A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells.

Nociceptin/Orphanin FQ (N/OFQ) is the endogenous opioid agonist for the N/OFQ receptor or NOP. This receptor system is involved in pain processing but also has a role in immune regulation. Indeed, polymorphonuclear cells (PMNs) express mRNA for N/OFQ precursor and are a potential source for circulating N/OFQ. Current measurements are based on ELISA and RIA techniques. In this study we have designed a bioassay to measure N/OFQ release from single PMNs. Chinese Hamster Ovary (CHO) cells transfected with the human (h) NOP receptor and Gαiq5 chimera force receptor coupling in biosensor cells to increase intracellular Ca2+; this can be measured with FLUO-4 dye. If isolated PMNs from healthy human volunteers are layered next to CHOhNOPGαiq5 biosensor cells then stimulated with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) we hypothesise that released N/OFQ will activate the biosensor. PMNs also release ATP and CHO cells express purinergic receptors coupled to elevated Ca2+. In a system where these receptors (P2Y1, P2Y2 and P2X7) are blocked with high concentrations of PPADS and oATP, PMN stimulation with fMLP increases Ca2+ in PMNs then shortly afterwards the biosensor cells. Our data therfore reports detection of single cell N/OFQ release from immune cells. This was absent when cells were preincubated with the selective NOP antagonist; SB-612111. Collectively this is the first description of single cell N/OFQ release. We will deploy this assay with further purified individual cell types and use this to further study the role of the N/OFQ-NOP system in disease; in particular sepsis where there is strong evidence for increased levels of N/OFQ worsening outcome.

Late Survival in Nonoperated Patients with Infrarenal Abdominal Aortic Aneurysm.

OBJECTIVE/BACKGROUND: Historical studies report high rupture rates in patients with nonoperated abdominal aortic aneurysms (AAAs) of > 5.5 cm diameter, although a recent audit has questioned this. METHODS: This was a retrospective review of 138/764 (18%) patients with AAAs evaluated in a preassessment anaesthetic clinic (PAC) between 2006 and 2012, who either did not undergo elective AAA repair or who underwent deferred repair. The remaining 626 underwent repair. Patients with severe comorbidities (dementia, advanced malignancy, life-expectancy < 1 year) and not referred to PAC were excluded. RESULTS: At a median of 27 months, 71 (52%) died, 36 (51%) following rupture. Cumulative survival, free from rupture or surgery for acute symptoms, was 96% at 1 year, 84% at 3 years, and 64% at 5 years, where baseline AAA diameters were 5.5-6.9 cm. For diameters ≥ 7 cm, survival, free from rupture, was 65% at 1 year, 29% at 3 years, and 0% at 5 years. Median interval to rupture was 47 months (AAA diameter 5.5-6.9 cm) and 21 months where baseline diameters were ≥ 7 cm. Rupture accounted for 32% of late deaths in patients with AAAs of 5.5-5.9 cm diameter, 46% in those with AAAs measuring 6.0-6.9 cm in diameter, and 71% in patients with AAA measuring ≥ 7 cm in diameter. CONCLUSION: Approximately half of all late deaths in this nonoperated cohort were not AAA related, suggesting that even had repair been undertaken, it would not have prolonged patient survival. The incidence of rupture in "high-risk" patients with an AAA < 7 cm diameter was < 5% at 1 year, thereby giving ample time to optimise risk factors and improve pre-existing medical conditions prior to undertaking a deferred intervention. Even if these patients did not undergo surgical repair, the risk of late rupture was relatively low. By contrast, nonoperated patients with AAAs ≥ 7 cm in diameter face a very high risk of rupture and will probably benefit from elective surgery, with the caveat that a higher procedural risk might have to be incurred.

Factors influencing short- and long-term mortality after lower limb amputation.

Mortality after lower limb amputation is high, with UK 30-day mortality rates of 9-17%. We performed a retrospective analysis of factors affecting early and late outcome after lower limb amputation for peripheral vascular disease or diabetic complications at a UK tertiary referral vascular centre between 2003 and 2010. Three hundred and thirty-nine patients (233 male), of median (IQR [range]) age 73 (62-79 [26-92]) years underwent amputation. Thirty-day mortality was 12.4%. On regression modelling, the risk of 30-day mortality was increased in patients of ASA grade ≥ 4 (OR 4.23, 95% CI 2.07-8.63), p < 0.001 and age between 74 and 79 years (OR 3.8, 95% CI 1.10-13.13), p = 0.04 and older than 79 years (OR 4.08, 95% CI 1.25-13.25), p = 0.02. Peri-operative (30-day) mortality for these groups was 23.2%, 13.7% and 18.8%, respectively. Survival and Cox regression analysis demonstrated that long-term mortality was associated with: age 74-79 years (HR 2.15, 95% CI 1.38-3.35), p = 0.001; age > 79 years (HR 2.78, 95% CI 1.82-4.25), p < 0.001; ASA grade ≥ 4 (HR 2.04, 95% CI 1.51-2.75), p < 0.001; out-of-hours operating (HR 1.51, 95% CI 1.08-2.10), p = 0.02; and chronic kidney disease stage 4-5 (1.57, 95% CI 1.07-2.30), p = 0.02. Anaesthetic technique was associated with long-term mortality on survival analysis (p = 0.04), but not when analysed using regression modelling. Mortality after lower limb amputation relates to patient age, ASA, out-of-hours surgery and renal dysfunction. These data support lower limb amputations' being performed during daytime hours and after modification replace with 'of ' correctable risk factors.

