Mycobacterium marinum Causes a Latent Infection that Can Be Reactivated by Gamma Irradiation in Adult Zebrafish

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (∼35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5–10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.     

Do allopatric male Calopteryx virgo damselflies learn species recognition?

There is a growing amount of empirical evidence that premating reproductive isolation of two closely related species can be reinforced by natural selection arising from avoidance of maladaptive hybridization. However, as an alternative for this popular reinforcement theory, it has been suggested that learning to prefer conspecifics or to discriminate heterospecifics could cause a similar pattern of reinforced premating isolation, but this possibility is much less studied. Here, we report results of a field experiment in which we examined (i) whether allopatric Calopteryx virgo damselfly males that have not encountered heterospecific females of the congener C. splendens initially show discrimination, and (ii) whether C. virgo males learn to discriminate heterospecifics or learn to associate with conspecifics during repeated experimental presentation of females. Our experiment revealed that there was a statistically nonsignificant tendency for C. virgo males to show initial discrimination against heterospecific females but because we did not use sexually naïve individuals in our experiment, we were not able to separate the effect of innate or associative learning. More importantly, however, our study revealed that species discrimination might be further strengthened by learning, especially so that C. virgo males increase their association with conspecific females during repeated presentation trials. The role of learning to discriminate C. splendens females was less clear. We conclude that learning might play a role in species recognition also when individuals are not naïve but have already encountered potential conspecific mates.     

GAGA protein is essential for male germ cell development in Drosophila

The Drosophila Trithorax‐like (Trl) gene encodes a GAGA factor which regulates a number of developmentally important genes. In this study, we identify a new function for Drosophila GAGA factor in male germ cell development. Trl mutants carrying strong hypomorphic alleles display loss of primordial germ cells during their migration in embryogenesis and severe disruption in mitochondria structure during early spermatogenesis. The mutation resulted in small testes formation, a deficit of germ cells, abnormal mitochondrial morphogenesis, spermatocyte death through autophagy, and partial or complete male sterility. Pleiotropic mutation effects can be explained by the misexpression of GAGA factor target genes, the products of which are required for germ cell progression into mature sperm. genesis 52:738–751, 2014. © 2014 Wiley Periodicals, Inc.     

Huntington disease in Finland: a molecular and genealogical study

Summary Huntington disease (HD) is found at exceptionally low frequency in the Finnish population. In this population, linkage disequilibrium was earlier established with markers from the D4S10 and D4S43 loci. We now report a continuation to the restriction fragment length polymorphism haplotype analysis, in combination with a genealogical study of all the Finnish HD families. When the HD pedigrees were systematically traced to the 18th century, only one consanguinity was found, and a high percentage (28%) of the families had foreign ancestors. The majority of the Finnish ancestors were localized to border regions or trade centers of the country following the old postal routes. The observed high risk haplotypes formed with markers from the D4S10 and D4S43 loci were evenly distributed among the HD families in different geographical locations. Consequently, the HD gene(s) has most probably arrived in Finland on several occasions via foreign immigrants during the last few centuries.     

Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport

The Na⁺/K⁺-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na⁺ for extracellular K⁺ to the hydrolysis of ATP. The asymmetric distribution of Na⁺ and K⁺ is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers–Post model. It involves the presence of gates alternatively exposing Na⁺/K⁺-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K⁺, information is lacking about Na⁺-occluded intermediates, as occluded Na⁺ was only detected in states incapable of performing a catalytic cycle, including two Na⁺-containing crystallographic structures. The current knowledge is that intracellular Na⁺ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na⁺-occluded intermediates, we isolated species with tightly bound Na⁺ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na⁺ becomes spontaneously occluded in the E1 dephosphorylated form of the Na⁺/K⁺-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na⁺ occlusion as it would have been expected; on the contrary, occluded Na⁺ transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers–Post model for explaining the Na⁺ transport pathway.     

