing season, with postdrainage changes in microbial communities, soil temperatures, and plant communities also contributing to this reduction. In contrast, we observed CH₄ emissions increased by 10% in the drained areas during the nongrowing season, although this difference was insignificant given the small magnitudes of fluxes. This study showed that long‐term drainage considerably reduced CH₄ fluxes through modified ecosystem properties.     

Pre-steady-state kinetic study of substrate specificity of Escherichia coli formamidopyrimidine--DNA glycosylase

Formamidopyrimidine-DNA glycosylase (Fpg) is responsible for removal of 8-oxoguanine (8-oxoG) and other oxidized purine lesions from DNA and can also excise some oxidatively modified pyrimidines [such as dihydrouracil (DHU)]. Fpg is also specific for a base opposite the lesion, efficiently excising 8-oxoG paired with C but not with A. We have applied stopped-flow kinetics using intrinsic tryptophan fluorescence of the enzyme and fluorescence of 2-aminopurine-labeled DNA to analyze the conformational dynamics of Escherichia coli Fpg during processing of good substrates (8-oxoG.C), poor substrates (8-oxoG.A), and substrates of unclear specificity (such as DHU and 8-oxoG opposite T or G). The analysis of fluorescence traces allows us to conclude that when the enzyme encounters its true substrate, 8-oxoG.C, the complex enters the productive catalytic reaction after approximately 50 ms, partitioning the substrate away from the competing dissociation process, while poor substrates linger in the initial encounter complex for longer. Several intermediate ES complexes were attributed to different structures that exist along the reaction pathway. A likely sequence of events is that the damaged base is first destabilized by the enzyme binding and then everted from DNA, followed by insertion of several amino acid residues into DNA and isomerization of the enzyme into a pre-excision complex. We conclude that rejection of the incorrect substrates occurs mostly at the early stage of formation of the pre-eversion recognition complex, supporting the role of indirect readout in damage recognition.     

Prebiotic Syntheses Under Shock in the Water – Formamide – Potassium Bicarbonate – Sodium Hydroxide System

Origins of Life and Evolution of Biospheres - Syntheses under shock in nitrogen bubbled samples of the water – formamide – bicarbonate – sodium hydroxide system at...     

Dental indicators of adaptation in the Sahara Desert during the Late Holocene

The present paper examines dental diseases and linear enamel hypoplasia among the Garamantes, a Late Holocene Saharan population, and aims to draw conclusions about nutrition and adaptation to a hyper-arid environment. Archaeological evidence suggests that the Garamantian diet included animal protein and local, Mediterranean and Near Eastern plants. Moreoever, although the Garamantes had developed urban centres, the size of these was not large enough to allow for particularly unhygienic conditions to appear. The above archaeological findings were partly corroborated by the current bioarchaeological study. At an intra-population level, the Garamantes showed limited sex differences in dental disease prevalence, while all dental conditions increased in frequency with age, as expected. At an inter-population level, the frequency of all dental conditions was comparable to that found among other North African groups, with the exception of ante-mortem tooth loss. The low frequency of most dental conditions is an indication that the Garamantian diet was overall balanced, while the high frequency of ante-mortem tooth loss may be related to factors such as oral hygiene, food preparation or eating mode, which cannot be controlled for osteologically. Finally, the low frequency of enamel hypoplasia suggests either that the Sahara did not inflict particular stresses on the population, or, more likely, that the Garamantes had developed effective mechanisms for coping with their natural environment.     

Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.     

Cryptococcus festucosus sp. nov. a new hymenomycetous yeast in the Holtermannia clade

Five yeast strains belonging to the genus Cryptococcus Vuillemin were isolated from steppe plants and turf collected in the Prioksko-terrasny biosphere reserve (Moscow region, Russia). Sequence analyses of the D1/D2 domains of the 26S rDNA and of the internal transcribed spacer region revealed that these yeast strains and strain CBS 8016 have almost identical sequences and belong to the Holtermannia clade of the Tremellomycetidae (Basidiomycota, Hymenomycetes). A novel species named Cryptococcus festucosus (type strain VKM Y-2930) is proposed to accommodate these strains. Physiological characteristics and mycocin sensitivity patterns distinguishing Cryptococcus festucosus from the other species of this clade are presented.     

