Heterogeneous human NK cell responses to Plasmodium falciparum-infected erythrocytes

Human NK cells can respond rapidly to Plasmodium falciparum-infected RBC (iRBC) to produce IFN-gamma. In this study, we have examined the heterogeneity of this response among malaria-naive blood donors. Cells from all donors become partially activated (up-regulating CD69, perforin, and granzyme) upon exposure to iRBC but cells from only a subset of donors become fully activated (additionally up-regulating CD25, IFN-gamma, and surface expression of lysosomal-associated membrane protein 1 (LAMP-1)). Although both CD56dim and CD56bright NK cell populations can express IFN-gamma in response to iRBC, CD25 and LAMP-1 are up-regulated only by CD56dim NK cells and CD69 is up-regulated to a greater extent in this subset; by contrast, perforin and granzyme A are preferentially up-regulated by CD56bright NK cells. NK cells expressing IFN-gamma in response to iRBC always coexpress CD69 and CD25 but rarely LAMP-1, suggesting that individual NK cells respond to iRBC either by IFN-gamma production or cytotoxicity. Furthermore, physical contact with iRBC can, in a proportion of donors, lead to NK cell cytoskeletal reorganization suggestive of functional interactions between the cells. These observations imply that individuals may vary in their ability to mount an innate immune response to malaria infection with obvious implications for disease resistance or susceptibility.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c(3) is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c(3). This work presents the NMR assignment of the haem substituents in type I cytochrome c(3) isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c(3) belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation

Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying.     

Sexing single bovine blastomeres using TSPY gene amplification

The testis-specific protein Y-encoded gene (TSPY) is a Y-specific gene present in variable copy number in many mammalian species, including cattle. We tested the applicability of the TSPY gene as a Y-specific marker to predict preimplantation embryo sex in Nelore (Bos indicus) cattle. Two blastomeres were removed from each embryo. A total of 36 single blastomeres and the remaining cells of their 18 matched in vitro conceived embryos were screened for TSPY amplification by nested-PCR. The results obtained from a single blastomere and the remaining cells of the same embryo were concordant in all cases. All blastomeres (16/16) from eight embryos produced with sexed sperm (specific for production of male embryos) were TSPY-positive. We conclude that TSPY is a good male-specific marker, the usefulness of which is probably enhanced by the high copy number. Other methods that are less time-consuming, such as real-time PCR, could be improved with the use of the TSPY gene sequences to generate primers and/or probes. This is the first report to demonstrate the applicability of the TSPY gene for sexing single cells in cattle.     

Phenol removal from hypersaline wastewaters in a Membrane Biological Reactor (MBR): operation and microbiological characterisation

In this study, two Membrane Biological Reactors (MBR) with submerged flat membranes, one at lab-scale conditions and the other at pilot-plant conditions, were operated at environmental temperature to treat an industrial wastewater characterised by low phenol concentrations (8-16 mg L⁻¹) and high salinity (∼150-160 mS cm⁻¹). During the operation of both reactors, the phenol loading rate was progressively increased and less than 1 mg phenol L⁻¹ was detected even at very low HRTs (0.5-0.7 days). Membrane fouling was minimized by the cross flow aeration rate inside the MBRs and by intermittent permeation. Microbial community analysis of both reactors revealed that members of the genera Halomonas and Marinobacter (gammaproteobacteria) were major components. Growth-linked phenol degradation by pure cultures of Marinobacter isolates demonstrated that this bacterium played a major role in the removal of phenol from the bioreactors.     

A novel role for BRCA1 in regulating breast cancer cell spreading and motility

BRCA1 C-terminal (BRCT) domains in BRCA1 are essential for tumor suppressor function, though the underlying mechanisms remain unclear. We identified ezrin, radixin, and moesin as BRCA1 BRCT domain-interacting proteins. Ezrin-radixin-moesin (ERM) and F-actin colocalized with BRCA1 at the plasma membrane (PM) of cancer cells, especially at leading edges and focal adhesion sites. In stably expressing cancer cells, high levels of enhanced green fluorescent protein (EGFP)-BRCA1(1634-1863) acted as a dominant-negative factor, displacing endogenous BRCA1 from the PM. This led to delayed cell spreading, increased spontaneous motility, and irregular monolayer wound healing. MCF-7 cells (intact BRCA1) showed lower motility than HCC1937 cells (truncated BRCA1), but expression of EGFP-BRCA1(1634-1863) in MCF-7 increased motility. Conversely, full-length BRCA1 expression in HCC1937 decreased motility but only if the protein retained ubiquitin ligase activity. We conclude that full-length BRCA1 is important for complete tumor suppressor activity via interaction of its BRCT domains with ERM at the PM, controlling spreading and motility of cancer cells via ubiquitin ligase activity.     

