Human Cytomegalovirus pUL37x1 Is Important for Remodeling of Host Lipid Metabolism.

Human cytomegalovirus (HCMV) replication requires host metabolism. Infection alters the activity in multiple metabolic pathways, including increasing fatty acid elongation and lipid synthesis. The virus-host interactions regulating the metabolic changes associated with replication are essential for infection. While multiple host factors, including kinases and transcription factors, important for metabolic changes that occur following HCMV infection have been identified, little is known about the viral factors required to alter metabolism. In this study, we tested the hypothesis that pUL37x1 is important for the metabolic remodeling that is necessary for HCMV replication using a combination of metabolomics, lipidomics, and metabolic tracers to measure fatty acid elongation. We observed that fibroblast cells infected with wild-type (WT) HCMV had levels of metabolites similar to those in cells infected with a mutant virus lacking the UL37x1 gene, subUL37x1. However, we found that relative to WT-infected cells, subUL37x1-infected cells had reduced levels of two host proteins that were previously demonstrated to be important for lipid metabolism during HCMV infection: fatty acid elongase 7 (ELOVL7) and the endoplasmic reticulum (ER) stress-related kinase PERK. Moreover, we observed that HCMV infection results in an increase in phospholipids with very-long-chain fatty acid tails (PL-VLCFAs) that contain 26 or more carbons in one of their two tails. The levels of many PL-VLCFAs were lower in subUL37x1-infected cells than in WT-infected cells. Overall, we conclude that although pUL37x1 is not necessary for network-wide metabolic changes associated with HCMV infection, it is important for the remodeling of a subset of metabolic changes that occur during infection.IMPORTANCE Human cytomegalovirus (HCMV) is a common pathogen that asymptomatically infects most people and establishes a lifelong infection. However, HCMV can cause end-organ disease that results in death in the immunosuppressed and is a leading cause of birth defects. HCMV infection depends on host metabolism, including lipid metabolism. However, the viral mechanisms for remodeling of metabolism are poorly understood. In this study, we demonstrate that the viral UL37x1 protein (pUL37x1) is important for infection-associated increases in lipid metabolism, including fatty acid elongation to produce very-long-chain fatty acids (VLCFAs). Furthermore, we found that HCMV infection results in a significant increase in phospholipids, particularly those with VLCFA tails (PL-VLCFAs). We found that pUL37x1 was important for the high levels of fatty acid elongation and PL-VLCFA accumulation that occur in HCMV-infected cells. Our findings identify a viral protein that is important for changes in lipid metabolism that occur following HCMV infection.

Biochemical Characterization of CYP505D6, a Self-Sufficient Cytochrome P450 from the White-Rot Fungus Phanerochaete chrysosporium.

The activity of a self-sufficient cytochrome P450 enzyme, CYP505D6, from the lignin-degrading basidiomycete Phanerochaete chrysosporium was characterized. Recombinant CYP505D6 was produced in Escherichia coli and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C9-18 carbon chain lengths as the substrates. Hydroxylation occurred at the ω-1 to ω-6 positions of such substrates with C9-15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at the ω-1 to ω-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active-site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid were analyzed. Compared with wild-type CYP505D6, its V51Y variant generated few products hydroxylated at the ω-4 to ω-6 positions. The products generated by wild-type CYP102A1 were hydroxylated at the ω-1 to ω-4 positions, whereas its Y51V variant generated ω-1 to ω-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regiospecificity of fatty acid hydroxylation, at least that at the ω-4 to ω-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry.IMPORTANCEPhanerochaete chrysosporium is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in this study, P. chrysosporium CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C9-15) and fatty acids (C9-15) at the ω-1 to ω-7 or ω-1 to ω-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than those of the known fused proteins CYP102A1 and CYP505A1. The substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications.

Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.