Opioids and immune modulation: more questions than answers.

Opioid addicts are more likely to present with infections suggesting opioids are immune modulators. The potential sites/mechanism(s) for this modulation are controversial and on close inspection not well supported by the current literature. It has long been assumed that opioid-induced immune modulation occurs via a combination of direct actions on the immune cell itself, via the hypothalamic-pituitary-adrenal (HPA) axis, or both. Opioid receptors are classified as MOP (µ, mu), DOP (δ, delta), and KOP (κ, kappa)--classical naloxone sensitive receptors--or NOP (the receptor for nociceptin/orphanin FQ), which is naloxone insensitive. Opioids currently used in clinical practice predominantly target the MOP receptor. There do not appear to be classical opioid receptors present on immune cells. The evidence for HPA activation is also poor and shows some species dependence. Most opioids used clinically or as drugs of abuse do not target the NOP receptor. Other possible target sites for immune modulation include the sympathetic nervous system and central sites. We are currently unable to accurately define the cellular target for immune modulation and suggest further investigation is required. Based on the differences observed when comparing studies in laboratory animals and those performed in humans we suggest that further studies in the clinical setting are needed.

Vitamin K: fracture prevention and beyond.

Morbidity and decreased function related to osteoporosis, fracture, cardiovascular disease, stroke, and peripheral vascular disease are encountered by clinicians daily. Although we have seen vast advancement in treatment and management of these conditions, preventative practice has unfortunately served a lesser role in patient care. Increasing the dietary intake of vitamin K may have substantial utility in the prevention of these disease states. Since the discovery of vitamin K in 1935, its primary role was thought to be involved in the synthesis of clotting factors II, VII, IX, and X. Recently, its function in other metabolic pathways has emerged, leading to exploration of its significance beyond coagulation. Vitamin K is essential to bone physiology and prevention of atherosclerosis. It is involved in bone remodeling, cell signaling, apoptosis, arterial calcification, and chemotaxis, and it has anti-inflammatory effects. Conversely, warfarin, a potent vitamin K inhibitor, has demonstrated adverse effects on bone remodeling and atherosclerosis. Natural forms of vitamin K are available in multiple dietary sources, and some structural forms are more readily available for use in metabolic pathways than are others. With regard to supplementation, the specific form of vitamin K is often not disclosed, and the recommended daily value is potentially less than what is physiologically required. On the basis of a review of the literature, it appears advantageous to encourage patients to eat a diet rich in vitamin K; however, the benefit of vitamin K supplementation alone is yet to be thoroughly conveyed.

Genomic sequences of blackberry chlorotic ringspot virus and strawberry necrotic shock virus and the phylogeny of viruses in subgroup 1 of the genus Ilarvirus.

Three members of subgroup 1 of the genus Ilarvirus: blackberry chlorotic ringspot (BCRV), strawberry necrotic shock (SNSV), and tobacco streak viruses (TSV), may infect Rubus and Fragaria species. All cause symptoms similar to those previously attributed to infection by TSV alone. Although similarities exist among the genomic sequences of the three, phylogenetic analysis shows them to be distinct viruses. These viruses and Parietaria mottle virus, the other currently accepted member of subgroup 1, appear to have evolved from a common ancestral virus, share conserved motifs in the products of the genomic RNAs, and constitute a distinct subgroup within the genus.

The complete nucleotide sequence of hydrangea ringspot virus. Brief report.

The complete nucleotide sequence of the genome of the potexvirus, hydrangea ringspot virus, has been determined. The sequence is 6,185 nt in length, excluding the poly (A) tail, and contains six ORFs coding for proteins of 156, 26, 12, 8, 24, and 16 kDa, respectively. ORF 6 is contained within ORF 5 and in this respect the virus is similar to the potexviruses CsCMV, NMV, and SMYEV. Phylogenetic analysis of the putative products of the ORFs and signature motifs contained within these products shows the virus to be most closely related to CsCMV. A similar analysis of data for the coat proteins of potexviruses did not support the previously reported serological relationships between HdRSV and other potexviruses. This is the first complete sequence published for the genome of a potexvirus infecting a dicotyledonous, temperate, deciduous woody species.