Sex chromosome evolution: life,death and repetitive DNA

Dimorphic sex chromosomes create problems. Males of many species, including Drosophila, are heterogametic, with dissimilar X and Y chromosomes. The essential process of dosage compensation modulates the expression of X-linked genes in one sex to maintain a constant ratio of X to autosomal expression. This involves the regulation of hundreds of dissimilar genes whose only shared property is chromosomal address. Drosophila males dosage compensate by up regulating X-linked genes 2 fold. This is achieved by the Male Specific Lethal (MSL) complex, which is recruited to genes on the X chromosome and modifies chromatin to increase expression. How the MSL complex is restricted to X-linked genes remains unknown. Recent studies of sex chromosome evolution have identified a central role for 2 types of repetitive elements in X recognition. Helitrons carrying sites that recruit the MSL complex have expanded across the X chromosome in at least one Drosophila species.¹ Our laboratory found that siRNA from an X-linked satellite repeat promotes X recognition by a yet unknown mechanism.² The recurring adoption of repetitive elements as X-identify elements suggests that the large and mysterious fraction of the genome called “junk” DNA is actually instrumental in the evolution of sex chromosomes.     

Genomic Evidence for Island Population Conversion Resolves Conflicting Theories of Polar Bear Evolution

Despite extensive genetic analysis, the evolutionary relationship between polar bears (Ursus maritimus) and brown bears (U. arctos) remains unclear. The two most recent comprehensive reports indicate a recent divergence with little subsequent admixture or a much more ancient divergence followed by extensive admixture. At the center of this controversy are the Alaskan ABC Islands brown bears that show evidence of shared ancestry with polar bears. We present an analysis of genome-wide sequence data for seven polar bears, one ABC Islands brown bear, one mainland Alaskan brown bear, and a black bear (U. americanus), plus recently published datasets from other bears. Surprisingly, we find clear evidence for gene flow from polar bears into ABC Islands brown bears but no evidence of gene flow from brown bears into polar bears. Importantly, while polar bears contributed <1% of the autosomal genome of the ABC Islands brown bear, they contributed 6.5% of the X chromosome. The magnitude of sex-biased polar bear ancestry and the clear direction of gene flow suggest a model wherein the enigmatic ABC Island brown bears are the descendants of a polar bear population that was gradually converted into brown bears via male-dominated brown bear admixture. We present a model that reconciles heretofore conflicting genetic observations. We posit that the enigmatic ABC Islands brown bears derive from a population of polar bears likely stranded by the receding ice at the end of the last glacial period. Since then, male brown bear migration onto the island has gradually converted these bears into an admixed population whose phenotype and genotype are principally brown bear, except at mtDNA and X-linked loci. This process of genome erosion and conversion may be a common outcome when climate change or other forces cause a population to become isolated and then overrun by species with which it can hybridize.     

Dual phosphorylations underlie modulation of unitary KCNQ K(+) channels by Src tyrosine kinase

Src tyrosine kinase suppresses KCNQ (M-type) K(+) channels in a subunit-specific manner representing a mode of modulation distinct from that involving G protein-coupled receptors. We probed the molecular and biophysical mechanisms of this modulation using mutagenesis, biochemistry, and both whole-cell and single channel modes of patch clamp recording. Immunoprecipitation assays showed that Src associates with KCNQ2-5 subunits but phosphorylates only KCNQ3-5. Using KCNQ3 as a background, we found that mutation of a tyrosine in the amino terminus (Tyr-67) or one in the carboxyl terminus (Tyr-349) abolished Src-dependent modulation of heterologously expressed KCNQ2/3 heteromultimers. The tyrosine phosphorylation was much weaker for either the KCNQ3-Y67F or KCNQ3-Y349F mutants and wholly absent in the KCNQ3-Y67F/Y349F double mutant. Biotinylation assays showed that Src activity does not alter the membrane abundance of channels in the plasma membrane. In recordings from cell-attached patches containing a single KCNQ2/3 channel, we found that Src inhibits the open probability of the channels. Kinetic analysis was consistent with the channels having two discrete open times and three closed times. Src activity reduced the durations of the longest open time and lengthened the longest closed time of the channels. The implications for the mechanisms of channel regulation by the dual phosphorylations on both channel termini are discussed.