Determinants of Eurasian otter (Lutra lutra) diet in a seasonally changing reservoir

Otter diet in reservoirs is known to experience seasonal changes. We selected a reservoir with a large population of exclusively wintering great cormorants and seasonal changes in stored water volume to test the relative influence of abiotic and biotic factors on otter foraging ecology. DNA metabarcoding of otter spraints revealed a dietary change from autumn to winter. Otters had a diet dominated by the exotic goldfish in autumn, but predated intensively on the native northern straight-mouth nase in winter. This change was likely caused by predation of cormorants on goldfish and to fish biology. Secondly, macroscopic analysis of spraints revealed that otters shifted from a diet dominated by fish (in terms of biomass) to a diet dominated by red swamp crayfish during spring–summer, when the latter became overabundant. As revealed by modelling, this second shift was most likely influenced by the sudden increase in stored water volume in spring, but also by the cumulative effect of cormorant predation on fish during autumn–winter. Macroscopic analyses of otter spraints collected in a second reservoir with no cormorants revealed a lack of seasonality. Hence, the combined influence of both biotic and abiotic factors explained otter diet seasonality in a lentic-water novel ecosystem.

     

DYRK1A promotes dopaminergic neuron survival in the developing brain and in a mouse model of Parkinson's disease

In the brain, programmed cell death (PCD) serves to adjust the numbers of the different types of neurons during development, and its pathological reactivation in the adult leads to neurodegeneration. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) is a pleiotropic kinase involved in neural proliferation and cell death, and its role during brain growth is evolutionarily conserved. Human DYRK1A lies in the Down syndrome critical region on chromosome 21, and heterozygous mutations in the gene cause microcephaly and neurological dysfunction. The mouse model for DYRK1A haploinsufficiency (the Dyrk1a^+/− mouse) presents neuronal deficits in specific regions of the adult brain, including the substantia nigra (SN), although the mechanisms underlying these pathogenic effects remain unclear. Here we study the effect of DYRK1A copy number variation on dopaminergic cell homeostasis. We show that mesencephalic DA (mDA) neurons are generated in the embryo at normal rates in the Dyrk1a haploinsufficient model and in a model (the mBACtgDyrk1a mouse) that carries three copies of Dyrk1a. We also show that the number of mDA cells diminishes in postnatal Dyrk1a^+/− mice and increases in mBACtgDyrk1a mice due to an abnormal activity of the mitochondrial caspase9 (Casp9)-dependent apoptotic pathway during the main wave of PCD that affects these neurons. In addition, we show that the cell death induced by 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), a toxin that activates Casp9-dependent apoptosis in mDA neurons, is attenuated in adult mBACtgDyrk1a mice, leading to an increased survival of SN DA neurons 21 days after MPTP intoxication. Finally, we present data indicating that Dyrk1a phosphorylation of Casp9 at the Thr125 residue is the mechanism by which this kinase hinders both physiological and pathological PCD in mDA neurons. These data provide new insight into the mechanisms that control cell death in brain DA neurons and they show that deregulation of developmental apoptosis may contribute to the phenotype of patients with imbalanced DYRK1A gene dosage.The total number of neurons in the brain, and ultimately the size of this organ, depends both on the number of cells that are produced during neurogenesis and the number of neurons that die due to physiological programmed cell death (PCD). Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) regulates brain growth in a dose-dependent manner,¹ and indeed, loss-of-function mutations in DYRK1A (minibrain in Drosophila melanogaster) cause microcephaly and several neurological alterations in humans,^{2, 3, 4, 5} mice⁶ and flies.⁷ Accordingly, it has been proposed that haploinsufficiency of DYRK1A is the cause of the microcephaly and developmental delay associated to partial monosomy of chromosome 21 involving DYRK1A.⁸ Moreover, triplication of the gene has been associated to the developmental brain dysfunctions and age-associated neurodegeneration observed in Down syndrome.^{9, 10, 11}Anatomical analysis of adult Dyrk1a mutant mice that model human diseases involving an imbalance in DYRK1A gene dosage (the Dyrk1a^+/− mouse and the mBACtgDyrk1a mouse, carrying one or three functional copies of Dyrk1a, respectively) revealed a positive correlation between Dyrk1a gene copy number, the overall size of the brain and the number of neurons in specific regions.