Investigation of ICP34.5 and Its Role in HSV Pathogenicity

The aims of this project were to determine the role of two antisense genes, RL1 and ORF P in herpes simplex virus (HSV) virulence and to identify the HSV-2 strain HG52 ICP34.5 expression. The first overall aim was to define individual phenotypes associated with ICP34.5 and ORF P in HSV-1 strain 17+. HSV-1 contains an ORF in the long repeat sequences named RL1 (also called ?134.5) which expresses the polypeptide ICP34.5, One RL1 deletion variant, 1716, is avirulent upon intracranial inoculation in the central nervous system (CNS) and the peripheral nervous system (PNS). Replication of 1716 in vitro was shown to be cell type and cell state dependent. The avirulent phenotype of 1716 based on deletion of this region of the HSV-1 genome was originally attributed solely to the RL1 gene. However, transcripts in the opposite direction and antisense to RL1 in HSV-1 were identified in 1994. From these transcripts two open reading frames (ORF O and ORF P) mapped antisense to RL1. This led to the hypothesis that ORF P may be at least partly responsible for the avirulent phenotype of ICP34.5 negative mutants. 1716 was used as the backbone to generate three recombinant viruses containing either RL1 or ORF P in the nonessential UL43 gene. Two lengths of RL1 were cloned due to uncertainty about the position of the putative RL1 promoter sequence and role of the 5' untranslated leader in expression. One RL1 fragment contained only the ORF while the other fragment contained the ORF and 134 bp additional upstream sequence. The RL1 or ORF P genes were individually sub-cloned downstream of the HSV-1 gD promoter. This promoter was used because it has similar kinetics to that of the natural ICP34.5 promoter. These RL1 or ORF P cassettes also contained the lacZ gene under the SV40 promoter in the opposite orientation for detection of recombinant virus. The recombinant virus, 1622, contained only the RL1 open reading frame and demonstrated an 8-fold overexpression of ICP34.5 by Western blotting and immunoprecipitation. 1622 regained wild type replication kinetics in vitro in cell lines non- permissive for ICP34.5 negative viruses. In certain cell types, an in vitro function of ICP34.5 is to maintain protein synthesis by using a protein kinase (PKR) pathway. 1716 showed host protein synthesis shut-off in human neuroblastoma and HeLa cells confirming the previously reported data for the HSV-1 strain F ICP34.5 deletion mutant, R3616. 1622 infected cells demonstrated protein synthesis similar to 17+ by SDS-PAGE. 1622 demonstrated a small reduction in neurovirulence compared with 17+ by intracranial inoculation but was 40-fold more virulent than its parent following infection via the snout and trigeminal ganglia as assayed by in vivo replication and immunohistochemsitry. The recombinant virus, 1623, by Western blotting expressed very low levels of ICP34.5 compared to 17+. Based on 2-fold dilutions of 1623 infected BHK cell extracts, ICP34.5 is expressed eight times less than in 17+ and thus 64-fold less than in 1622. Interestingly, this low level of expression still enabled 1623 to maintain wild type levels of protein synthesis in neuroblastoma and HeLa cells. However, the replication kinetics in vitro from 1623 was slower than 1716. This suggests that there may be a second mutation elsewhere in the genome. The recombinant virus, 1624, contained ORF P under the HSV-1 gD promoter. Due to difficulties in cloning the ORF P gene, final purification and characterisation of this mutant was not carried out. However, expression of ORF P from 17+ infected cells was analysed by an antiserum from a GST/ORF P fusion protein. By Western blotting, ORF P was detected in 17+ infected cells during normal lytic infection in vitro. Cells infected with a RL1 epitope tagged virus, 1621, demonstrated expression of a slightly larger form of ORF P. The temperature sensitive virus, tsK, when grown at 39.

An integrated workflow for 2D and 3D posture analysis during vestibular system testing in mice

IntroductionPosture extraction from videos is fundamental to many real-world applications, including health screenings. In this study, we extend the utility and specificity of a well-established protocol, the balance beam, for examining balance and active motor coordination in adult mice of both sexes.ObjectivesThe primary objective of this study is to design a workflow for analyzing the postures of mice walking on a balance beam.MethodsWe developed new tools and scripts based on the FluoRender architecture, which can interact with DeepLabCut (DLC) through Python code. Notably, twenty input videos were divided into four feature point groups (head, body, tail, and feet), based on camera positions relative to the balance beam (left and right), and viewing angles (90° and 45° from the beam). We determined key feature points on the mouse to track posture in a still video frame. We extracted a standard walk cycle (SWC) by focusing on foot movements, which were computed by a weighted average of the extracted walk cycles. The correlation of each walk cycle to the SWC was used as the weight.ResultsWe learned that positions of the camera angles significantly improved the performance of 2D pose estimation (90°) and 3D (45°). Comparing the SWCs from age-matched mice, we found a consistent pattern of supporting feet on the beam. Two feet were consistently on the beam followed by three feet and another three feet in a 2-3-3 pattern. However, this pattern can be mirrored among individual subjects. A subtle phase shift of foot movement was also observed from the SWCs. Furthermore, we compared the SWCs with speed values to reveal anomalies in mouse walk postures. Some anomalies can be explained as the start or finish of the traversal, while others may be correlated to the distractions of the test environment, which will need further investigation.ConclusionOur posture analysis workflow improves the classical behavioral testing and analysis, allowing the detection of subtle, but significant differences in vestibular function and motor coordination