Human cytomegalovirus (HCMV) replication depends on the activities of several host regulators of metabolism. Hypoxia-inducible factor 1α (HIF1α) was previously proposed to support virus replication through its metabolic regulatory function. HIF1α protein levels rise in response to HCMV infection in nonhypoxic conditions, but its effect on HCMV replication was not investigated. We addressed the role of HIF1α in HCMV replication by generating primary human cells with HIF1α knocked out using CRISPR/Cas9. When HIF1α was absent, we found that HCMV replication was enhanced, showing that HIF1α suppresses viral replication. We used untargeted metabolomics to determine if HIF1α regulates metabolite concentrations in HCMV-infected cells. We discovered that in HCMV-infected cells, HIF1α suppresses intracellular and extracellular concentrations of kynurenine. HIF1α also suppressed the expression of indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme in kynurenine synthesis. In addition to its role in tryptophan metabolism, kynurenine acts as a signaling messenger by activating aryl hydrocarbon receptor (AhR). Inhibiting AhR reduces HCMV replication, while activating AhR with an exogenous ligand increases virus replication. Moreover, we found that feeding kynurenine to cells promotes HCMV replication. Overall, our findings indicate that HIF1α reduces HCMV replication by regulating metabolism and metabolite signaling.IMPORTANCE Viruses, including human cytomegalovirus (HCMV), reprogram cellular metabolism using host metabolic regulators to support virus replication. Alternatively, in response to infection, the host can use metabolism to limit virus replication. Here, our findings show that the host uses hypoxia-inducible factor 1α (HIF1α) as a metabolic regulator to reduce HCMV replication. Further, we found that HIF1α suppresses kynurenine synthesis, a metabolite that can promote HCMV replication by signaling through the aryl hydrocarbon receptor (AhR). In infected cells, the rate-limiting enzyme in kynurenine synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is suppressed by a HIF1α-dependent mechanism. Our findings describe a functional connection between HIF1α, IDO1, and AhR that allows HIF1α to limit HCMV replication through metabolic regulation, advancing our understanding of virus-host interactions.

Public Stranger Violence-What Makes You Vulnerable?A Comparison of Perspectives From Young Adults and Industry Experts.

The purpose of this study was to determine the situational and individual factors that influence vulnerability to public stranger violence (PSV) from the perspective of young adults and industry professionals. In total, 25 young adults aged between 18 and 29 years formed one sample and participated in one of four focus groups. The second sample consisted of 10 industry professionals with backgrounds in policing, corrections, and forensic psychology. Each professional participated in an individual semistructured interview. Both samples were asked questions regarding vulnerability and safety in public, where responses were analyzed using a thematic analysis. Multiple themes were identified and categorized into situational and individual factors associated with victimization. Situational factors referred to aspects of the environment that may influence vulnerability to PSV and included visibility, location, and level of support for potential victims. Individual factors referred to aspects about the person that may influence vulnerability to PSV and included unpredictability, ease of target, stereotypes, in-groups versus out-groups, distractions, and personality traits. Although both samples identified similar situational factors, young adults were found to be either unaware of individual factors identified by industry professionals as influencing vulnerability or at least misunderstood some of these factors. This is problematic as young adults may be basing their public behaviors on misinformation that may in fact increase, rather than decrease, vulnerability to PSV. The findings from this study have implications for the design of personal safety programs as well as community-based interventions to reduce vulnerability, the prevalence of PSV, and the negative outcomes associated with PSV, including anxiety and fear of crime.

Shear-Mediated Platelet Activation is Accompanied by Unique Alterations in Platelet Release of Lipids.

INTRODUCTION: Platelet activation by mechanical means such as shear stress exposure, is a vital driver of thrombotic risk in implantable blood-contacting devices used in the treatment of heart failure. Lipids are essential in platelets activation and have been studied following biochemical activation. However, little is known regarding lipid alterations occurring with mechanical shear-mediated platelet activation. METHODS: Here, we determined if shear-activation of platelets induced lipidome changes that differ from those associated with biochemically-mediated platelet activation. We performed high-resolution lipidomic analysis on purified platelets from four healthy human donors. For each donor, we compared the lipidome of platelets that were non-activated or activated by shear, ADP, or thrombin treatment. RESULTS: We found that shear activation altered cell-associated lipids and led to the release of lipids into the extracellular environment. Shear-activated platelets released 21 phospholipids and sphingomyelins at levels statistically higher than platelets activated by biochemical stimulation. CONCLUSIONS: We conclude that shear-mediated activation of platelets alters the basal platelet lipidome. Further, these alterations differ and are unique in comparison to the lipidome of biochemically activated platelets. Many of the released phospholipids contained an arachidonic acid tail or were phosphatidylserine lipids, which have known procoagulant properties. Our findings suggest that lipids released by shear-activated platelets may contribute to altered thrombosis in patients with implanted cardiovascular therapeutic devices. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00692-x.

