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HCMV infection in pregnant women continues to be the leading cause of virus-induced neurologic birth defects. The mechanism through which congenital HCMV (cCMV) infection induces pathological changes to the developing fetal central nervous system (CNS) remains unclear.

Nidogen 1 (NID1) is an important basement membrane protein secreted by many cell types. We previously found that human cytomegalovirus (HCMV) infection rapidly induced chromosome 1 breaks and that the basement membrane protein NID1, encoded near the 1q42 break site, was downregulated. We have now determined that the specific breaks in and of themselves did not regulate NID1, rather interactions between several viral proteins and the cellular machinery and DNA regulated NID1. We screened a battery of viral proteins present by 24 hours postinfection (hpi) when regulation was induced, including components of the incoming virion and immediate early (IE) proteins. Adenovirus (Ad) delivery of the tegument proteins pp71 and UL35 and the IE protein IE1 influenced steady-state (ss) NID1 levels. IE1’s mechanism of regulation was unclear, while UL35 influenced proteasomal regulation of ss NID1. Real-time quantitative PCR (RT-qPCR) experiments determined that pp71 downregulated NID1 transcription. Surprisingly, WF28-71, a fibroblast clone that expresses minute quantities of pp71, suppressed NID1 transcription as efficiently as HCMV infection, resulting in the near absence of ss NID1. Sequence analysis of the region surrounding the 1q42 break sites and NID1 promoter revealed CCCTC-binding factor (CTCF) binding sites. Chromatin immunoprecipitation experiments determined that pp71 and CTCF were both bound at these two sites during HCMV infection. Expression of pp71 alone replicated this binding. Binding was observed as early as 1 hpi, and colocalization of pp71 and CTCF occurred as quickly as 15 min postinfection (pi) in infected cell nuclei. In fibroblasts where CTCF was knocked down, Adpp71 infection did not decrease NID1 transcription nor ss NID1 protein levels. Our results emphasize another aspect of pp71 activity during infection and identify this viral protein as a key contributor to HCMV’s efforts to eliminate NID1. Further, we show, for the first time, direct interaction between pp71 and the cellular genome. IMPORTANCE We have found that human cytomegalovirus (HCMV) utilizes multiple viral proteins in multiple pathways to regulate a ubiquitous cellular basement membrane protein, nidogen-1 (NID1). The extent of the resources and the redundant methods that the virus has evolved to affect this control strongly suggest that its removal provides a life cycle advantage to HCMV. Our discoveries that one of the proteins that HCMV uses to control NID1, pp71, binds directly to the cellular DNA and can exert control when present in vanishingly small quantities may have broad implications in a wide range of infection scenarios. Dysregulation of NID1 in an immunocompetent host is not known to manifest complications during infection; however, in the naive immune system of a developing fetus, disruption of this developmentally critical protein could initiate catastrophic HCMV-induced birth defects.

Digital fringe projection is a surface-profiling technique used for highly accurate non-contact measurements. As with any measurement technique, a variety of sources degrade to the measurement accuracy of the method. This paper presents an analytically-derived probability density function that explicitly models the surface height measurement error due to inevitable phase measurement error, and it includes the specific case of pixel noise inducing the phase measurement error that ultimately leads to the height estimation error. The accuracy of the model was validated through Monte-Carlo simulations of resultant height distributions subject to arbitrarily correlated pixel intensity noise and experimental digital fringe projection measurements where the pixel-by-pixel height uncertainty estimations were compared to the predictions of the derived model.

In 2000, we reported that human cytomegalovirus (HCMV) induced specific damage on chromosome 1. The capacity of the virus to induce DNA breaks indicated potent interaction between viral proteins and these loci. We have fine mapped the 1q42 breaksite. Transcriptional analysis of genes encoded in close proximity revealed virus-induced downregulation of a single gene, nidogen 1 (NID1). Beginning between 12 and 24 hours postinfection (hpi) and continuing throughout infection, steady-state (ss) NID1 protein levels were decreased in whole-cell lysates and secreted supernatants of human foreskin fibroblasts. Addition of the proteasomal inhibitor MG132 to culture medium stabilized NID1 in virus-infected cells, implicating infection-activated proteasomal degradation of NID1. Targeting of NID1 via two separate pathways highlighted the virus’ emphasis on NID1 elimination. NID1 is an important basement membrane protein secreted by many cell types, including the endothelial cells (ECs) lining the vasculature. We found that ss NID1 was also reduced in infected ECs and hypothesized that virus-induced removal of NID1 might offer HCMV a means of increased distribution throughout the host. Supporting this idea, transmigration assays of THP-1 cells seeded onto NID1-knockout (KO) EC monolayers demonstrated increased transmigration. NID1 is expressed widely in the developing fetal central and peripheral nervous systems (CNS and PNS) and is important for neuronal migration and neural network excitability and plasticity and regulates Schwann cell proliferation, migration, and myelin production. We found that NID1 expression was dramatically decreased in clinical samples of infected temporal bones. While potentially beneficial for virus dissemination, HCMV-induced elimination of NID1 may underlie negative ramifications to the infected fetus. IMPORTANCE We have found that HCMV infection promotes the elimination of the developmentally important basement membrane protein nidogen 1 (NID1) from its host. The virus both decreased transcription and induced degradation of expressed protein. Endothelial cell (EC) secretion of basement membrane proteins is critical for vascular wall integrity, and infection equivalently affected NID1 protein levels in these cells. We found that the absence of NID1 in an EC monolayer allowed increased transmigration of monocytes equivalent to that observed after infection of ECs. The importance of NID1 in development has been well documented. We found that NID1 protein was dramatically reduced in infected inner ear clinical samples. We believe that HCMV’s attack on host NID1 favors viral dissemination at the cost of negative developmental ramifications in the infected fetus.