Persistent expression of chemokine and chemokine receptor RNAs at primary and latent sites of herpes simplex virus 1 infection

Inflammatory cytokines and infiltrating T cells are readily detected in herpes simplex virus (HSV) infected mouse cornea and trigeminal ganglia (TG) during the acute phase of infection, and certain cytokines continue to be expressed at lower levels in infected TG during the subsequent latent phase. Recent results have shown that HSV infection activates Toll-like receptor signaling. Thus, we hypothesized that chemokines may be broadly expressed at both primary sites and latent sites of HSV infection for prolonged periods of time. Real-time reverse transcriptase-polymrease chain reaction (RT-PCR) to quantify expression levels of transcripts encoding chemokines and their receptors in cornea and TG following corneal infection. RNAs encoding the inflammatory-type chemokine receptors CCR1, CCR2, CCR5, and CXCR3, which are highly expressed on activated T cells, macrophages and most immature dendritic cells (DC), and the more broadly expressed CCR7, were highly expressed and strongly induced in infected cornea and TG at 3 and 10 days postinfection (dpi). Elevated levels of these RNAs persisted in both cornea and TG during the latent phase at 30 dpi. RNAs for the broadly expressed CXCR4 receptor was induced at 30 dpi but less so at 3 and 10 dpi in both cornea and TG. Transcripts for CCR3 and CCR6, receptors that are not highly expressed on activated T cells or macrophages, also appeared to be induced during acute and latent phases; however, their very low expression levels were near the limit of our detection. RNAs encoding the CCR1 and CCR5 chemokine ligands MIP-1α, MIP-1β and RANTES, and the CCR2 ligand MCP-1 were also strongly induced and persisted in cornea and TG during the latent phase. These and other recent results argue that HSV antigens or DNA can stimulate expression of chemokines, perhaps through activation of Toll-like receptors, for long periods of time at both primary and latent sites of HSV infection. These chemokines recruit activated T cells and other immune cells, including DC, that express chemokine receptors to primary and secondary sites of infection. Prolonged activation of chemokine expression could provide mechanistic explanations for certain aspects of HSV biology and pathogenesis

WASP family members and formin proteins coordinate regulation of cell protrusions in carcinoma cells

We examined the role of the actin nucleation promoters neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE2 in cell protrusion in response to epidermal growth factor (EGF), a key regulator in carcinoma cell invasion. We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect. However, simultaneous KD of both proteins results in the formation of large jagged protrusions with lamellar properties and increased filopod formation. This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF. A mammalian Diaphanous–related formin, mDia1, localizes at the jagged protrusions in double KD cells. Constitutively active mDia1 recapitulated the phenotype, whereas inhibition of mDia1 blocked the formation of these protrusions. Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype. These data show that coordinate regulation between the WASP family and mDia proteins controls the balance between lamellar and lamellipodial protrusion activity

Spontaneous and Acetylcholine Evoked Calcium Transients in the Developing Mouse Utricle

Spontaneous calcium transients are present during early postnatal development in the mouse retina and cochlea, and play an important role in maturation of the sensory organs and neural circuits in the central nervous system (CNS). It is not known whether similar calcium transients occur during postnatal development in the vestibular sensory organs. Here we demonstrate spontaneous intracellular calcium transients in sensory hair cells (HCs) and supporting cells (SCs) in the murine utricular macula during the first two postnatal weeks. Calcium transients were monitored using a genetically encoded calcium indicator, GCaMP5G (G5), at 100 ms-frame−1 in excised utricle sensory epithelia, including HCs, SCs, and neurons. The reporter line expressed G5 and tdTomato (tdT) in a Gad2-Cre dependent manner within a subset of utricular HCs, SCs and neurons. Kinetics of the G5 reporter limited temporal resolution to calcium events lasting longer than 200 ms. Spontaneous calcium transients lasting 1-2 s were observed in the expressing population of HCs at birth and slower spontaneous transients lasting 10-30 s appeared in SCs by P3. Beginning at P5, calcium transients could be modulated by application of the efferent neurotransmitter acetylcholine (ACh). In mature mice, calcium transients in the utricular macula occurred spontaneously, had a duration 1-2 s, and could be modulated by the exogenous application of acetylcholine (ACh) or muscarine. Long-lasting calcium transients evoked by ACh in mature mice were blocked by atropine, consistent with previous reports describing the role of muscarinic receptors expressed in calyx bearing afferents in efferent control of vestibular sensation. Large spontaneous and ACh evoked transients were reversibly blocked by the inositol trisphosphate receptor (IP3R) antagonist aminoethoxydiphenyl borate (2-APB). Results demonstrate long-lasting calcium transients are present in the utricular macula during the first postnatal week, and that responses to ACh mature over this same time period