Viruses in subgroup 2 of the genus Ilarvirus share both serological relationships and characteristics at the molecular level.

Sequence data have been determined for 5 members of subgroup 2 of the genus Ilarvirus. These data support the known serological relationships among accepted members of this group and indicate that the ilarvirus Hydrangea mosaic virus (HdMV) is an isolate of Elm mottle virus (EMoV). The close relationships between members of this subgroup, exhibited through the coat proteins coded on RNA 3, extend to the other genomic molecules. Primers designed from the sequences of RNA 1 and RNA 2 of EMoV amplified fragments from all other subgroup 2 viruses but not from other ilarviruses. Although closely related, members of this subgroup occur naturally in distinctly different host species. The possible origins of the viruses are discussed in relation to similarities among the genomic molecules, in particular RNA 3.

Comparison of the amino acid sequences of RNA-dependent RNA polymerases of cypoviruses in the family Reoviridae.

The nucleotide sequences of the genome segment S2 of Bombyx mori cypovirus 1, S2 of Lymantria dispar cypovirus 1, S1 of Lymantria dispar cypovirus 14 and S1 of a proposed new electropherotype of Trichoplusia ni cypovirus 15 were determined. These segments encoded putative RNA-dependent RNA polymerases (RDRPs). The deduced amino acid sequences of RDRPs within the genus Cypovirus showed 32% to 94% identities, while extent of homology between RDRPs in the genera Cypovirus and Oryzavirus, a genus most closely related, was approximately 26% identity. Both the genera Cypovirus and Oryzavirus might have originated from a common insect virus ancestor.

First Report of Tomato ringspot virus in Butterfly Bush (Buddleia davidii).

Leaves displaying bright yellow or light green line pattern symptoms were collected from individual, large, mature buddleias in a home garden in Clemson, SC, a botanical garden in Knoxville, TN, and a container-grown plant on sale in a retail home and garden store in Seneca, SC. Buddleias grown in the southeastern United States frequently display virus-like symptoms, but the line pattern symptom displayed by these plants was atypical of the mosaic, mottling, and leaf deformation seen when buddleias are infected with Alfalfa mosaic virus (AMV) or Cucumber mosaic virus (CMV) (2,4). Line pattern symptoms are frequently seen in woody species infected by ilarviruses or nepoviruses (2). No ilarviruses are reported to infect buddleia and only the nepovirus, Strawberry latent ringspot virus, which is restricted mainly to Europe, is reported to infect this species (1,2). The nepoviruses Tomato ringspot virus (ToRSV) and Tobacco ringspot virus (TRSV) are frequently found infecting plants of many species in the southeastern United States (3). Total RNA was extracted from the three symptomatic plants and used in reverse transcription-polymerase chain reactions (RT-PCR) to detect ToRSV and TRSV using primer pairs developed in this laboratory, which amplify regions around the amino terminus of the coat protein of the respective viruses. The expected amplification product for ToRSV of 327 base pairs was obtained from samples tested from each plant, and the nucleotide sequence of the product showed 96% identity with the corresponding fragment of GenBank Accession No. NC_003839 that the primers were designed to amplify. Repeated attempts to isolate a virus from symptomatic leaves using sap inoculation to Chenopodium amaranticolor Coste & Reyne, C. quinoa Willd, Nicotiana clevelandii Gray, and N. tabacum L. have failed. Repeated testing by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of leaves from the plant growing in Clemson consistently produced absorbance values at 405 nm in the range of 0.47 to 0.55 (mean of 8 separate samples per test) for symptomatic and asymptomatic leaves. The range of values for the positive control (ToRSV-G growing in N. clevelandii) was 1.3 to 1.5. The ranges of values for the noninfected controls (noninfected N. clevelandii and leaf tissue from a buddleia known to be infected with AMV and CMV but in which ToRSV or TRSV had never been detected by RT-PCR) were 0.102 to 0.104 and 0.102 to 0.106, respectively. The extraction buffer produced absorbance readings in the range of 0.098 to 0.102. RT-PCR of RNA extracted from other portions of the leaves used in the ELISA consistently amplified the 327-bp product from symptomatic leaves and from the positive control but not from noninfected control tissues. RNA from asymptomatic leaves on the infected plant also produced the 327-bp product in RT-PCR. Isolation of viruses from woody hosts is frequently difficult, and although, we have yet to succeed to confirm the association between the observed symptom and ToRSV, the evidence from PCR and ELISA would indicate ToRSV is present in these plants. To our knowledge, this is the first report of ToRSV, a member of the genus Nepovirus, in buddleia. References: (1) J. Albouy and J. C. Devergne. Maladies á Virus des Plants Ornementales. INRA Editions, Paris, 1998. (2) J. I. Cooper. Virus Diseases of Trees and Shrubs. 2nd ed. Chapman and Hill, London, 1993. (3) J. R. Edwards and R. G. Christie. Pages 352-353 in: Handbook of Viruses Infecting Legumes. CRC Press, Boca Raton, FL, 1991. (4) C. J. Perkins and R. G. T. Hicks. Plant Pathol. 38:443, 1989.