VibroSleeve: A wearable vibro-tactile feedback device for arm guidance.

Vibro-tactile feedback offers a complementary augmentation cue for motor guidance and training. Motivated by the needs of prosthetic vision rehabilitation, we explore the use of the cylindrical forearm surface to deliver arm guidance cues via vibro-tactile stimulation. We present 'VibroSleeve', a novel wearable arm motion guidance aid made up of 4x4 arrangement of vibration coin motors embedded within an elastic sleeve for delivering vibro-tactile patterns to the forearm. In this paper, we present the concept and design of VibroSleeve, along with results of preliminary evaluation. We outline key insights gained into the perceptual aspects of calibration and usable intensity bandwidth critical for reliable interpretation of encoded information. Our results demonstrate feasibility of the approach, and provide foundations for future work on developing the sleeve as a rehabilitation aid for guiding the arm towards visually perceived targets.

Peer learning, lectures and online learning--putting it all together.

INTRODUCTION: This paper evaluates usage of discussion forums forming part of an integrated online learning environment that supplements a face to face general practice registrar training program. METHOD: Basic general practice registrars were given training in the use of the online learning environment, which was explicitly linked to their other educational activities. A group of advanced registrars was given access to the online environment without any formal training or support and without specific curriculum linkage. Usage data were in the form of quantitative analysis of site logs, posts to discussion forums before and after a website redesign, and qualitative feedback from focus groups. RESULTS: The majority of basic registrars accessed forums to read messages. Only one third posted messages. The number of registrars to access forums was similar before and after site redesign. Log data indicated greater exploration of resources in the redesigned environment. The group of advanced registrars used the online resources significantly less than the basic registrars. DISCUSSION: These data underscore the importance of linking curriculum to online learning resources, and of providing adequate information technology training and support.

Defining Cyberbullying: A Multiple Perspectives Approach.

To date, there has been a lack of consensus among researchers, practitioners, and laypersons about the definition of cyberbullying. Researchers have typically applied the key characteristics of intent to harm, power imbalance, and repetition from the definition of traditional bullying to cyberbullying, but how these characteristics transfer from the real world to a technology-mediated environment remains ambiguous. Moreover, very few studies have specifically investigated how cyberbullying is defined from the perspective of bullies, victims and bystanders. To this end, this article will propose a three-part definition of cyberbullying, which incorporates the perspective of bullies, victims and bystanders.

Chasing The 'Like': Adolescent Use Of Social Networking Sites In Australia.

The current study investigated how adolescents behave on Social Networking Sites (SNSs) and how they interpret the feedback they receive online from others. Thirty-four Australian adolescents (26 girls, 8 boys) aged 13 to 17 years participated in the study. Five semi-structured focus groups (3 mixed groups, 2 all-girl groups) were conducted to explore how adolescents perceive their own and others' SNS behaviours, the motivation underlying these behaviours, and the expected outcomes related to particular behaviours. Teenagers reported that they spend a good deal of time planning their SNS posts, felt that the information they posted was a true reflection of them as a person, and thus interpreted feedback ("likes") as measuring their self-worth. In contrast, some teenagers were perceived as "chasing the like" for status and popularity while not caring about how accurately their posts represented them as a person. A potential gender bias in these findings is discussed.

What do we mean by social networking sites?

The purpose of this study was to explore people's conceptual understanding of Social Networking Sites (SNSs) through exploring the combined use of a range of popular SNSs, including Facebook, Twitter, Myspace, Instagram, Tumblr, LinkedIn and Google Plus. Seventy-three adults, aged 18 to 63, participated in an online survey that used open-ended questions to ask how participants define and use different SNSs. Four themes were identified, including the explicit presentation and interpretation of different selves, the love-hate relationship with SNSs, privacy and danger concerns, and limited SNS knowledge. The findings from this study suggest that researchers need to consider how people use SNSs in combination as this influences the decisions people make about which SNS accounts they use and how they present themselves on these sites.