Oral contraceptive use and risk of melanoma in premenopausal women

Melanoma has been increasing in white populations. Incidence rates rise steeply in women until about age 50, suggesting oestrogen as a possible risk factor. Oestrogens can increase melanocyte count and melanin content and cause hyperpigmentation of the skin. We examined prospectively the association between oral contraceptive (OC) use and diagnoses of superficial spreading and nodular melanoma among 183 693 premenopausal white women in the Nurses’ Health Study (NHS) and the Nurses’ Health Study II (NHS II) cohorts. One hundred and forty six cases were confirmed in NHS during follow-up from 1976 to 1994, and 106 cases were confirmed in NHS II from 1989 to 1995. Skin reaction to sun exposure, sunburn history, mole counts, hair colour, family history of melanoma, parity, height and body mass index were also assessed and included in logistic regression models. A significant twofold increase in risk of melanoma (relative risk (RR) = 2.0, 95% confidence interval (CI) 1.2–3.4) was observed among current OC users compared to never users. Risk was further increased among current users with 10 or more years of use (RR = 3.4, 95% CI 1.7–7.0). Risk did not appear elevated among past OC users, even among those with longer durations of use, and risk did not decline linearly with time since last use. In conclusion, risk of premenopausal melanoma may be increased among women who are current OC users, particularly among those with longer durations of use. Further research is needed to determine whether low-dose oestrogen pills in particular are associated with an increase in risk and to describe possible interactions between OC use and sun exposure or other risk factors for melanoma. © 1999 Cancer Research Campaig

A pooled analysis of 10 case–control studies of melanoma and oral contraceptive use

Data regarding the effects of oral contraceptive use on women's risk of melanoma have been difficult to resolve. We undertook a pooled analysis of all case–control studies of melanoma in women completed as of July 1994 for which electronic data were available on oral contraceptive use along with other melanoma risk factors such as hair colour, sun sensitivity, family history of melanoma and sun exposure. Using the original data from each investigation (a total of 2391 cases and 3199 controls), we combined the study-specific odds ratios and standard errors to obtain a pooled estimate that incorporates inter-study heterogeneity. Overall, we observed no excess risk associated with oral contraceptive use for 1 year or longer compared to never use or use for less than 1 year (pooled odds ratio (pOR)=0.86; 95% CI=0.74–1.01), and there was no evidence of heterogeneity between studies. We found no relation between melanoma incidence and duration of oral contraceptive use, age began, year of use, years since first use or last use, or specifically current oral contraceptive use. In aggregate, our findings do not suggest a major role of oral contraceptive use on women's risk of melanoma

Sun exposure and melanoma risk at different latitudes: a pooled analysis of 5700 cases and 7216 controls

Background Melanoma risk is related to sun exposure; we have investigated risk variation by tumour site and latitude

Comparison of risk patterns in carcinoma and melanoma of the skin in men: a multi-centre case–case–control study

We directly compared risk factors between 214 histologically confirmed melanomas (CMM), 215 basal-cell carcinomas (BCC) and 139 squamous-cell carcinomas (SCC) in a multiple case–case–control study with 349 controls from patients without dermatological disease admitted to the same hospitals. Subjects with fair hair had a significant risk increase for all types of tumours at a comparable level (ORadj for blonde hair: CMM 2.3; SCC 2.4; BCC 2.3). The effect of pale eyes was significant and similar for CMM and BCC (ORadj 2.6). Intermittent sun exposure measured in hours spent at beach during holidays was significant for both CMM (ORadj 2.6 for more than 7000 lifelong hours) and BCC (ORadj 2.1 for more than 7000 lifelong hours), while SCC exhibited a significant risk increase for chronic exposure to sunlight measured in hours of outdoor work (ORadj 2.2 for more than 6000 lifelong hours). In the case–case comparison using a multinomial logistic regression model, we found a statistically significant risk difference for pale eyes, and number of naevi in the CMM group, compared to other skin cancers. For intermittent sun exposure, there was a significant risk difference of BCC when compared to the risk of SCC. Factors influencing risk of SCC are different, with chronic exposure to sun playing a major role in causing this type of carcinoma

New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk.

Levels of circulating glucose are tightly regulated. To identify new loci influencing glycemic traits, we performed meta-analyses of 21 genome-wide association studies informative for fasting glucose, fasting insulin and indices of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 nondiabetic participants. Follow-up of 25 loci in up to 76,558 additional subjects identified 16 loci associated with fasting glucose and HOMA-B and two loci associated with fasting insulin and HOMA-IR. These include nine loci newly associated with fasting glucose (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and C2CD4B) and one influencing fasting insulin and HOMA-IR (near IGF1). We also demonstrated association of ADCY5, PROX1, GCK, GCKR and DGKB-TMEM195 with type 2 diabetes. Within these loci, likely biological candidate genes influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Our results demonstrate that genetic studies of glycemic traits can identify type 2 diabetes risk loci, as well as loci containing gene variants that are associated with a modest elevation in glucose levels but are not associated with overt diabetes