Dual Function of the pUL7-pUL51 Tegument Protein Complex in Herpes Simplex Virus 1 Infection

The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes. $\textbf{IMPORTANCE}$: The $\textit{Herpesviridae }$ is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus 1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells.This work was supported by the Leverhulme Trust (grant RPG-2012-793 to C.M.C.), the Royal Society (University Research Fellowship UF090010 to C.M.C.), and the Royal Society and the Wellcome Trust (Sir Henry Dale Fellowship 098406/Z/12/Z to S.C.G.). L.D. was supported by Wellcome Trust Ph.D. Programme funding (086158/Z/08/Z). D.J.O. was supported by a John Lucas Walker studentship. M.F.A. was supported by a Commonwealth Scholarship Commission PhD scholarship (BDCA-2014-7)

Report of the ICES\NAFO Joint Working Group on Deep-water Ecology (WGDEC), 11–15 March 2013, Floedevigen, Norway.

On 11 February 2013, the joint ICES/NAFO WGDEC, chaired by Francis Neat (UK) and attended by ten members met at the Institute for Marine Research in Floedevi-gen, Norway to consider the terms of reference (ToR) listed in Section 2. WGDEC was requested to update all records of deep-water vulnerable marine eco-systems (VMEs) in the North Atlantic. New data from a range of sources including multibeam echosounder surveys, fisheries surveys, habitat modelling and seabed imagery surveys was provided. For several areas across the North Atlantic, WGDEC makes recommendations for areas to be closed to bottom fisheries for the purposes of conservation of VMEs

Why do bilaterally symmetrical flowers orient vertically? Flower orientation influences pollinator landing behaviour

Protein lysine posttranslational modification by an increasing number of different acyl groups is becoming appreciated as a regulatory mechanism in cellular biology. Sirtuins are class III histone deacylases that use NAD(+) as a co-substrate during amide bond hydrolysis. Several studies have described the sirtuins as sensors of the NAD(+)/NADH ratio, but it has not been formally tested for all the mammalian sirtuins in vitro. To address this problem, we first synthesized a wide variety of peptide-based probes, which were used to identify the range of hydrolytic activities of human sirtuins. These probes included aliphatic ϵ-N-acyllysine modifications with hydrocarbon lengths ranging from formyl (C(1)) to palmitoyl (C(16)) as well as negatively charged dicarboxyl-derived modifications. In addition to the well established activities of the sirtuins, “long chain” acyllysine modifications were also shown to be prone to hydrolytic cleavage by SIRT1–3 and SIRT6, supporting recent findings. We then tested the ability of NADH, ADP-ribose, and nicotinamide to inhibit these NAD(+)-dependent deacylase activities of the sirtuins. In the commonly used 7-amino-4-methylcoumarin-coupled fluorescence-based assay, the fluorophore has significant spectral overlap with NADH and therefore cannot be used to measure inhibition by NADH. Therefore, we turned to an HPLC-MS-based assay to directly monitor the conversion of acylated peptides to their deacylated forms. All tested sirtuin deacylase activities showed sensitivity to NADH in this assay. However, the inhibitory concentrations of NADH in these assays are far greater than the predicted concentrations of NADH in cells; therefore, our data indicate that NADH is unlikely to inhibit sirtuins in vivo. These data suggest a re-evaluation of the sirtuins as direct sensors of the NAD(+)/NADH ratio

