David Hawkes

An Antiviral Response Directed by PKR Phosphorylation of the RNA Helicase A

The double-stranded RNA-activated protein kinase R (PKR) is a key regulator of the innate immune response. Activation of PKR during viral infection culminates in phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) to inhibit protein translation. A broad range of regulatory functions has also been attributed to PKR. However, as few additional PKR substrates have been identified, the mechanisms remain unclear. Here, PKR is shown to interact with an essential RNA helicase, RHA. Moreover, RHA is identified as a substrate for PKR, with phosphorylation perturbing the association of the helicase with double-stranded RNA (dsRNA). Through this mechanism, PKR can modulate transcription, as revealed by its ability to prevent the capacity of RHA to catalyze transactivating response (TAR)–mediated type 1 human immunodeficiency virus (HIV-1) gene regulation. Consequently, HIV-1 virions packaged in cells also expressing the decoy RHA peptides subsequently had enhanced infectivity. The data demonstrate interplay between key components of dsRNA metabolism, both connecting RHA to an important component of innate immunity and delineating an unanticipated role for PKR in RNA metabolism.

Labeling of Multiple HIV-1 Proteins with the Biarsenical-Tetracysteine System

Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection.

Central injection of relaxin-3 receptor (RXFP3) antagonist peptides reduces motivated food seeking and consumption in C57BL/6J mice

Behavioural arousal in mammals is regulated by various interacting central monoamine- and peptide-neurotransmitter/receptor systems, which function to maintain awake, alert and active states required for performance of goal-directed activities essential for survival, including food seeking. Existing anatomical and functional evidence suggests the highly-conserved neuropeptide, relaxin-3, which signals via its cognate Gi/o-protein coupled receptor, RXFP3, contributes to behavioural arousal and feeding behaviour in rodents. In studies to investigate this possibility further, adult male C57BL/6J mice were treated with the selective RXFP3 antagonist peptides, R3(B1-22)R/I5(A) and R3(B1-22)R, and motivated food seeking and consumption was assessed as a reflective output of behavioural arousal. Compared to vehicle treatment, intracerebroventricular (icv) injection of RXFP3 antagonists reduced: (i) food anticipatory activity before meal time during food restriction; (ii) consumption of highly palatable food; (iii) consumption of regular chow during the initial dark phase, and; (iv) consumption of regular chow after mild (∼4-h) food deprivation. Effects were not due to sedation and appeared to be specifically mediated via antagonism of relaxin-3/RXFP3 signalling, as RXFP3 antagonist treatment did not alter locomotor activity in wild-type mice or reduce palatable food intake in relaxin-3 deficient (knock-out) mice. Notably, in contrast to similar studies in the rat, icv injection of RXFP3 agonists and infusion into the paraventricular hypothalamic nucleus did not increase food consumption in mice, suggesting species differences in relaxin-3/RXFP3-related signalling networks. Together, our data provide evidence that endogenous relaxin-3/RXFP3 signalling promotes motivated food seeking and consumption, and in light of the established biological and translational importance of other arousal systems, relaxin-3/RXFP3 networks warrant further experimental investigation.

Revisiting adverse reactions to vaccines: A critical appraisal of Autoimmune Syndrome Induced by Adjuvants (ASIA).

In 2011 Shoenfeld and Agmon-Levin proposed a new syndrome as a way of grouping together a range of emerging autoimmune diseases with possible adjuvant-associated causes, Autoimmune/Auto-inflammatory Syndrome Induced by Adjuvants (ASIA). At present, there is no evidence to suggest that ASIA syndrome is a viable explanation for unusual autoimmune diseases. Since the initial paper, over 80 publications have discussed ASIA. This systematic review examines the research that has been done to investigate whether ASIA is a broad umbrella term with little clinical significance, or whether there is some underlying mechanism which could be utilised to reduce the occurrence of adjuvant mediated disease. Twenty-seven animal, epidemiological and case studies were reviewed. Unfortunately, a robust animal model of ASIA using biologically relevant doses of adjuvants has yet to be defined. It is also apparent that the broadness of the current ASIA criteria lack stringency and, as a result, very few cases of autoimmune disease could be excluded from a diagnosis of ASIA. The current studies involving human cases are so diverse, in both external stimuli and in resulting conditions, that there is currently a lack of reproducible evidence for any consistent relationship between adjuvant and autoimmune condition. The addition of a mandatory criterion requiring temporal association and clinically relevant adjuvant dose would allow better definition of what constitutes a diagnosis of ASIA.