Positive-feedback mechanisms such as fatigue induce a self-accelerating behavior, captured by models displaying infinite limit-state asymptotics, collectively known as the failure forecast method (FFM). This paper presents a Bayesian model parameter estimation approach to the fully nonlinear FFM implementation and compares the results to the classic linear regression formulation, including a regression uncertainty model. This process is demonstrated in a cyclic loading fatigue crack propagation application, both on a synthetic data set and on a full fatigue experiment. A novel ”switch point” parameter is included in the Bayesian formulation to account for nonstationary changes in the growth parameter.

Additive manufacturing is a technology transforming traditional production timelines. Specifically, metal additive manufacturing (MAM) has been increasingly adopted by a variety of industries, not only to prototype, but also to fulfill full production scale applications with much lower lead times. Like any maturing manufacturing technology, developments in verifying and validating processes are necessary to support continuous growth. Due to the complex nature of MAM, part quality and repeatability remain integral challenges that inhibit further adoption of MAM for critical component production. In this study, we present data taken from a developing in-process monitoring system designed to measure and detect powder bed defects (PBDs) in powder bed fusion MAM systems using surface height maps created with structured light illumination. We showcase the feasibility of the monitoring technique for in-process implementation by detecting streak PBDs with varying severities (height, width) created in a lab environment. We present results of powder bed measurements for varying experimental parameters of the structured light system such as illumination angle, illumination pattern, and number of illuminations. We also present an expression used to determine experimental height noise based on input parameters for PBD detection based on the instrument transfer function of the structured light monitoring system for arbitrary pixel intensity noise contributions. With the results of PBD detection across across multiple experimental measurement parameters, we provide a best practices approach to in-process implementation of the monitoring system in powder bed fusion manufacturing.

Congenital human cytomegalovirus (HCMV) infection causes a broad spectrum of central and peripheral nervous system disorders, ranging from microcephaly to hearing loss. These ramifications mandate the study of virus-host interactions in neural cells. Neural progenitor cells are permissive for lytic infection. We infected two induced pluripotent stem cell (iPSC) lines and found these more primitive cells to be susceptible to infection but not permissive. Differentiation of infected iPSCs induced de novo expression of viral antigens. iPSCs can be cultured in three dimensions to generate cerebral organoids, closely mimicking in vivo development. Mock- or HCMV-infected iPSCs were subjected to a cerebral organoid generation protocol. HCMV IE1 protein was detected in virus-infected organoids at 52 days postinfection. Absent a significant effect on organoid size, infection induced regions of necrosis and the presence of large vacuoles and cysts. Perhaps more in parallel with the subtler manifestations of HCMV-induced birth defects, infection dramatically altered neurological development of organoids, decreasing the number of developing and fully formed cortical structure sites, with associated changes in the architectural organization and depth of lamination within these structures, and manifesting aberrant expression of the neural marker β-tubulin III. Our observations parallel published descriptions of infected clinical samples, which often contain only sparse antigen-positive foci yet display areas of focal necrosis and cellular loss, delayed maturation, and abnormal cortical lamination. The parallels between pathologies present in clinical specimens and the highly tractable three-dimensional (3D) organoid system demonstrate the utility of this system in modeling host-virus interactions and HCMV-induced birth defects. IMPORTANCE Human cytomegalovirus (HCMV) is a leading cause of central nervous system birth defects, ranging from microcephaly to hearing impairment. Recent literature has provided descriptions of delayed and abnormal maturation of developing cortical tissue in infected clinical specimens. We have found that infected induced pluripotent stem cells can be differentiated into three-dimensional, viral protein-expressing cerebral organoids. Virus-infected organoids displayed dramatic alterations in development compared to those of mock-infected controls. Development in these organoids closely paralleled observations in HCMV-infected clinical samples. Infection induced regions of necrosis, the presence of larger vacuoles and cysts, changes in the architectural organization of cortical structures, aberrant expression of the neural marker β-tubulin III, and an overall reduction in numbers of cortical structure sites. We found clear parallels between the pathologies of clinical specimens and virus-infected organoids, demonstrating the utility of this highly tractable system for future investigations of HCMV-induced birth defects.