Specific connectivity and molecular diversity of mouse rubrospinal neurons

While much progress has been made in understanding the development, differentiation, and organization of the spinal motor system, the complex circuitry that is integrated to determine a motor behavior has yet to be fully understood. The activity of motor neurons is influenced by sensory feedback, excitatory and inhibitory interneurons, and supraspinal control from higher brain regions in the CNS. Descending pathways from the cortex and midbrain are involved in the control of voluntary motor output. This is made possible by their projections onto spinal interneurons and, to a degree that varies between species, directly onto motor neurons. However, the somatotopic organization and molecular diversity of supraspinal projection neurons, and the circuitry that underlies their contribution to motor output, remain incompletely understood. The evolutionary emergence of direct descending projections onto motor neurons has been considered to reflect a specialized level of organization for precise control of individual forelimb muscles. Unlike their polysynaptic counterparts, monosynaptic connections represent direct, unfiltered access to the motor neuron circuit. The direct circuit is thought to represent a neural specialization for the increase in fractionated digit movements exhibited by primates and humans. The progressive realization that rodents have a greater degree of manual dexterity than was previously thought has evoked renewed interest in the role of direct supraspinal projections in other mammalian species. Lesion studies in the rodent indicated that, of the two major supraspinal pathways involved in the control of voluntary movement, the rubrospinal tract had a greater role in control of distal forelimb musculature. However, the degree to which this reflected direct projections onto motor neurons was not clear. Earlier anatomical tracing studies in the rat indicated that there are close appositions between labeled rubrospinal axons and motor neurons projecting to intermediate and distal forelimb muscles. To confirm that these contacts correspond to synapses, I developed a viral tracing strategy to visualize projections from the midbrain. Using an established technique of high-magnification confocal imaging combined with co-localization of the rubrospinal synaptic terminal marker, vglut2, I established the existence of monosynaptic connections from the ventral midbrain at the level of the red nucleus onto a restricted population of forelimb motor neurons at a single spinal level (C7-C8) in the rodent. To determine whether the motor neurons that receive synaptic input correspond to specific motor pool(s), I first established a positional map of forelimb muscle motor pools in the cervical enlargement of the mouse spinal cord. A single motor pool, that which innervates the extensor digitorum muscle, appeared to be situated in the dense dorsolateral termination zone of rubrospinal ventral fibers. The extensor digitorum muscle plays a key role in digit extension and arpeggio movements during skilled reaching. Anterograde labeling of rubrospinal descending fibers combined with retrograde labeling of extensor digitorum motor neurons revealed a direct circuit from the red nucleus onto this population of motor neurons. Surprisingly, neighboring motor pools innervating digit flexor muscles did not receive rubrospinal inputs. Moreover, other modulatory inputs onto motor neurons, including corticospinal, proprioceptive, and cholinergic interneuron afferents did not distinguish between extensor and flexor digitorum motor neurons. My data therefore reveal a previously unrecognized level of motor pool specificity in the direct rubrospinal circuit. The identification of a small number of rubrospinal fibers that project onto extensor digitorum motor neurons suggested a considerable degree of heterogeneity between rubrospinal neurons. I therefore investigated the anatomical and molecular organization of subpopulations of rubrospinal neurons using retrograde labeling to identify subpopulations of rubrospinal neurons projecting, respectively, to cervical and lumbar levels of the spinal cord. Two rubrospinal populations could be identified within the red nucleus: a rostral population of intermingled cervical and lumbar projection neurons which express the Pou transcription factor Brn3a, and a caudal population containing segregated cervical and lumbar domains, which co-express Brn3a and a novel member of the C1q/TNF protein family, C1qL2. Following laser capture microdissection and genetic profiling of these three populations, I identified and validated molecular correlates of the topographic domains within the rodent red nucleus. The transcription factors tshz3 and mafB are expressed in the caudal cervical domain, whereas the chemokine fam19a4 is restricted to the caudal lumbar domain. KitL is an axon guidance molecule that is expressed in both the rostral population and the caudal cervical population. Finally, I identified two genes, cxcl13 and gpr88, that characterize subpopulations within these topographic divisions. Although the functional role of these genes in the establishment of the rubrospinal circuit remains to be determined, the data reveal a high level of molecular heterogeneity within the red nucleus. I hypothesize that this diversity allows rubrospinal neurons to form circuits in a precise and specific manner during development. Overall, my data provide evidence for a novel organization within the rodent motor system in which direct projections from the rubrospinal tract onto motor neurons appear to control a very specific aspect of skilled movement: the stereotypic extension and separation of the digits in preparation for a task requiring digit manipulation. Identifying molecular correlates of the direct rubrospinal population is the logical next step in further understanding the specific circuitry that encodes descending motor commands. My results will provide a basis for the dissection of the rubro-motoneuronal circuit, enabling the establishment of a direct link between neural connectivity and individual muscle control during a skilled movement

Pregnant women and infants as sentinel populations to monitor prevalence of malaria: results of pilot study in Lake Zone of Tanzania