Nucleus incertus promotes cortical desynchronization and behavioral arousal.

Arousal and vigilance are essential for survival and relevant regulatory neural circuits lie within the brainstem, hypothalamus and forebrain. The nucleus incertus (NI) is a distinct site within the pontine periventricular gray, containing a substantial population of GABAergic neurons with long-range, ascending projections. Existing neuroanatomical data and functional studies in anesthetized rats, suggest the NI is a central component of a midline behavioral control network well positioned to modulate arousal, vigilance and exploratory navigation, yet none of these roles have been established experimentally. We used a chemogenetic approach—clozapine-N-oxide (CNO) activation of virally delivered excitatory hM3Dq-DREADDs—to activate the NI in rats and examined the behavioral and physiological effects, relative to effects in naïve rats and appropriate viral-treated controls. hM3Dq activation by CNO resulted in long-lasting depolarization of NI neurons with action potentials, in vitro. Peripheral injection of CNO significantly increased c-Fos immunoreactivity in the NI and promoted cortical electroencephalograph (EEG) desynchronization. These brain changes were associated with heightened arousal, and increased locomotor activity in the homecage and in a novel environment. Furthermore, NI activation altered responses in a fear conditioning paradigm, reflected by increased head-scanning, vigilant behaviors during conditioned fear recall. These findings provide direct evidence that the NI promotes general arousal via a broad behavioral activation circuit and support early hypotheses, based on its connectivity, that the NI is a modulator of cognition and attention, and emotional and motivated behaviors.

Research Trends in Evidence-Based Medicine: A Joinpoint Regression Analysis of More than 50 Years of Publication Data

Background Evidence-based medicine (EBM) has developed as the dominant paradigm of assessment of evidence that is used in clinical practice. Since its development, EBM has been applied to integrate the best available research into diagnosis and treatment with the purpose of improving patient care. In the EBM era, a hierarchy of evidence has been proposed, including various types of research methods, such as meta-analysis (MA), systematic review (SRV), randomized controlled trial (RCT), case report (CR), practice guideline (PGL), and so on. Although there are numerous studies examining the impact and importance of specific cases of EBM in clinical practice, there is a lack of research quantitatively measuring publication trends in the growth and development of EBM. Therefore, a bibliometric analysis was constructed to determine the scientific productivity of EBM research over decades. Methods NCBI PubMed database was used to search, retrieve and classify publications according to research method and year of publication. Joinpoint regression analysis was undertaken to analyze trends in research productivity and the prevalence of individual research methods. Findings Analysis indicates that MA and SRV, which are classified as the highest ranking of evidence in the EBM, accounted for a relatively small but auspicious number of publications. For most research methods, the annual percent change (APC) indicates a consistent increase in publication frequency. MA, SRV and RCT show the highest rate of publication growth in the past twenty years. Only controlled clinical trials (CCT) shows a non-significant reduction in publications over the past ten years. Conclusions Higher quality research methods, such as MA, SRV and RCT, are showing continuous publication growth, which suggests an acknowledgement of the value of these methods. This study provides the first quantitative assessment of research method publication trends in EBM.

Regulation of angiotensin II receptors in the prostate of the transgenic (mRen-2)27 rat: effect of angiotensin-converting enzyme inhibition.