Our electron microscopy study (Kuan et al., 2016) found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introduction of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion.

Human Cytomegalovirus (HCMV) infection is compromised in cells lacking p53, a transcription factor that mediates cellular stress responses. In this study we have investigated compromised functional virion production in cells with p53 knocked out (p53KOs). Infectious center assays found most p53KOs released functional virions. Analysis of electron micrographs revealed modestly decreased capsid production in infected p53KOs compared to wt. Substantially fewer p53KOs displayed HCMV-induced infoldings of the inner nuclear membrane (IINMs). In p53KOs, fewer capsids were found in IINMs and in the cytoplasm. The deficit in virus-induced membrane remodeling within the nucleus of p53KOs was mirrored in the cytoplasm, with a disproportionately smaller number of capsids re-enveloped. Reintroduction of p53 substantially recovered these deficits. Overall, the absence of p53 contributed to inhibition of the formation and function of IINMs and re-envelopment of the reduced number of capsids able to reach the cytoplasm.

Digital fringe projection is a surface-profiling technique that is gaining popularity due to the increasing availability and quality of low-cost projection equipment and digital cameras. Noise in the pixel field of imaged targets induces error in the reconstructed phase and ultimately the surface profile measurement. In this paper, we present an approximate analytical probability density function for the estimated phase given an arbitrarily-correlated Gaussian pixel noise structure. This probability density function can be used to estimate the single point phase measurement uncertainty from easily obtainable pixel intensity noise statistics. We confirm the accuracy of the new model by comparing it to a Monte-Carlo simulation of the phase distribution. A complimentary graphics model is proposed which simulates the physical process of full-field phase measurement using a pin-hole camera model and three-dimensional point clouds of the measurement surface, allowing for another level of model verification.

The adherens junction couples the actin cytoskeletons of neighboring cells to provide the foundation for multicellular organization. The core of the adherens junction is the cadherin-catenin complex that arose early in the evolution of multicellularity to link cortical actin to intercellular adhesions. Over time, evolutionary pressures have shaped the signaling and mechanical functions of the adherens junction to meet specific developmental and physiological demands. Evolutionary rate covariation (ERC) identifies genes with correlated fluctuations in evolutionary rate that can reflect shared selective pressures and functions. Here we use ERC to identify genes with evolutionary histories similar toshotgun (shg), which encodes the Drosophila E-cadherin (DE-Cad) ortholog. Core adherens junction components α-catenin and p120-catenin displayed strong ERC correlations withshg, indicating that they evolved under similar selectivepressures during evolution between Drosophila species. Further analysis of theshgERC profile revealed a collection of genes not previously associated withshgfunction or cadherin-mediated adhesion. We then analyzed the function of a subset of ERC-identified candidate genes by RNAi during border cell (BC) migration and identified novel genes that function to regulate DE-Cad. Among these, we found that the geneCG42684, which encodes a putative GTPase activating protein (GAP), regulates BC migration and adhesion. We namedCG42684 raskol(“to split” in Russian) and show that it regulates DE-Cad levels and actin protrusions in BCs. We propose that Raskol functions with DE-Cad to restrict Ras/Rho signaling and help guide BC migration. Our results demonstrate that a coordinated selective pressure has shaped the adherens junction and this can be leveraged to identify novel components of the complexes and signaling pathways that regulate cadherin-mediated adhesion.Author SummaryThe establishment of intercellular adhesions facilitated the genesis of multicellular organisms. The adherens junction, which links the actin cytoskeletons of neighboring cells, arose early in the evolution of multicellularity and selective pressures have shaped its function and molecular composition over time. In this study, we used evolutionary rate covariation (ERC) analysis to examine the evolutionary history of the adherens junction and to identify genes that coevolved with the adherens junction geneshotgun, which encodes the Drosophila E-cadherin (DE-Cad). ERC analysis of shotgunrevealed a collection of genes with similar evolutionary histories. We then tested the role of these genes in border cell migration in the fly egg chamber, a process that requires the coordinated regulation of cell-cell adhesion and cell motility. Among these, we found that a previously uncharacterized geneCG42684, which encodes a putative GTPase activating protein (GAP), regulates the collective cell migration of border cells, stabilizes cell-cell adhesions and regulates the actin dynamics. Our results demonstrate that components of the adherens junction share an evolutionary history and that ERC analysis is a powerful method to identify novel components of cell adhesion complexes inDrosophila.

