Structural analysis of ligand‐bound states of the Salmonella type III secretion system ATPase InvC

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram‐negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N‐terminal domains. Using size‐exclusion chromatography coupled to multi‐angle light scattering and native mass spectrometry, we show that in the absence of the N‐terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP‐binding site by co‐crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP‐analogue recognition, these structures reveal remodeling of the ATP‐binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo‐oligomerization process and ATP‐dependent conformational changes underlying the T3SS ATPase activity.     

Variability in the Effect of 5-HTTLPR on Depression in a Large European Population: The Role of Age,Symptom Profile,Type and Intensity of Life Stressors

Background

Although 5-HTTLPR has been shown to influence the risk of life stress-induced depression in the majority of studies, others have produced contradictory results, possibly due to weak effects and/or sample heterogeneity.

Methods

In the present study we investigated how age, type and intensity of life-stressors modulate the effect of 5-HTTLPR on depression and anxiety in a European population cohort of over 2300 subjects. Recent negative life events (RLE), childhood adversity (CHA), lifetime depression, Brief Symptoms Inventory (BSI) depression and anxiety scores were determined in each subject. Besides traditional statistical analysis we calculated Bayesian effect strength and relevance of 5-HTTLPR genotypes in specified models.

Results

The short (s) low expressing allele showed association with increased risk of depression related phenotypes, but all nominally significant effects would turn to non-significant after correction for multiple testing in the traditional analysis. Bayesian effect strength and relevance analysis, however, confirmed the role of 5-HTTLPR. Regarding current (BSI) and lifetime depression 5-HTTLPR-by-RLE interactions were confirmed. Main effect, with other words direct association, was supported with BSI anxiety. With more frequent RLE the prevalence or symptoms of depression increased in ss carriers. Although CHA failed to show an interaction with 5-HTTLPR, in young subjects CHA sensitized towards the depression promoting effect of even mild RLE. Furthermore, the direct association of anxiety with the s allele was driven by young (≤30) individuals.

Limitations

Our study is cross-sectional and applies self-report questionnaires.

Conclusions

Albeit 5-HTTLPR has only weak/moderate effects, the s allele is directly associated with anxiety and modulates development of depression in homogeneous subgroups.     

Tamoxifen and raloxifene produce conditioned taste avoidance in female rats: A microstructural analysis of licking patterns

AimsEstrogen receptor activation has been shown to reduce body weight and produce conditioned taste avoidance (CTA) when estradiol administration is paired with a novel tastant. This study determined if the selective estrogen receptor modulators tamoxifen and raloxifene, which effectively prevent and treat breast cancer, can induce a CTA and alter body weight in ovariectomized (OVX)-female rats.Main methodsDuring conditioning, OVX-female rats were injected with tamoxifen, raloxifene, 17β-estradiol or vehicle, or were uninjected, prior to drinking 0.3 M sucrose in a lickometer. Immediately following sucrose access, alterations in locomotor activity and thigmotaxis (anxiety) were assessed in an open field apparatus. Conditioned drug effects on drinking, locomotor activity and anxiety were examined on a separate test day.Key findingsOur results suggest that both tamoxifen and raloxifene produce CTA that is similar to that produced by estradiol. Both the number and size of bursts of licking were significantly reduced, as well as body weight gain, in OVX-female rats treated with tamoxifen or raloxifene.SignificanceThe results of the present study suggest that tamoxifen and raloxifene may have the potential to produce CTA in breast cancer patients receiving chemoprevention care.     

The desire for variety: Italian wall lizard (Podarcis siculus) populations introduced to the United States via the pet trade are derived from multiple native-range sources

Tests of invasion success often require comparisons between introduced and native populations, but determining the native-range sources for introduced populations can be difficult. Molecular markers can help clarify the geographic extent of native-range sources, helping to identify which populations are appropriate for comparative studies. The Italian Wall Lizard (Podarcis siculus) was introduced multiple times to the United States with extant populations in California, Kansas, New Jersey, and New York. We used phylogeographic analysis of mtDNA sequences (cytb gene) for individuals sampled from these introduced populations and across the native range to identify the number of independent introductions and the location of the source populations. Haplotypes sampled from introduced populations were nested within three geographically distinct, well-supported clades that together encompassed a large portion of the native range. Combining these phylogeographic results with documentation of the introductions revealed putative sources: California individuals are derived from Sicily; Kansas and New York populations are from Tuscany near Florence; and the New Jersey population is likely from the Adriatic coastal region, but a more specific locality is not possible. The pet trade dominates the invasion pathway for P. siculus introductions to the US. The genetically and geographically diverse sampling of its native range may be driven by the desire for phenotypic variety in the pet trade, a hypothesis that needs future testing.     