As malaria control interventions are scaled-up, rational approaches are needed for monitoring impact over time. One proposed approach includes monitoring the prevalence of malaria infection among pregnant women and children at the time of routine preventive health facility (HF) visits. This pilot explored the feasibility and utility of tracking the prevalence of malaria infection in pregnant women attending their first antenatal care (ANC) visit and infants presenting at 9-12 months of age for measles vaccination.; Pregnant women attending first ANC and infants nine to 12 months old presenting for measles vaccination at a non-probability sample of 54 HFs in Tanzania's Lake Zone (Mara, Mwanza and Kagera Regions) were screened for malaria infection using a malaria rapid diagnostic test (RDT) from December 2012 to November 2013, regardless of symptoms. Participants who tested positive were treated for malaria per national guidelines. Data were collected monthly.; Overall 89.9 and 78.1 % of expected monthly reports on malaria infection prevalence were received for pregnant women and infants, respectively. Among 51,467 pregnant women and 35,155 infants attending routine preventive HF visits, 41.2 and 37.3 % were tested with RDT, respectively. Malaria infection prevalence was 12.8 % [95 % confidence interval (CI) 11.3-14.3] among pregnant women and 11.0 % (95 % CI 9.5-12.5) among infants, and varied by month. There was good correlation of the prevalence of malaria among pregnant women and infants at the HF level (Spearman rho = 0.6; p < 0.001). This approach is estimated to cost $1.28 for every person tested, with the RDT accounting for 72 % of the cost.; Malaria infection was common and well correlated among pregnant women and infants attending routine health services. Routine screening of these readily accessible populations may offer a practical strategy for continuously tracking malaria trends, particularly seasonal variation. Positivity rates among afebrile individuals presenting for routine care offer an advantage as they are unaffected by the prevalence of other causes of febrile illness, which could influence positivity rates among febrile patients presenting to outpatient clinics. The data presented here suggest that in addition to contributing to clinical management, ongoing screening of pregnant women could be used for routine surveillance and detection of hotspots

A Clinically Relevant Variant of the Human Hydrogen Sulfide-Synthesizing Enzyme Cystathionine β -Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5′-phosphate- (PLP-) dependent cystathionine β-synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (H2S). CBS activity, contributing to cellular redox homeostasis, is positively regulated by S-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenic CBS mutations is not yet fully understood. Here we found that the ferrous heme of the reportedly mild p.P49L CBS variant has altered spectral properties and markedly increased affinity for CO, making the protein much more prone than wild type (WT) CBS to inactivation at physiological CO levels. The higher CO affinity could result from the slightly higher flexibility in the heme surroundings revealed by solving at 2.80-Å resolution the crystallographic structure of a truncated p.P49L. Additionally, we report that p.P49L displays impaired H2S-generating activity, fully rescued by PLP supplementation along the purification, despite a minor responsiveness to AdoMet. Altogether, the results highlight how increased propensity to CO inactivation of an otherwise WT-like variant may represent a novel pathogenic mechanism in classical homocystinuria

Digital strategies for nitrogen management in grain production systems: lessons from multi-method assessment using on-farm experimentation

During the past few decades, a range of digital strategies for Nitrogen (N) management using various types of input data and recommendation frameworks have been developed. Despite much research, the benefits accrued from such technology have been equivocal. In this work, thirteen methods for mid-season N recommendations in cereal production systems were evaluated simultaneously, ranging from simple mass balance through to non-mechanistic approaches based on machine learning. To achieve this, an extensive field research program was implemented, comprising twenty-one N strip trials implemented in wheat and barley fields across Australia over four cropping seasons. A moving window regression approach was used to generate crop response functions to applied N and calculate economically optimal N rates along the length of the strips. The N recommendations made using various methods were assessed based on the error against the optimal rate and expected profitability. The root mean squared error of the recommendations ranged from 15 to 57 kg/ha. The best performing method was a data-driven empirical strategy in which a multivariate input to characterise field and season conditions was abundantly available and used to predict optimal N rates using machine learning. This was the only approach with potential to substantially outperform the existing farmer management, reducing the recommendation error from 42 to 15 kg/ha and improving profitability by up to A$47/ha. Despite being reliant on extensive historical databases, such a framework shows a promising pathway to drive production systems closer towards season- and site-specific economically optimum recommendations. Automated on-farm experimentation is a key enabler for building the necessary crop response databases to run empirical data-driven decision tool