This paper explores the use of a steady-state scanning laser Doppler vibrometer (LDV) system for the identification of transition areas between solid, liquid, and gaseous substances in an enclosed container. This technique images lateral surface velocity under the excitation of a single-frequency ultrasonic tone, produced by a piezoelectric actuator. Differences in measured spatial wavenumber at discrete measurement points of a surface scan can be used to detect the boundaries between solid, liquid and gaseous regions of material. We used the LDV system to compare the relative distributions of solid wax, liquid wax, and air in a cylindrical container based on local changes in wavenumber. Through the same methodology, we were able to distinguish the transition between solid and liquid epoxy in a container. Finally, by repeatedly scanning the container during a phase-changing reaction within the container, we established that the system can be used to monitor reactions as they progress.

The adherens junction couples the actin cytoskeletons of neighboring cells to provide the foundation for multicellular organization. The core of the adherens junction is the cadherin-catenin complex that arose early in the evolution of multicellularity to link actin to intercellular adhesions. Over time, evolutionary pressures have shaped the signaling and mechanical functions of the adherens junction to meet specific developmental and physiological demands. Evolutionary rate covariation (ERC) identifies proteins with correlated fluctuations in evolutionary rate that can reflect shared selective pressures and functions. Here we use ERC to identify proteins with evolutionary histories similar to the Drosophila E-cadherin (DE-cad) ortholog. Core adherens junction components α-catenin and p120-catenin displayed positive ERC correlations with DE-cad, indicating that they evolved under similar selective pressures during evolution between Drosophila species. Further analysis of the DE-cad ERC profile revealed a collection of proteins not previously associated with DE-cad function or cadherin-mediated adhesion. We then analyzed the function of a subset of ERC-identified candidates by RNAi during border cell (BC) migration and identified novel genes that function to regulate DE-cad. Among these, we found that the gene CG42684, which encodes a putative GTPase activating protein (GAP), regulates BC migration and adhesion. We named CG42684 raskol ("to split" in Russian) and show that it regulates DE-cad levels and actin protrusions in BCs. We propose that Raskol functions with DE-cad to restrict Ras/Rho signaling and help guide BC migration. Our results demonstrate that a coordinated selective pressure has shaped the adherens junction and this can be leveraged to identify novel components of the complexes and signaling pathways that regulate cadherin-mediated adhesion.

We investigated whether anthropometric measurements or metabolic risk factors correlated more with vascular changes associated with obesity.One hundred never smoking subjects (71 women, 29 men) without vascular events, with blood pressure (BP)140/90 mm Hg, LDL cholesterol4 mmol/l, glucose6.2 mmol/l participated. Anthropometric measurements (body mass index (BMI), waist/hip ratio (WHR), waist circumference (WC) and Waist/height ratio WHTR) and metabolic risk factors (glucose, insulin, lipid and uric acid levels plus BP) were assessed. Subjects underwent vascular measurements (Carotid intima-media thickness (IMT) using duplex ultrasonography, vascular stiffness assessment (Augmentation Index) by applanation tonometry and brachial artery reactivity tests).Risk factors were in the 'normal distribution'. BMI, WHR, WC, WHTR correlated significantly with triglyceride, HDL, LDL, insulin, glucose, uric acid and systolic BP levels (P0.001). IMT correlated with WHTR, BMI, WC, Glucose (P0.001), Homoeostasis Model Assessment (HOMA) and cholesterol levels (P0.05). Only Age, WHTR or BMI were significant correlates of IMT in a multivariate analysis (P0.01) including WHTR or BMI, with age, sex, systolic BP, HDLc and HOMA. Augmentation Index correlated with age (P0.0001), WHTR and WC (P0.0005) but with age only in a multivariate analysis. Brachial reactivity did not correlate with any anthropometric or metabolic parameters. Anthropometric cutoff points, (BMIor =25, WCor =102 cm men,or =88 cm women, WHRor =0.9 men,or =0.8 women and WHTRor =0.5 men and women) significantly differentiated normal from abnormal metabolic and vascular measurements. The WHTR ratioor =0.5 was as reliable as the BMI cutoffor =25 in determining metabolic and vascular abnormalities. BMI and WHTR were strongly associated with 89% agreement (P0.0001).These results demonstrated that in 'healthy individuals', anthropometric parameters and metabolic risk factors correlated with each other, but anthropometric parameters were the only significant correlates of carotid IMT. A waist/height ratioor =0.5 predicts both early vascular and metabolic changes. These data support a risk factor independent vasculotrophic effect of obesity.

