An in vitro network of intermolecular interactions between viral RNA segments of an avian H5N2 influenza A virus: comparison with a human H3N2 virus

The genome of influenza A viruses (IAV) is split into eight viral RNAs (vRNAs) that are encapsidated as viral ribonucleoproteins. The existence of a segment-specific packaging mechanism is well established, but the molecular basis of this mechanism remains to be deciphered. Selective packaging could be mediated by direct interaction between the vRNA packaging regions, but such interactions have never been demonstrated in virions. Recently, we showed that the eight vRNAs of a human H3N2 IAV form a single interaction network in vitro that involves regions of the vRNAs known to contain packaging signals in the case of H1N1 IAV strains. Here, we show that the eight vRNAs of an avian H5N2 IAV also form a single network of interactions in vitro, but, interestingly, the interactions and the regions of the vRNAs they involve differ from those described for the human H3N2 virus. We identified the vRNA sequences involved in five of these interactions at the nucleotide level, and in two cases, we validated the existence of the interaction using compensatory mutations in the interacting sequences. Electron tomography also revealed significant differences in the interactions taking place between viral ribonucleoproteins in H5N2 and H3N2 virions, despite their canonical ‘7 + 1’ arrangement.     

When size does matter: organelle size influences the properties of transport mediated by molecular motors

Background

Organelle transport is driven by the action of molecular motors. In this work, we studied the dynamics of organelles of different sizes with the aim of understanding the complex relation between organelle motion and microenvironment.

Methods

We used single particle tracking to obtain trajectories of melanosomes (pigmented organelles in Xenopus laevis melanophores). In response to certain hormones, melanosomes disperse in the cytoplasm or aggregate in the perinuclear region by the combined action of microtubule and actin motors.

Results and conclusions

Melanosome trajectories followed an anomalous diffusion model in which the anomalous diffusion exponent (α) provided information regarding the trajectories' topography and thus of the processes causing it. During aggregation, the directionality of big organelles was higher than that of small organelles and did not depend on the presence of either actin or intermediate filaments (IF). Depolymerization of IF significantly reduced α values of small organelles during aggregation but slightly affect their directionality during dispersion.

General significance

Our results could be interpreted considering that the number of copies of active motors increases with organelle size. Transport of big organelles was not influenced by actin or IF during aggregation showing that these organelles are moved processively by the collective action of dynein motors. Also, we found that intermediate filaments enhance the directionality of small organelles suggesting that this network keeps organelles close to the tracks allowing their efficient reattachment. The higher directionality of small organelles during dispersion could be explained considering the better performance of kinesin-2 vs. dynein at the single molecule level.     

Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase

Background

Ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, is highly regulated by antizymes (AZs), small proteins that bind and inhibit ODC and increase its proteasomal degradation. Early studies delimited the putative AZ-binding element (AZBE) to the region 117-140 of ODC. The aim of the present work was to study the importance of certain residues of the region 110-142 that includes the AZBE region for the interaction between ODC and AZ1 and the ODC functionality.

Methods

Computational analysis of the protein sequences of the extended AZBE site of ODC and ODC paralogues from different eukaryotes was used to search for conserved residues. The influence of these residues on ODC functionality was studied by site directed mutagenesis, followed by different biochemical techniques.

Results

The results revealed that: a) there are five conserved residues in ODC and its paralogues: K115, A123, E138, L139 and K141; b) among these, L139 is the most critical one for the interaction with AZs, since its substitution decreases the affinity of the mutant protein towards AZs; c) all these conserved residues, with the exception of A123, are critical for ODC activity; d) substitutions of K115, E138 or L139 diminish the formation of ODC homodimers.

Conclusions

These results reveal that four of the invariant residues of the AZBE region are strongly related to ODC functionality.

General significance

This work helps to understand the interaction between ODC and AZ1, and describes various new residues involved in ODC activity, a key enzyme for cell growth and proliferation.     

