Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples

We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0519-7) contains supplementary material, which is available to authorized users.     

Interaction of a Partially Disordered Antisigma Factor with Its Partner,the Signaling Domain of the TonB-Dependent Transporter HasR

Bacteria use diverse signaling pathways to control gene expression in response to external stimuli. In Gram-negative bacteria, the binding of a nutrient is sensed by an outer membrane transporter. This signal is then transmitted to an antisigma factor and subsequently to the cytoplasm where an ECF sigma factor induces expression of genes related to the acquisition of this nutrient. The molecular interactions involved in this transmembrane signaling are poorly understood and structural data on this family of antisigma factor are rare. Here, we present the first structural study of the periplasmic domain of an antisigma factor and its interaction with the transporter. The study concerns the signaling in the heme acquisition system (Has) of Serratia marcescens. Our data support unprecedented partially disordered periplasmic domain of an anti-sigma factor HasS in contact with a membrane-mimicking environment. We solved the 3D structure of the signaling domain of HasR transporter and identified the residues at the HasS−HasR interface. Their conservation in several bacteria suggests wider significance of the proposed model for the understanding of bacterial transmembrane signaling.     

Release of Interleukin-1α or Interleukin-1β Depends on Mechanism of Cell Death

The cytokine interleukin-1 (IL-1) has two main pro-inflammatory forms, IL-1α and IL-1β, which are central to host responses to infection and to damaging sterile inflammation. Processing of IL-1 precursor proteins to active cytokines commonly occurs through activation of proteases, notably caspases and calpains. These proteases are instrumental in cell death, and inflammation and cell death are closely associated, hence we sought to determine the impact of cell death pathways on IL-1 processing and release. We discovered that apoptotic regulation of caspase-8 specifically induced the processing and release of IL-1β. Conversely, necroptosis caused the processing and release of IL-1α, and this was independent of IL-1β processing and release. These data suggest that the mechanism through which an IL-1-expressing cell dies dictates the nature of the inflammatory mechanism that follows. These insights may allow modification of inflammation through the selective targeting of cell death mechanisms during disease.     

Zinc depletion regulates the processing and secretion of IL-1β

Sterile inflammation contributes to many common and serious human diseases. The pro-inflammatory cytokine interleukin-1β (IL-1β) drives sterile inflammatory responses and is thus a very attractive therapeutic target. Activation of IL-1β in sterile diseases commonly requires an intracellular multi-protein complex called the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome. A number of disease-associated danger molecules are known to activate the NLRP3 inflammasome. We show here that depletion of zinc from macrophages, a paradigm for zinc deficiency, also activates the NLRP3 inflammasome and induces IL-1β secretion. Our data suggest that zinc depletion damages the integrity of lysosomes and that this event is important for NLRP3 activation. These data provide new mechanistic insight to how zinc deficiency contributes to inflammation and further unravel the mechanisms of NLRP3 inflammasome activation.     

SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.     

The contribution of Kv7 channels to pregnant mouse and human myometrial contractility

Premature birth accounts for approximately 75% of neonatal mortality and morbidity in the developed world. Despite this, methods for identifying and treating women at risk of preterm labour are limited and many women still present in preterm labour requiring tocolytic therapy to suppress uterine contractility. The aim of this study was to assess the utility of Kv7 channel activators as potential uterine smooth muscle (myometrium) relaxants in tissues from pregnant mice and women. Myometrium was obtained from early and late pregnant mice and from lipopolysaccharide (LPS)‐injected mice (day 15 of gestation; model of infection in pregnancy). Human myometrium was obtained at the time of Caesarean section from women at term (38–41 weeks). RT‐PCR/qRT‐PCR detected KCNQ and KCNE expression in mouse and human myometrium. In mice, there was a global suppression of all KCNQ isoforms, except KCNQ3, in early pregnancy (n= 6, P < 0.001 versus late pregnant); expression subsequently increased in late pregnancy (n= 6). KCNE isoforms were also gestationally regulated (P < 0.05). KCNQ and KCNE isoform expression was slightly down‐regulated in myometrium from LPS‐treated‐mice versus controls (P < 0.05, n= 3–4). XE991 (10 μM, Kv7 inhibitor) significantly increased spontaneous myometrial contractions in vitro in both human and mouse myometrial tissues (P < 0.05) and retigabine/flupirtine (20 μM, Kv7 channel activators) caused profound myometrial relaxation (P < 0.05). In summary, Kv7 activators suppressed myometrial contraction and KCNQ gene expression was sustained throughout gestation, particularly at term. Consequently, activation of the encoded channels represents a novel mechanism for treatment of preterm labour.     

Repeatability of events during spontaneous and gonadotrophin-induced oestrus in bitches.