Previously, we reported that the absence of the ataxia telangiectasia mutated (ATM) kinase, a critical DNA damage response (DDR) signaling component for double-strand breaks, caused no change in HCMV Towne virion production. Later, others reported decreased AD169 viral titers in the absence of ATM. To address this discrepancy, human foreskin fibroblasts (HFF) and three ATM − lines (GM02530, GM05823, and GM03395) were infected with both Towne and AD169. Two additional ATM − lines (GM02052 and GM03487) were infected with Towne. Remarkably, both previous studies' results were confirmed. However, the increased number of cell lines and infections with both lab-adapted strains confirmed that ATM was not necessary to produce wild-type-level titers in fibroblasts. Instead, interactions between individual virus strains and the cellular microenvironment of the individual ATM − line determined efficiency of virion production. Surprisingly, these two commonly used lab-adapted strains produced drastically different titers in one ATM − cell line, GM05823. The differences in titer suggested a rapid method for identifying genes involved in differential virion production. In silico comparison of the Towne and AD169 genomes determined a list of 28 probable candidates responsible for the difference. Using serial iterations of an experiment involving virion entry and input genome nuclear trafficking with a panel of related strains, we reduced this list to four (UL129, UL145, UL147, and UL148). As a proof of principle, reintroduction of UL148 largely rescued genome trafficking. Therefore, use of a battery of related strains offers an efficient method to narrow lists of candidate genes affecting various virus life cycle checkpoints. IMPORTANCE Human cytomegalovirus (HCMV) infection of multiple cell lines lacking ataxia telangiectasia mutated (ATM) protein produced wild-type levels of infectious virus. Interactions between virus strains and the microenvironment of individual ATM − lines determined the efficiency of virion production. Infection of one ATM − cell line, GM05823, produced large titer differentials dependent on the strain used, Towne or AD169. This discrepancy resolved a disagreement in the literature of a requirement for ATM expression and HCMV reproduction. The titer differentials in GM08523 cells were due, in part, to a decreased capacity of AD169 virions to enter the cell and traffic genomes to the nucleus. In silico comparison of the Towne, AD169, and related variant strains' genomes was coupled with serial iterations of a virus entry experiment, narrowing 28 candidate proteins responsible for the phenotype down to 4. Reintroduction of UL148 significantly rescued genome trafficking. Differential behavior of virus strains can be exploited to elucidate gene function.

A recombinant strain of attenuated Salmonella enterica serovar Typhi surface-expressing Yersinia pestis F1 antigen was generated by transforming strain BRD1116 (aroA aroC htrA) with plasmid pAH34L encoding the Y. pestis caf operon. BRD1116/pAH34L was stable in vitro and in vivo. An immunisation regimen of two intranasal doses of 1 x 10(8) cfu of BRD1116/pAH34L given intranasally to mice 7 days apart induced the strongest immune response compared to other regimens and protected 13 out of 20 mice from lethal challenge with Y. pestis. Intranasal immunisation of mice constitutes a model for oral immunisation with Salmonella vaccines in humans. Thus, the results demonstrate that attenuated strains of S. enterica serovar Typhi which express Y. pestis F1 antigen may be developed to provide an oral vaccine against plague suitable for use in humans.

The aim of this work was to investigate if the presence of Aeromonas salmonicida A-layer had a suppressive effect on the development of antibodies in Atlantic salmon to outer membrane protein antigens expressed when A. salmonicida is grown under iron-restricting conditions. Atlantic salmon were immunised with an A-layer positive (A + ) or A-layer negative (A − ) A. salmonicida iron-restricted whole cell bacterin, and were boosted 8 weeks later with homologous bacterin, heterologous bacterin, or phosphate buffered saline. Serum antibody titres to iron-restricted outer membrane protein antigens were measured for a period of 8 months. It was found that the presence of A-layer at the time of priming, or the introduction of A-layer 8 weeks post-prime, had no significant effect on the development of antibody responses to iron-restricted outer membrane protein antigens. In addition, it was found that boosting fish when antibody levels are high gave an enhanced antibody response compared to unboosted fish, and this response was characterised by both an increase in specific antibody titres and an increase in the percentage of responding fish.

The ability of peripheral blood leucocytes (PBL) of Atlantic salmon to bind immune complexes in an antibody-dependent fashion was investigated. Immune complexes were labelled with fluorescein isothiocyanate and binding of these complexes to isolated PBL was determined by flow cytometry. The data show that a high proportion (up to 65%) of PBL were capable of binding immune complexes, and this binding did not occur when immune serum was replaced with normal serum. The presence of fresh normal serum inhibited or abrogated immune complex-binding of PBL. This is the first report of high levels of immune complex receptors on leucocytes in fish, and the dependence of complex binding on the presence of antibody suggests that these receptors may be similar to Fc receptors which are widely distributed on immunocytes of mammals.