Towards defining dental drilling competence, part 2: a study of cues and factors in bone drilling.

The study of expertise in surgery aims to facilitate the development of improved training methods by understanding the characteristics of expert practitioners. In this article and its companion, we present our study of the characteristics of competence and expertise in the field of oral surgery. We observed participants of different skill levels as they performed an ex vivo drilling task designed to test the psychomotor skill of distinguishing the material boundaries between tooth and bone. Part 1 of this study examined the physical characteristics of drilling performance, while this article examines the cognitive aspects of performance. In this article we investigate the psychomotor cues used for decision making during drilling and explore other factors that affect a participant's ability to distinguish tooth from bone. Our results suggest that visual and tactile cues were the most important cues guiding drilling performance in all participant groups. Our results also suggest that when compared to experts, novices relied more on visual cues rather than tactile cues and lacked the psychomotor skills required to utilize the broader range of cues used by experts.

Towards defining dental drilling competence, part 1: a study of bone drilling technique.

Technical skills are critical for dentists. Computer-based simulation offers a range of potential benefits for surgical training, but to date the development of simulators has not been characterized by a structured investigation of specific mechanisms by which trainees attain competence. This two-part study contributes to the understanding of the manner in which surgical psychomotor skills are acquired so that this knowledge can be incorporated into the design of training simulations. We studied participant groups of varying skill levels as they performed a drilling task in oral surgery. In this first part of our study, we investigated the elements of surgical technique and differences in the drilling performance of novice, competent, and expert dentists. Our results indicate that novice dentists employ a technique that differs considerably in drilling stroke length and duration from that employed by experts. Expert dentists perform faster, apply more force, lift the bur off the bone less, and produce superior results compared with novices.

Control of vertebrate skeletal mineralization by polyphosphates.

BACKGROUND: Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO(3)(-))(n)) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization. PRINCIPAL FINDINGS/METHODOLOGY: The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO(4)(3-)) concentration while permitting the accumulation of a high total PO(4)(3-) concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4',6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation. CONCLUSIONS/SIGNIFICANCE: We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.

Simulation-based medical error disclosure training for pediatric healthcare professionals.

Ethical and regulatory guidelines recommend disclosure of medical errors to patients and families. Yet few studies examine how to effectively train healthcare professionals to deliver communications about adverse events to family members of affected pediatric patients. This pilot study uses a preintervention-postintervention study design to investigate the effects of medical error disclosure training in a simulated setting for pediatric oncology nurses (N=16). The results of a paired t test showed statistically significant increases in nurses' communication self-efficacy to carry out medical disclosure (t = 6.68, p < .001). Ratings of setting "realism" and simulation effectiveness were high (21 out of 25 composite score). Findings provide preliminary support for further research on simulation-based disclosure training for healthcare professionals.

Absence of the proapoptotic Bax protein extends fertility and alleviates age-related health complications in female mice.

The menopausal transition in human females, which is driven by a loss of cyclic ovarian function, occurs around age 50 and is thought to underlie the emergence of an array of health problems in aging women. Although mice do not undergo a true menopause, female mice exhibit ovarian failure long before death because of chronological age and subsequently develop many of the same age-associated health complications observed in postmenopausal women. Here we show in mice that inactivation of the proapoptotic Bax gene, which sustains ovarian lifespan into advanced age, extends fertile potential and minimizes many age-related health problems, including bone and muscle loss, excess fat deposition, alopecia, cataracts, deafness, increased anxiety, and selective attention deficit. Further, ovariectomy studies show that the health benefits gained by aged females from Bax deficiency reflect a complex interplay between ovary-dependent and -independent pathways. Importantly, and contrary to popular belief, prolongation of ovarian function into advanced age by Bax deficiency did not lead to an increase in tumor incidence. Thus, the development of methods for postponing ovarian failure at menopause may represent an attractive option for improving the quality of life in aging females.