Untersuchungen zum Bekämpfungsrichtwert bei der Haferblattlaus,Rhopalosiphum padi (L.), an Sommergerste

In unseren Untersuchungen prüften wir den Einfluß einer Saatgutbehandlung des Maises mit Imidacloprid (Gaucho) sowohl auf die Übertragung des persistenten barley yellow dwarf virus (BYDV) als auch auf die nichtpersistenten Potyviren maize dwarf mosaic virus (MDMV) und sugarcane mosaic virus (SCMV). Dazu wurden Freiland‐ und Gewächshausversuche sowie Experimente in einer Klimakammer angestellt. Für die Virusübertragungen nutzten wir die Blattlausarten Rhopalosiphum padi L. und weniger häufig Sitobion avenae F. Im Vergleich zu S. avenae wanderte R. padi schneller von den behandelten Pflanzen ab. In Gewächshausversuchen zeigte sich, daß das SCMV nach einer Saatgutbehandlung in deutlich geringerem Umfang übertragen wurde, wenn die Inokulation bis 3 Wochen nach dem Auslegen erfolgte. Inokulationen von älteren Pflanzen ließen keine insektizide Wirkung erkennen. Ebenso ergaben Freilandversuche keinen eindeutigen Zusammenhang zwischen Saatgutbehandlung und Virusbefall. Im Falle des BYDV konnte bei einer Inokulation bis 9 Tage nach dem Auslegen durch einen Imidacloprid‐Einsatz eine signifikante Reduzierung der Übertragung erreicht werden. Spätere Inokulationen verliefen in der Regel negativ. Die Ergebnisse des Feldversuches 1992 bestätigten die im Gewächshaus gewonnenen Resultate. Im Freiland war die natürliche Besiedlung des Maises durch Blattläuse in den Parzellen mit behandeltem Saatgut deutlich vermindert. Hinzu kam eine zeitliche Verzögerung des Massenwechselgeschehens um ca. 2 Wochen.

Für eine abschließende Beurteilung von Imidacloprid sind weiterführende Untersuchungen sowohl zum Saugverhalten der Blattläuse als auch zu seiner Wirkung auf die Virusübertragung unter Einbeziehung verschiedener Sorten erforderlich.     

Progesterone and a phospholipase inhibitor increase the endosomal bis(monoacylglycero)phosphate content and block HIV viral particle intercellular transmission

Progesterone, the cationic amphiphile U18666A and a phospholipase inhibitor (Methyl Arachidonyl Fluoro Phosphonate, MAFP) inhibited by 70%–90% HIV production in viral reservoir cells, i.e. human THP-1 monocytes and monocyte-derived macrophages (MDM). These compounds triggered an inhibition of fluid phase endocytosis (macropinocytosis) and modified cellular lipid homeostasis since endosomes accumulated filipin-stained sterols and Bis(Monoacylglycero)Phosphate (BMP). BMP was quantified using a new cytometry procedure and was increased by 1.25 times with MAFP, 1.7 times with U18666A and 2.5 times with progesterone. MAFP but not progesterone or U18666A inhibited the hydrolysis of BMP by the Pancreatic Lipase Related Protein 2 (PLRP2) as shown by in-vitro experiments. The possible role of sterol transporters in steroid-mediated BMP increase is discussed.     

Antibacterial and leishmanicidal activities of temporin-SHd,a 17-residue long membrane-damaging peptide

Temporins are a family of short antimicrobial peptides (8–17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF_amide), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins.     