Osseous haemophilic pseudotumours are uncommon. The commonest sites of involvement are the femur and the pelvis. Trauma is the initiating factor in most reported cases and repeated bleeding into the lesion contributes to their growth. Most lesions grow slowly and are often asymptomatic. Complications include massive haemorrhage, infection and pathological fracture. We present an extremely unusual presentation where a large haemophilic pseudotumour of the pelvis extended to impinge the adjacent colon, resulting in large bowel obstruction.

Background. Differences in the epidemiology and treatment outcome of stomach cancer have led to the suggestion that in Japan, this disease may be biologically less aggressive than that found in the West. The authors compared p53b and c-erb-B2 expression, trying to identify genetic differences in Japanese compared with Western stomach cancers. Methods. Paraffin embedded formalin fixed tissues from 89 British and 89 matched Japanese patients were examined by immunohistochemistry after microwave treatment. Cases were matched for T-stage, year of surgery, and histopathologic grade. Results. Tumors from 48 British and 46 Japanese patients expressed p53, whereas those of 27 British and 28 Japanese patients expressed c-erb-B2. No significant difference in the density or distribution of protein expression was found between the two populations. The distribution of expression between diffuse and intestinal types and the proportion of cases expressing both antigens were similar in the two groups. Conclusions. p53 and c-erb-B2 are expressed in the same way in stomach carcinomas from Japanese and British patients. This study found no evidence of genetic differences in the cancers from the two countries. Cancer 1995;75:920-5.

Micrometastases have been detected by immunocytochemical means in the lymph nodes of patients with otherwise node-negative cancer of the colon and rectum. This study examines the incidence and prognostic significance of nodal micrometastases in Dukes' B carcinoma. Five hundred and fifty-nine lymph nodes from 77 cases of Dukes' B carcinoma were examined for lymph node micrometastases by immunocytochemical staining for cytokeratin AE1:AE3. Micrometastases were detected in 19 cases (25 per cent). Cell clusters were present in ten cases, the remaining nine cases displaying only single cells. The presence of micrometastases was unrelated to age (P = 0.06), sex (P = 0.32), tumour site (P = 0.37), tumour size (P = 0.67), or tumour differentiation (P = 0.66). Ten-year survival estimates by the Kaplan-Meier lifetable method was 47 per cent in patients with and without micrometastases (chi 2 = 0.35 and 1 df, P = ns). The presence of nodal micrometastases detectable only by immunocytochemistry in patients with Dukes' B colorectal cancer does not justify reassignment to a more advanced disease stage.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, largely manifested as central nervous system (CNS) disorders. The principal site of manifestations in the mouse model is the fetal brain's neural progenitor cell (NPC)-rich subventricular zone. Our previous human NPC studies found these cells to be fully permissive for HCMV and a useful in vitro model system. In continuing work, we observed that under culture conditions favoring maintenance of multipotency, infection caused NPCs to quickly and abnormally differentiate. This phenotypic change required active viral transcription. Whole-genome expression analysis found rapid downregulation of genes that maintain multipotency and establish NPCs’ neural identity. Quantitative PCR, Western blot, and immunofluorescence assays confirmed that the mRNA and protein levels of four hallmark NPC proteins (nestin, doublecortin, sex-determining homeobox 2, and glial fibrillary acidic protein) were decreased by HCMV infection. The decreases required active viral replication and were due, at least in part, to proteasomal degradation. Our results suggest that HCMV infection causes in utero CNS defects by inducing both premature and abnormal differentiation of NPCs.

Brachial artery reactivity following compression is used as a surrogate marker of endothelial function. Brachial diameter increases exceeding 10% indicate normal endothelial function. We investigated if basal brachial artery tone influenced brachial artery reactivity. One hundred never smoking, healthy, normotensive, normolipidaemic subjects (41+9 yrs, 71F, 29M) underwent brachial artery assessments. Basal, Reactive (endothelial dependant diameter change following 4minutes of cuff compression) and Post GTN (endothelial independent dilatation) diameters were recorded. Tone was calculated as the percent difference in Post GTN and Basal brachial diameters. Basal diameter 3.6+0.05mm, Reactive diameter 4.0+0.06mm, Post GTN Diameter 4.4+0.1mm There was a significant correlation between Brachial artery reactivity and basal brachial artery tone (All r=0.60, p