Retinotopische Beziehungen und Struktur rezeptiver Felder im Tectum opticum und Praetectum der Katze

Zusammenfassung 1.Von 600 Neuronen des Colliculus superior und Praetectums der Katze wurde mit Stahlmikroelektroden abgeleitet und der Ableitort markiert. Die Lage der rezeptiven Felder wurde mit bewegten und stationären Lichtreizen bestimmt und dem Ableitort zugeordnet.2.Im Colliculus superior und Praetectum fanden sich richtungsspezifische und richtungsunspezifische Bewegungsneurone. Ein Teil der praetectalen Neurone reagierte richtungsspezifisch auf Bewegungen vom Tier weg und auf das Tier zu (S-Neurone).3.Innerhalb einer senkrecht zur Oberfläche des Colliculus verlaufenden Penetrationssäule nahm die Feldgröße bei gleichbleibender Feldposition mit zunehmender Tiefe zu. Zwischen Ableitort und Feldposition bestand eine systematische retinotopische Beziehung. Die Projektion des vertikalen O-Meridians des Gesichtsfeldes verlief im rostralen Drittel des Colliculus von medial nach lateral, die des horizontalen O-Meridians in der Mitte des Colliculus von rostral nach caudal. Das Projektionsschema eines Colliculus enthält einen nasalen Teil der ipsilateralen Gesichtsfeldhälfte.4.Im Praetectum verlief die Projektion vertikaler Meridiane am caudalen Ende von medial nach lateral und überlappte sich teilweise mit dem Projektionsgebiet des vertikalen O-Meridians im Colliculus. Die horizontalen Meridiane verliefen so von caudal nach rostral, daß das Projektionsschema des Praetectums spiegelbildlich zu dem des Colliculus superior angeordnet war. Dieses Projektionsschema galt nur für den Nucleus tractus optici und die Area praetectalis. Die übrigen praetectalen Kerne mit zum Teil sehr großen rezeptiven Feldern und spezifischen Reaktionsweisen erhielten keine retinotopische Projektion.5.Rezeptive Felder der oberflächennahen Schichten waren uniform on-, off-oder on-off strukturiert, Felder tiefergelegener Einheiten waren ungeordnet aus on-, off- und on-off Bezirken zusammengesetzt. Binocular erregbare Neurone zeigten für beide Augen gleiche Position und Struktur der rezeptiven Felder.6.Die Ergebnisse wurden mit den an anderen Tierarten erhobenen Befunden verglichen. Ihre mögliche funktionelle Bedeutung wurde diskutiert.

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Retinotopic relationship and structure of receptive fields in the optic tectum and pretectum of the cat

Summary 1.600 neurons of the cat's superior colliculus and pretectum were recorded and marked with stainless-steel microelectrodes. The position and structure of receptive fields were tested with stationary flickering and moving stimuli. The position of the stimuli in the visual field was determined by the direction of the lamp projecting the light-points because animal and lamp were arranged in a fixed relationship to the screen. The positions of the stimuli were described in a coordinate system based on the horizontal-and vertical zeromeridean of the retina.2.About 55% of tectal neurons are directionally selective and signal mainly movements directed to the periphery of the visual field. Neurons of the pretectum have the same response characteristics as neurons of the superior colliculus but in addition some are selectively responsive to movements towards the animal or away from it (S-neurons).3.Neurons in one functional column (diameter 0.5 mm, length 3.6 mm) perpendicular to the surface of the superior colliculus react to the same position and preferred direction of a moving stimulus. The size, complexity and directional selectivity of the receptive fields increase with the depth of the recorded neurons. The projection of the vertical zero-meridean passes across the rostral part of the colliculus but does not form the rostral border of the superior colliculus. The nasal part of the ipsilateral visual field projects to the most rostral part of the superior colliculus. The projection of the horizontal zeromeridean passes rostro-caudally in a nearly sagittal plane down the middle of the colliculus. Along this projection-line the resolving power is 13°/column in the caudal part and 6°/column in the rostral part of the superior colliculus. The size of the receptive fields increase with their excentricity in the visual field. (Average of field diameters: 26±13°).4.The diameter of receptive fields in the pretectum was 21±11°, except for a few very large fields (70° and larger). Along the medio-lateral axis of the pretectum there was a retinotopic organization identical to that in the colliculus. Along the caudo-rostral axis, the retinotopic organization was the mirror image of that in the colliculus. No retinotopic organization was observed in the so-called deep pretectal nucleus or in the nucleus of the posterior commissure. Neurons of these nuclei may represent more complex levels in the visual pathway.5.The more superficial neurons of the colliculus (0.1–1.8 mm deep) react mainly with uniform on-, off- or on-off responses to stationary flickered stimuli, i.e. their receptive fields (7–20° in diameter) are uniformly on-, off- or on-off. The deeper neurons (2 mm and deeper) have receptive fields (20–40° in diameter) with compound but not antagonistic structure. No receptive fields showed on- or off-inhibition. Binocularly driven neurons have the same position and structure of their receptive fields for both eyes.6.A survey of the literature reveals that all vertebrates so far investigated show small differences in the destination and retinotopic organization of their retinofugal fibre projections and in the types of tectal receptive fields. These differences seem to indicate an adaption to the development of binocular representation of the center of the visual field, of a specialized area of the retina and of a retino-cortical system.