Oestrus induction using equine chorionic gonadotrophin and human chorionic gonadotrophin was successful in five out of six bitches, although the first day of increased plasma progestagen concentration differed considerably between bitches. Induced oestrous periods differed from spontaneous cycles in the timing of vaginal epithelial cell cornification; plasma oestrogen concentrations were generally greater and progestogen concentrations were less in induced cycles. These results suggest that this schedule of oestrus induction would not be suitable for allowing mating on a predetermined day.     

Comparison of community based service with hospital based service for people with acute,severe psychiatric illness.

OBJECTIVE--To compare the burden on relatives and outcome of people treated for severe acute psychiatric illness by a community service and a traditional hospital based service. DESIGN--Follow up of patients aged 16-65 who required admission to hospital or home treatment for psychiatric illness during January 1990 to February 1991. SETTING--Two Birmingham electoral wards, Sparkbrook and Small Heath; Sparkbrook has a community based service and Small Heath a traditional hospital based service. SUBJECTS--69 patients from Sparkbrook and 55 from Small Health. MAIN OUTCOME MEASURES--Scores on present state examination, social behaviour assessment schedule, and general health questionnaire. RESULTS--24 (35%) of Sparkbrook patients received some treatment in hospital during the initial episodes. Relatives of Sparkbrook patients were less distressed by their burden at the initial assessment than relatives of Small Health patients (mean score 0.11 v 0.29, p < 0.01). Relatives were also more satisfied with the support they received and the treatment received by patients. More patients from Sparkbrook than Small Health were in contact with a psychiatrist (81% (95% confidence interval 71% to 91%) v 62% (44% to 68%)) and community nurse (56% (44% to 68%) v 14% (13% to 24%)) one year after the initial episode. Sparkbrook patients spent significantly fewer days in hospital during the initial episode (8 days v 59 days) and the first year (20.6 v 67.9 days). CONCLUSION--The community based service is as effective as the hospital based service and is preferred by relatives. It is more effective in keeping people in long term contact with psychiatrists.     

Synthesis and characterization of soluble dextran-adenosine phosphate complexes: kinetic effects of coenzyme loading.

Soluble dextran-ATP complexes have been synthesized using a bifunctional oxirane as the coupling agent. The degree of coupling is time-dependent, allowing materials of varying coenzyme loadings to be produced very simply. Characterization studies have shown that at the maximum coenzyme loading obtained (34 molecules per complex) all coenzyme moieties were coenzymically active with hexokinase. The extent of coenzyme loading was shown to have a considerable influence on the values of Km and Vmax of the complex as a substrate for hexokinase. Enzyme activity was also found with acetate kinase and myokinase, and coenzyme recycling (ATP, ADP) was demonstrated in an ultrafiltration reactor.     

Male-female differences in effects of parental absence on glucocorticoid stress response

This study examines the family environments and hormone profiles of 316 individuals aged 2 months-58 years residing in a rural village on the east coast of Dominica, a former British colony in the West Indies. Fieldwork was conducted over an eight-year period (1988–1995). Research methods and techniques include radioimmunoassay of cortisol and testosterone from saliva samples (N=22,340), residence histories, behavioral observations of family interactions, extensive ethnographic interview and participant observation, psychological questionnaires, and medical examinations. Analyses of data indicate complex, sex-specific effects of family environment on endocrine function. Male endocrine profiles exhibit greater sensitivity to presence of father than do female endocrine profiles. Father-absent males tend to have (a) low cortisol levels during infancy, (b) high or abnormal cortisol profiles during childhood and adolescence, and (c) high cortisol and low testosterone levels during adulthood compared with those of males raised with a resident father. These results indicate that early family environment has significant effects on endocrine response throughout male life histories. Mark V. Flinn is an Associate Professor of Anthropology at the University of Missouri, Columbia. He studies family relationships, endocrine stress response, and child health from a mix of evolutionary and developmental psychology perspectives. Robert J. Quinlan is a graduate student in the Department of Anthropology at the University of Missouri, Columbia. His interests include time allocation, family relationships, and medical anthropology. He is planning a long-term ethnographic study of cross-cousin marriage among the E’nyepa of Venezuela. Mark T. Turner is a graduate student in the Department of Anthropology at Northwestern University. He studies covariance of mother and infant hormone and immune function in naturalistic settings using assays from saliva and breast milk samples and ethnographic observations. Seamus A. Decker is a graduate student in the Department of Anthropology at Emory University. He has studied social factors associated with daily variations of salivary cortisol and testosterone levels among males in a Caribbean village. He is currently investigating levels of stress in rural and urban populations in Botswana. Barry G. England is an Associate Professor of Pathology and director of the ligand assay laboratories of the University of Michigan Hospitals. His primary interests concern reproductive endocrinology.