Human cytomegalovirus (HCMV) is a common cause of morbidity and mortality in immunocompromised and immunosuppressed individuals. During infection, HCMV is known to employ host transcription factors to facilitate viral gene expression. To further understand the previously observed delay in viral replication and protein expression in p53 knockout cells, we conducted microarray analyses of p53 +/+ and p53 −/− immortalized fibroblast cell lines. At a multiplicity of infection (MOI) of 1 at 24 h postinfection (p.i.), the expression of 22 viral genes was affected by the absence of p53. Eleven of these 22 genes (group 1) were examined by real-time reverse transcriptase, or quantitative, PCR (q-PCR). Additionally, five genes previously determined to have p53 bound to their nearest p53-responsive elements (group 2) and three control genes without p53 binding sites in their upstream sequences (group 3) were also examined. At an MOI of 1, >3-fold regulation was found for five group 1 genes. The expression of group 2 and 3 genes was not changed. At an MOI of 5, all genes from group 1 and four of five genes from group 2 were found to be regulated. The expression of control genes from group 3 remained unchanged. A q-PCR time course of four genes revealed that p53 influences viral gene expression most at immediate-early and early times p.i., suggesting a mechanism for the reduced and delayed production of virions in p53 −/− cells.

The lead time between diagnosis of Crohn's disease and presentation with a Crohn's related malignancy is generally twenty years from diagnosis. This case outlines that of a young man who presented to the emergency department with abdominal pain and was subsequently discovered to have a malignant stricture complicating underlying Crohn's disease that was previously quiescent and undiagnosed. It demonstrates that a new diagnosis of Crohn's disease does not rule out previously quiescent underlying disease and therefore risk of colrectal carcinoma.

Binding of a fluorescent-labelled soluble immune complex to different types of Atlantic salmon leucocytes was investigated using flow cytometry. Peripheral blood leucocytes (PBL) were separated into sIg+ and sIg− enriched populations by magnetic activated cell sorting, blood neutrophils were identified by electronic gating, and kidney macrophages were selected by plastic-adherence. About 60% of both sIg+ and sIg− enriched PBL, 44% of neutrophils and 34% of macrophages bound the soluble immune complexes.

1. Faecal excretion of the leucocyte primary granule component, myeloperoxidase, and of the secondary granule component, lactoferrín, were compared in inflammatory bowel disease and infective diarrhoea. 2. Faecal lactoferrín correlated with faecal myeloperoxidase in both inflammatory bowel disease (P = 0.0018; n = 32) and infective diarrhoea (P = 0.00013; n = 37), but inflammatory bowel disease was associated with a much higher faecal excretion of lactoferrín but lower excretion of myeloperoxidase than infective diarrhoea. As a consequence, the median ratio of lactoferrin/myeloperoxidase excretion (both expressed as ng/mg of protein) for inflammatory bowel disease was 7.5 (range 3.5–21.3) with similar values for ulcerative colitis (n = 18) and Crohn's disease (n = 14) compared with only 0.9 (range 0.4–2.3; P < 0.0001) for infective diarrhoea. In inflammatory bowel disease faecal lactoferrín and myeloperoxidase excretion remained increased even in clinical remission. 3. In subsequent immunohistochemical studies to assess the possible explanation for these findings, lactoferrín and myeloperoxidase were demonstrated within crypt abscesses and surface mucus, both in inflammatory bowel and in infective diarrhoea mucosal samples. There was a slight increase in the number of lactoferrin-containing cells in the mucosal samples from ulcerative colitis and in the submucosa of samples from Crohn's disease compared with infective diarrhoea, but these changes were not sufficient to account for the marked increase in faecal lactoferrín excretion in inflammatory bowel disease. 4. In all mucosal samples, including those from normal mucosa, lactoferrín was also shown to be contained within mast cells. 5. These results could best be explained by a different mechanism for leucocyte activation in inflammatory bowel disease compared with infective diarrhoea, and are compatible with selective secretion of secondary granule components, which include lactoferrín but not myeloperoxidase, as a result of leucocyte activation by N-formylated bacterial peptides in inflammatory bowel disease.

Differences in the epidemiology and treatment outcome of stomach cancer have led to the suggestion that in Japan, this disease may be biologically less aggressive than that found in the West. The authors compared p53b and c-erb-B2 expression, trying to identify genetic differences in Japanese compared with Western stomach cancers.Paraffin embedded formalin fixed tissues from 89 British and 89 matched Japanese patients were examined by immunohistochemistry after microwave treatment. Cases were matched for T-stage, year of surgery, and histopathologic grade.Tumors from 48 British and 46 Japanese patients expressed p53, whereas those of 27 British and 28 Japanese patients expressed c-erb-B2. No significant difference in the density or distribution of protein expression was found between the two populations. The distribution of expression between diffuse and intestinal types and the proportion of cases expressing both antigens were similar in the two groups.p53 and c-erb-B2 are expressed in the same way in stomach carcinomas from Japanese and British patients. This study found no evidence of genetic differences in the cancers from the two countries.