¹ DYRK1A regulates several fundamental neurodevelopmental processes, including proliferation, neuron differentiation and PCD.¹² Overexpression of DYRK1A in neural precursors attenuates proliferation and promotes the differentiation of neurons in different model systems.^{13, 14, 15} Conversely, treatment of neural progenitors with DYRK1A kinase inhibitors increases proliferation.¹⁵ Although these data are consistent with some of the defects in cellularity identified in specific brain regions of Dyrk1a gene copy number mutants, they cannot explain the severe microcephaly evident in mice and humans carrying one functional copy of DYRK1A, or the overall macrocephaly in the mBACtgDyrk1a model carrying three Dyrk1a alleles.^{1, 5} Thus, deregulation of other DYRK1A functions might also contribute to the defects in brain cellularity in these Dyrk1a gene copy number mutants, such as those described in retinal neurons that restrain developmental PCD.¹⁶Dopaminergic (DA) neurons in the substantia nigra (SN) and ventral tegmental area (VTA) have an important role in controlling fine motor actions, as well as in motivation and reward behaviours, and their loss is associated with Parkinson''s disease.¹⁷ In aged Dyrk1a^+/− mice the SN is smaller and contains fewer DA neurons than in wild-type mice.¹⁸ These mutant animals are hypoactive, with altered gait dynamics, and as these defects are evident preweaning and in young animals,^{6, 18, 19} as well as in children with heterozygous mutations in DYRK1A,^{3, 4, 5} they might arise during development.To provide insight into the aetiology of the neurological phenotype caused by DYRK1A haploinsufficiency, here we studied the development of mesencephalic DA (mDA) neurons in Dyrk1a^+/− and mBACtgDyrk1a mouse models. The results obtained show that Dyrk1a copy number variation does not affect the generation of DA neurons, but rather it modifies the number of these neurons that undergo physiological PCD due to an inhibitory effect of the Dyrk1a kinase on the apoptotic activity of caspase9 (Casp9), the initiator caspase in the mitochondrial-dependent apoptotic pathway.²⁰ Thus, deregulation of Casp9-dependent PCD during development may contribute to the brain size defects observed in aneuploidies involving DYRK1A.As inappropriate re-activation of the mitochondrial-dependent apoptotic pathway in mature mDA neurons contributes to the neurodegeneration associated with Parkinson''s disease,²¹ we used the mBACtgDyrk1a mouse model to assess whether basal Dyrk1a-dependent inhibition of Casp9 apoptotic activity could restrain the neurodegeneration induced in vivo by the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our results show that the apoptotic response to the toxin in mBACtgDyrk1a mice is significantly attenuated, leading to an increase in the number of SN pars compacta DA neurons that resist the pathological insult.     

Bacteriophage-receptor binding proteins for multiplex detection of Staphylococcus and Enterococcus in blood

Healthcare-associated infections (HCAIs) affect hundreds of millions of patients, representing a significant burden for public health. They are usually associated to multidrug resistant bacteria, which increases their incidence and severity. Bloodstream infections are among the most frequent and life-threatening HCAIs, with Enterococcus and Staphylococcus among the most common isolated pathogens. The correct and fast identification of the etiological agents is crucial for clinical decision-making, allowing to rapidly select the appropriate antimicrobial and to prevent from overuse and misuse of antibiotics and the consequent increase in antimicrobial resistance. Conventional culture methods are still the gold standard to identify these pathogens, however, are time-consuming and may lead to erroneous diagnosis, which compromises an efficient treatment. (Bacterio)phage receptor binding proteins (RBPs) are the structures responsible for the high specificity conferred to phages against bacteria and thus are very attractive biorecognition elements with high potential for specific detection and identification of pathogens. Taking into account all these facts, we have designed and developed a new, fast, accurate, reliable and unskilled diagnostic method based on newly identified phage RBPs and spectrofluorometric techniques that allows the multiplex detection of Enterococcus and Staphylococcus in blood samples in less than 1.5 hr after an enrichment step.