Do Insectivorous Birds use Volatile Organic Compounds from Plants as Olfactory Foraging Cues? Three Experimental Tests

Some insectivorous birds orient towards insect‐defoliated trees even when they do not see the foliar damage or the herbivores. There are, however, only a few studies that have examined the mechanisms behind this foraging behaviour. Previous studies suggest that birds can use olfactory foraging cues (e.g. volatile organic compounds (VOCs) emitted by defoliated plants), indirect visual cues or a combination of the two sensory cues. VOCs from insect‐defoliated plants are known to attract natural enemies of herbivores, and researchers have hypothesized that VOCs could also act as olfactory foraging cues for birds. We conducted three experiments across a range of spatial scales to test this hypothesis. In each experiment, birds were presented with olfactory cues and their behavioural responses or foraging outcomes were observed. In the first experiment, two different VOC blends, designed to simulate the volatile emissions of mountain birch (Betula pubescens ssp. czerepanovii) after defoliation by autumnal moth (Epirrita autumnata) larvae, were used in behavioural experiments in aviaries with pied flycatchers (Ficedula hypoleuca). The second experiment was a field‐based trial of bird foraging efficiency; the same VOC blends were applied to mountain birches, silver birches (B. pendula) and European white birches (B. pubescens) with plasticine larvae attached to the trees to serve as artificial prey for birds and provide a means to monitor predation rate. In the third experiment, the attractiveness of silver birch saplings defoliated by autumnal moth larvae versus intact controls was tested with great tits (Parus major) and blue tits (Cyanistes caeruleus) in an aviary. Birds did not orient towards either artificial or real trees with VOC supplements or towards herbivore‐damaged saplings when these saplings and undamaged alternatives were hidden from view. These findings do not support the hypothesis that olfactory foraging cues are necessary in the attraction of birds to herbivore‐damaged trees.     

The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression

The phosphatase and actin regulator 1 (PHACTR1) locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI). However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively) at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks’ follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550). Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio.     

Bloom forming Alexandrium ostenfeldii (Dinophyceae) in shallow waters of the Åland Archipelago,Northern Baltic Sea

In the past years, late summer blooms of the bioluminescent dinoflagellate Alexandrium ostenfeldii have become a recurrent phenomenon in coastal waters of the central and Northern Baltic Sea. This paper reports exceptionally high cell concentrations (10⁵ to 10⁶ cells L^?1) of the species found during bioluminescent blooms in 2003 and 2004 in a shallow embayment of the Åland archipelago at the SW coast of Finland. Clonal cultures were established for morphological, molecular, toxicological and ecophysiological investigations to characterize the Finnish populations and compare them to other global A. ostenfeldii isolates. The Finnish isolates exhibited typical morphological features of A. ostenfeldii such as large size, a prominent ventral pore and an orthogonally bent first apical plate. However, unambiguous differentiation from closely related Alexandrium peruvianum was difficult due to considerable variation of sulcal anterior plate shapes. The Finnish strains were genetically distinct from other isolates of the species, but phylogenetic analyses revealed a close relationship to isolates from southern England and an A. peruvianum morphotype from the Spanish Mediterranean. Together these isolates formed a distinct clade which was separated from a clade containing other Northern European, North American and New Zealand populations. Toxin analyses confirmed the presence of the PSP toxins GTX2, GTX3 and STX in both Finnish isolates with GTX3 being the dominant toxin. Total relative PSP toxin contents were moderate, ranging from approximately 6 to 15 fmol cell^?1 at local salinities of 5 and 10 psu, respectively. Spirolides were not detected. Salinity tolerance experiments showed that the Finnish isolates were well adapted to grow at the low salinities of the Baltic Sea. With a salinity range of approximately 6 to 20–25 psu, Baltic populations are physiologically distinct from their marine relatives. Vigorous production of different cyst types in the cultures suggest that cysts may play a crucial role in the survival and retainment of A. ostenfeldii populations in the Baltic Sea.     

Identification of amino acid residues at the active site of endosialidase that dissociate the polysialic acid binding and cleaving activities in Escherichia coli K1 bacteriophages

Endosialidase (endo-N-acetylneuraminidase) is a tailspike enzyme of bacteriophages specific for human pathogenic Escherichia coli K1, which specifically recognizes and degrades polySia (polysialic acid). polySia is also a polysaccharide of the capsules of other meningitis- and sepsis-causing bacteria, and a post-translational modification of the NCAM (neural cell-adhesion molecule). We have cloned and sequenced three spontaneously mutated endosialidases of the PK1A bacteriophage and one of the PK1E bacteriophage which display lost or residual enzyme activity but retain the binding activity to polySia. Single to triple amino acid substitutions were identified, and back-mutation constructs indicated that single substitutions accounted for only partial reduction of enzymic activity. A homology-based structural model of endosialidase revealed that all substituted amino acid residues localize to the active site of the enzyme. The results reveal the importance of non-catalytic amino acid residues for the enzymatic activity. The results reveal the molecular background for the dissociation of the polySia binding and cleaving activities of endosialidase and for the evolvement of 'host range' mutants of E. coli K1 bacteriophages.