The neurobiology of adaptation to seasons: Relevance and correlations in bipolar disorders

ABSTRACT

Bipolar disorders (BDs) are severe and common psychiatric disorders. BD pathogenesis, clinical manifestations and relapses are associated with numerous circadian rhythm abnormalities. In addition, infradian fluctuations of mood, social activity, weight and sleep patterns are very frequent in BD. Disease course with a seasonal pattern (SP) occurs in approximately 25% of depressive and 15% of manic episodes, which is coupled to a more severe disease symptomatology. The pathophysiological mechanisms of seasonal effects in BD await clarification, with likely important clinical consequences. This review aims at synthesizing available data regarding the underlying pathophysiological mechanisms of seasonality in BD patients, with implications for future research directions in the study of seasonality in BD. Three factors are suggested to play significant roles in BD with SP, namely the suprachiasmatic nuclei, as well as the melatonergic and photoperiodism systems. It is proposed that BD with SP may be considered as a complex disorder resulting from the interaction of clock gene vulnerabilities and biological clock neuroplasticity, with environmental factors, such as the response to light. Light seems to play a key role in BD with SP, mainly due to two seasonal signaling pathways: a light to cortex serotonin transporter pathway, as well as a pathway connecting light to melatonin synthesis. This provides a theoretical framework for BD with SP, including for future research and clinical management. The review proposes that future research should explore markers of seasonality in BD, such as plasma melatonin, sleep–wake rhythms (with actigraphy) and genetic or epigenetic variants within the melatonin synthesis pathway. The role of light in driving BD with SP is an active area of research. Seasonality may also be intimately linked to wider aspects of BD, including via interactions with the gut microbiome, the gut–liver axis, cholesterol regulation, aspects of metabolic syndrome, vitamin D, decreased longevity, suicide risk and medication treatment targets. Further research on the role of seasonality in BD is likely to clarify the etiology, course and treatment of BD more widely.     

Myotonic dystrophytype 1 – report of non-24-h sleep-wake disorder with excessive daytime sleepiness

ABSTRACT

Myotonic dystrophy (MD) is a neuromuscular disease with myotonia, progressive weakness, and involvement of CNS, heart, and gastrointestinal system. Excessive daytime sleepiness (EDS) in myotonic dystrophy type 1 (MD1) is related to sleep breathing diseases, restless leg syndrome, periodic limb movements during sleep and narcoleptic-like phenotype. However, authors highlight a central dysfunction of sleep regulation.

We describe a 26-year-old, female, MD1 patient with EDS. Sleep diary/actigraphy evidenced two different circadian periods with values of 1442 and 1522 min. Agomelatine, 50 mg at night, was prescribed with improvement of the circadian rhythm and complaints of sleepiness.

The identification of unanticipated causes of EDS, such as circadian rhythm disorders permits an appropriated treatment. As we know, it is the first relate of non-24-h sleep-wake disorder in patient with MD1. Sleep diary and actigraphy could be good options to investigate sleep-wake cycle disorder in patients with MD and EDS.     

The multidisciplinary therapy in binge eating disorder is able to influence the interdaily stability and sleep quality?

ABSTRACT

We have recently shown that rest-activity circadian rhythm significantly differed in women with Binge Eating Disorder (BED) compared to the Ctrl group. In details, patients with BED exhibited significantly reduced levels of MESOR and Amplitude with respect to the Ctrl group. In addition, in this previous study, the results of the actigraphic sleep monitoring provided no evidence of differences in sleep parameters between the two groups. We expanded the original sample obtaining a total of 28 volunteered women, 14 BED women, and 14 Ctrl. We recorded in all 28 participants a 5-day actigraphic monitoring to detect the rhythmometric parameters, interdaily stability, intradaily variability, L5, M10, and sleep parameters. During the study, BED’s women group kept an individual multidisciplinary therapy lasting five weekly days, from Monday to Friday, consisting in cognitive-behavioral therapy and nutritional program, administered in outpatient care from 8:00 a.m. at 5:00 p.m. The combination of both our previous and current study supports the conclusion that the sleep quality of the BED group is significantly better compared to Ctrl. The non-parametric indexes showed how interdaily stability, significantly correlated to sleep efficiency, was higher in BED group compared to the Ctrl group, indicating a better synchronization of rest-activity circadian rhythm. In conclusion, the maintenance of a regular lifestyle, such as imposed by the multidisciplinary therapy, is important to avoid alterations in the sleep-wake cycle, particularly in patients with eating disorders.