We study a combined sample of 264 star-forming, 51 composite, and 73 active galaxies using optical spectra from the Sloan Digital Sky Survey (SDSS) and mid-infrared (mid-IR) spectra from the Spitzer Infrared Spectrograph. We examine optical and mid-IR spectroscopic diagnostics that probe the amount of star formation and relative energetic contributions from star formation and an active galactic nucleus (AGN). Overall we find good agreement between optical and mid-IR diagnostics. Misclassifications of galaxies based on the SDSS spectra are rare despite the presence of dust obscuration. The luminosity of the [Ne II] 12.8 μm emission line is well correlated with the star formation rate measured from the SDSS spectra, and this holds for the star-forming, composite, and AGN-dominated systems. AGNs show a clear excess of [Ne III] 15.6 μm emission relative to star-forming and composite systems. We find good qualitative agreement between various parameters that probe the relative contributions of the AGN and star formation, including the mid-IR spectral slope, the ratio of the [Ne V] 14.3 μm to [Ne II] μm 12.8 fluxes, the equivalent widths of the 7.7 μm, 11.3 μm, and 17 μm polycyclic aromatic hydrocarbon (PAH) features, and the optical D parameter which measures the distance at which a source lies from the locus of star-forming galaxies in the optical BPT emission-line diagnostic diagram. We also consider the behavior of the three individual PAH features by examining how their flux ratios depend upon the degree of AGN dominance. We find that the PAH 11.3 μm feature is significantly suppressed in the most AGN-dominated systems.

We report a composite nodular ganglioneuroblastoma, distinctive for the presence of coarsely granular black intracellular pigment, which was histochemically indistinguishable from.melanin. Ultrastructurally, the pig-ment was resolved as numerous heterogeneous lyso-somes, containing material compatible with oxidized lipid residues, the accumulation of which may reflect. acquired lysosomal dysfunction in individual tumour cells-a tumour cell ‘storage disease’.

Venous thromboembolism (VTE) after vaginal delivery is a major cause of maternal mortality. The most recent Confidential Enquiries provided guidelines for thromboprophylaxis in vaginal deliveries. The aims of this study were (i) to determine the prevalence of risk factors for VTE in women delivering vaginally, and (ii) to implement a strategy of thromboprophylaxis in vaginal delivery. Information about risk factors for VTE and the use of postnatal thromboprophylaxis was collected prospectively from each woman delivering vaginally in our unit over a 10-week period. The Confidential Enquiries guidelines for thromboprophylaxis in vaginal deliveries were then introduced after an education programme. Women categorised as moderate or high risk for VTE were to receive enoxaparin. Adherence to the guideline was then audited over a further 10 weeks. During the initial audit, 262 women delivered vaginally. Fifty-seven (21%) of these women were categorised as either moderate or high risk for VTE, but only four women...

This third edition of O'Rourke and Boyle's Medical Ethics: Sources of Catholic Teaching is a useful and comprehensive collection of statements published, for the most part, by the central authorities of the Roman Catholic Church, the National Conference of Catholic Bishops in the United States and the bishops' conferences of individual US states on a wide range of issues in the area of medical ethics. …

Since whole rapeseed is a high energy feed source but only moderate in protein content and value it is desirable to combine it with another feedstuff such as peas so that the combined product has an improved balance of amino acids and a more favourable protein to energy ratio. Extrusion is a useful method of processing a rape:pea mixture since this results in effective release of oil from the rape and its absorption by the pea. The heat generated by extrusion could also lead to the destruction of heat labile anti-nutritive factors in both rapeseed and peas.

Frequent measurement of pH and PCO2 levels of fetal blood during labour provides an insight into the presence and nature of fetal asphyxia, effectiveness of in utero resuscitation and the need for operative delivery. A pH measuring system utilizing a hydrogen ion-selective polymer membrane built into a fetal probe suitable for making intermittent measurements on the fetal scalp during labour is described. Probe design, construction of electrodes and preliminary results obtained using the system in vitro are presented.

Summary. In a prospective study, 200 patients with accurate menstrual data, assessed clinically and b y early ultrasound, recorded their date of quickening. Fetal movements were first noted by primigravidae at a mean gestation of 19.04 weeks (SD 1.5) and by multigravidae at 17–4 weeks (SD 1.7). Since the range in both groups was wide: 15–22 weeks for primigravidae and 14–22 weeks for multigravidae, quickening was of limited value in estimating gestational age.

Several simplifications in the current realization methods for third-order paramount matrices are presented, based on classroom experiences in the teaching of resistance network synthesis.