The RNA 5 of Prunus necrotic ringspot virus is a biologically inactive copy of the 3'-UTR of the genomic RNA 3.

In addition to the four RNAs known to be encapsidated by Prunus necrotic ringspot virus (PNRSV) and Apple mosaic virus (ApMV), an additional small RNA (RNA 5) was present in purified preparations of several isolates of both viruses. RNA 5 was always produced following infection of a susceptible host by an artificial mixture of RNAs 1, 2, 3, and 4 indicating that it was a product of viral replication. RNA 5 does not activate the infectivity of mixtures that contain the three genomic RNAs (RNA 1 + RNA 2 + RNA 3) nor does it appear to modify symptom expression. Results from hybridization studies suggested that RNA 5 had partial sequence homology with RNAs 1, 2, 3, and 4. Cloning and sequencing the RNA 5 of isolate CH 57/1-M of PNRSV, and the 3' termini of the RNA 1, RNA 2 and RNA 3 of this isolate indicated that it was a copy of the 3' untranslated terminal region (3'-UTR) of the genomic RNA 3.

Common multiple dsRNAs are present in populations of the fungus Discula destructiva originating from widely separated geographic locations.

DsRNAs were detected in 85/108 isolates of Discula destructiva, the cause of dogwood anthracnose, collected in South Carolina, Idaho, and Alabama. The eastern isolates contained a greater diversity of dsRNA than did Idaho isolates, but most isolates, irrespective of state of origin, contained two small bands (ca. 1.5-2.5 kb) with sequence homology indicated by Northern hybridization. Differences in the banding patterns suggest that genetic diversity of dsRNA in D. destructiva is generated rapidly and that D. destructiva can be simultaneously infected by multiple dsRNA viruses.

Differences between the coat protein amino acid sequences of English and Scottish serotypes of Raspberry ringspot virus exposed on the surface of virus particles.

The region of the RNA 2 coding for the putative helper/movement protein and the coat protein (CP) of each of six isolates of Raspberry ringspot virus was sequenced and these sequences were compared with the published sequence of the Scottish type isolate. Minimal differences were detected among the putative translations of the helper/movement proteins, however, multiple alignment and phylogenetic analysis of the putative CPs separated the English and Scottish serotypes into two distinct clades. Superimposing the amino acid sequences of the CPs of these two serotypes on the 3D model for the CP of a comovirus/nepovirus, showed that eight of the differences identified between the two serotypes occurred on the surface of the protein. Inspection of the recently reported structure of the capsid protein of Tobacco ringspot virus, the type member of the genus Nepovirus, indicated identical locations for these differences. The change of H (Scottish isolates) to R (English isolates) at position 219 in the amino acid sequences of the viruses occurred on an exposed, erect surface loop. The potential role of this change, and other unique differences between the amino acid sequences of the two serotypes, in the specificity of nematode transmission of the virus is discussed.

Utilization of human blood and sugar as nutrients by female Aedes aegypti (Diptera: Culicidae).

We examined the utilization of sugar and human blood as nutrient sources for small and large female Aedes aegypti (L.) when they were fed blood 2 or 5 d after emergence. Laboratory-reared mosquitoes were fed human blood alone or sugar plus human blood and assayed at 4, 12, 24, and 48 h after the blood meal. Starved and well-fed mosquitoes were obtained by holding teneral females (< or = 1 d old) with 0, 5, 10, and 15% sucrose solutions ad libidum from emergence. Both small and large mosquitoes increased their glycogen and sugar levels significantly by feeding on blood only or on blood plus sugar when they imbibed a human blood meal on day 2 after emergence. Mosquitoes only fed blood on day 2 had the highest lipid levels of any treatment group. Both size classes and all feeding regimes failed to increase the total amount of glycogen, lipid, or sugar when they fed on blood 5 d after emergence. We conclude that there is an energetic advantage to Ae. aegypti when they feed on blood early in adult life (< or = day 2 after emergence).