     

Characterization of gamma-tocopherol methyltransferases from Capsicum annuum L and Arabidopsis thaliana.

Tocopherols are essential micronutrients in human and animal nutrition due to their function as lipophilic antioxidants. They are exclusively synthesized by photosynthetic organisms including higher plants. Despite the attributed beneficial health effects and many industrial applications, research on the tocopherol biosynthetic pathway and its regulation in plants is still limited. In the work presented here we performed a detailed biochemical characterization of a gamma-tocopherol methyltransferase (gamma-TMT) from Arabidopsis thaliana and of a gamma-TMT purified from Capsicum annuum fruits, a tissue with high accumulation of tocopherols. The biochemical characteristics of both enzyme preparations were remarkably similar including substrate specificities. Both enzymes converted delta- and gamma- into beta- and alpha-tocopherol, respectively, but beta-tocopherol was not accepted as a substrate, pointing to a specific methylation at the C(5)-position of the tocopherol aromatic head group. A kinetic analysis performed with the Arabidopsis enzyme was consistent with an iso-ordered bi-bi type reaction mechanism. Our results emphasize the role of gamma-TMT in regulating the spectrum of accumulated tocopherols in plants.     

Neuraminidase-associated plasminogen recruitment enables systemic spread of natural avian Influenza viruses H3N1

Repeated outbreaks due to H3N1 low pathogenicity avian influenza viruses (LPAIV) in Belgium were associated with unusually high mortality in chicken in 2019. Those events caused considerable economic losses and prompted restriction measures normally implemented for eradicating high pathogenicity avian influenza viruses (HPAIV). Initial pathology investigations and infection studies suggested this virus to be able to replicate systemically, being very atypical for H3 LPAIV. Here, we investigate the pathogenesis of this H3N1 virus and propose a mechanism explaining its unusual systemic replication capability. By intravenous and intracerebral inoculation in chicken, we demonstrate systemic spread of this virus, extending to the central nervous system. Endoproteolytic viral hemagglutinin (HA) protein activation by either tissue-restricted serine peptidases or ubiquitous subtilisin-like proteases is the functional hallmark distinguishing (H5 or H7) LPAIV from HPAIV. However, luciferase reporter assays show that HA cleavage in case of the H3N1 strain in contrast to the HPAIV is not processed by intracellular proteases. Yet the H3N1 virus replicates efficiently in cell culture without trypsin, unlike LPAIVs. Moreover, this trypsin-independent virus replication is inhibited by 6-aminohexanoic acid, a plasmin inhibitor. Correspondingly, in silico analysis indicates that plasminogen is recruitable by the viral neuraminidase for proteolytic activation due to the loss of a strongly conserved N-glycosylation site at position 130. This mutation was shown responsible for plasminogen recruitment and neurovirulence of the mouse brain-passaged laboratory strain A/WSN/33 (H1N1). In conclusion, our findings provide good evidence in natural chicken strains for N1 neuraminidase-operated recruitment of plasminogen, enabling systemic replication leading to an unusual high pathogenicity phenotype. Such a gain of function in naturally occurring AIVs representing an established human influenza HA-subtype raises concerns over potential zoonotic threats.     

Human tyrosinase is able to oxidize both enantiomers of rhododendrol

Racemic RS‐4‐(4‐hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was used as a topical skin‐whitening agent until it was recently reported to induce leukoderma. We then showed that oxidation of RD with mushroom tyrosinase rapidly produces RD‐quinone, which is quickly converted to RD‐cyclic quinone and RD‐hydroxy‐p‐quinone. In this study, we examined whether either or both of the enantiomers of RD can be oxidized by human tyrosinase. Using a chiral HPLC column, racemic RD was resolved optically to R(?)‐RD and S(+)‐RD enantiomers. In the presence of a catalytic amount of l ‐dopa, human tyrosinase, which can oxidize l ‐tyrosine but not d ‐tyrosine, was found to oxidize both R(?)‐ and S(+)‐RD to give RD‐catechol and its oxidation products. S(+)‐RD was more effectively oxidized than l ‐tyrosine, while R(?)‐RD was less effective. These results support the notion that the melanocyte toxicity of RD depends on its tyrosinase‐catalyzed conversion to toxic quinones and the concomitant production of reactive oxygen species.