We examined the regulation/expression of angiotensin II (Ang II) receptors in the transgenic (TG) (mRen-2)27 rat compared to the normal Sprague-Dawley (SD) rat. Ang II receptor binding and mRNA expression were determined by quantitative autoradiography and real-time PCR, respectively. Ang II receptors in the rat prostate rat were of the AT(1) receptor subtype and were significantly reduced in the prostate of the TG rat compared to the normal SD rat. However, AT(1) receptor binding was significantly higher in the prostate of the TG rat treated with the ACE inhibitor lisinopril compared to the untreated TG rat and comparable to the control SD rat. In contrast to the protein, AT(1) receptor mRNA expression was not reduced in the prostate of the TG rat compared to the SD rat. However, AT(1) receptor mRNA was markedly reduced in the prostate of the lisinopril-treated TG rat compared to the untreated TG rat or control SD rat. In conclusion, the findings suggest that AT(1) receptors are present in the rat prostate at a protein level and are subject to down-regulation in the TG rat which may be due to receptor internalisation as a consequence of receptor hyper-stimulation by increased local tissue levels of Ang II. Moreover, AT(1) receptor protein and mRNA expression in the prostate may be inversely modulated.

Pharmacogenetic stimulation of neuronal activity increases myelination in an axon-specific manner

Mounting evidence suggests that neuronal activity influences myelination, potentially allowing for experience-driven modulation of neural circuitry. The degree to which neuronal activity is capable of regulating myelination at the individual axon level is unclear. Here we demonstrate that stimulation of somatosensory axons in the mouse brain increases proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) within the underlying white matter. Stimulated axons display an increased probability of being myelinated compared to neighboring non-stimulated axons, in addition to being ensheathed with thicker myelin. Conversely, attenuating neuronal firing reduces axonal myelination in a selective activity-dependent manner. Our findings reveal that the process of selecting axons for myelination is strongly influenced by the relative activity of individual axons within a population. These observed cellular changes are consistent with the emerging concept that adaptive myelination is a key mechanism for the fine-tuning of neuronal circuitry in the mammalian CNS.Neuronal activity is known to increase myelination by oligodendrocytes in the mouse brain. Here, Mitew et al. demonstrate that chemogenetic manipulations of somatosensory axon activity both increase the generation of new oligodendrocytes and preferentially enhance myelination of the activated axons.

Human papillomavirus vaccination and primary ovarian insufficiency: an association based on ideology rather than evidence.

A recent review by Gruber and Shoenfeld [1] drew attention to the possibility of a link between the quadrivalent human papillomavirus (HPV) vaccine (Gardasil) and primary ovarian insufficiency [(POI), also known as premature ovarian failure]. This is the most recent in a long line of conditions for which HPV vaccination has been proposed as a causative factor [2,3]. This editorial will examine the background on which the proposed link between HPV vaccination and POI is built. In the nearly 9 years since the widespread introduction of HPV vaccination, including over 170 million doses of Gardasil, there have been only six cases reporting POI in association with HPV vaccination across three publications. The first was published in 2012 by Little and Ward [4] and featured a single case study of a 16-year-old girl with POI. Two years later the same authors published a second study featuring three case studies, including the case from the original manuscript [5]. A second series of three case studies was published in 2013 by Colafrancesco et al. [6]. Gruber and Shoenfeld [1] provide a good overview of the six cases but omit to mention the time between HPV vaccination and the appearance of the first symptoms. None of the women presented with irregular menses until after the third dose of the HPV vaccination, apart from Colafrancesco case 1 who had irregular periods prior to the first HPV vaccination. Of the other five cases only one had an interval between vaccination and first irregular menses of less than a few months (this is the terminology given in [6]). Molecular Microbiology Laboratory, Victorian Cytology Service, Carlton, Department of Pharmacology and Therapeutics, University of Melbourne, Parkville, SAEFVIC, Murdoch Children’s Research Institute, Parkville, Department of Paediatrics, Monash University, Infection and Immunity, Monash Children’s Hospital and Monash Immunisation, Monash Health, Clayton, Australia

Answering human papillomavirus vaccine concerns; a matter of science and time.

Since the introduction of the HPV vaccine, questions have been asked about its efficacy in preventing cancer linked with HPV. Concerns about the HPV vaccine safety profile have also been raised. This paper highlights the rapidly growing body of evidence (including clinical trials and post-marketing surveillance) illustrating both the safety of the HPV vaccine, through a detailed investigation of reported adverse events, and its efficacy in reducing both HPV infections rates and the resulting drop in cervical lesions, which have been demonstrated to be good predictors of cervical cancer risk.