Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion

Herpes simplex virus entry into cells requires four glycoproteins, gB, gD, gH, and gL. Binding of gD to one of its receptors triggers steps requiring the core fusion proteins, gB and the gH/gL heterodimer. There is evidence that gH/gL initiates hemifusion of cells, but whether this complex interacts physically with gB to cause complete fusion is unknown. We used bimolecular complementation (BiMC) of enhanced yellow fluorescent protein (EYFP) to detect glycoprotein interactions during cell-cell fusion. The N- or C-terminal half of EYFP was fused to the C terminus of gD, gB, and gH to form six chimeric proteins (Dn, Dc, Bn, Bc, Hn, and Hc). BiMC was detected by confocal microscopy. Receptor-bearing (C10) cells cotransfected with Dn and Bc or Dn, Hc, and untagged gL exhibited EYFP fluorescence, indicative of interactions between gD and gB and between gD and gH/gL. EYFP complementation did not occur in cells transfected with gL, Bc, and Hn. However, when gD was coexpressed with these other three proteins, cell-cell fusion occurred and the syncytia exhibited bright EYFP fluorescence. To separate glycoprotein expression from fusion, we transfected C10 cells with gL, Bc, and Hn for 20 h and then added soluble gD to trigger fusion. We detected fluorescent syncytia within 10 min, and both their number and size increased with exposure time to gD. Thus, when gD binds its receptor, the core fusion machinery is triggered to form a multiprotein complex as a step in fusion and possibly virus entry.     

Novel hepatic progenitor cell surface markers in the adult rat liver

Hepatic progenitor/oval cells appear in injured livers when hepatocyte proliferation is impaired. These cells can differentiate into hepatocytes and cholangiocytes and could be useful for cell and gene therapy applications. In this work, we studied progenitor/oval cell surface markers in the liver of rats subjected to 2-acetylaminofluorene treatment followed by partial hepatectomy (2-AAF/PH) by using rat genome 230 2.0 Array chips and subsequent RT-PCR, immunofluorescent (IF), immunohistochemical (IHC) and in situ hybridization (ISH) analyses. We also studied expression of the identified novel cell surface markers in fetal rat liver progenitor cells and FAO-1 hepatoma cells. Novel cell surface markers in adult progenitor cells included tight junction proteins, integrins, cadherins, cell adhesion molecules, receptors, membrane channels and other transmembrane proteins. From the panel of 21 cell surface markers, 9 were overexpressed in fetal progenitor cells, 6 in FAO-1 cells and 6 are unique for the adult progenitors (CD133, claudin-7, cadherin 22, mucin-1, ros-1, Gabrp). The specificity of progenitor/oval cell surface markers was confirmed by ISH and double IF analyses. Moreover, study of progenitor cells purified with Ep-CAM antibodies from D-galactosamine injured rat liver, a noncarcinogenic model of progenitor cell activation, verified that progenitor cells expressed these markers. CONCLUSION: We identified novel cell surface markers specific for hepatic progenitor/oval cells, which offers powerful tool for their identification, isolation and studies of their physiology and pathophysiology. Our studies also reveal the mesenchymal/epithelial phenotype of these cells and the existence of species diversity in the hepatic progenitor cell identity.     

Sixty Day Continuous Use of Subdermal Wire Electrodes for EEG Monitoring During Treatment of Status Epilepticus

Introduction  

Use of continuous EEG in the ICU setting is increasing. The EEG electrode continues to be a weak link in the chain from recording to interpretation. The technical difficulties of maintaining artifact-free, low impedance data collection are magnified by the ICU environment and prolonged duration of monitoring often required for these patients.     

Public health educators' participation in teams: implications for preparation and practice

Collaboration among public health organizations is essential to ensuring the health of the public. Much of the day-to-day work of public health educators is done in groups or teams or in consultation with others. This study examined the extent of health educators' work in teams as a proxy for collaboration. Health educators participated in an average of four teams per individual; three of these were interorganizational teams. Moreover, 40% of the respondents participated in five or more teams. Health educators supervised by other health educators were more likely to work in interorganizational teams than were those supervised by other professionals. Certified Health Education Specialists were more likely to participate in intraorganizational teams. Curricula in academic programs should reflect the extensive teamwork in which health educators are involved. Employers need to provide health educators with grounding in organizational priorities and support to carry out their collaborative work.     

Correction of phenotype in a thalassemia mouse model using a nonmyeloablative marrow transplantation regimen.

Gene therapy, the replacement of normal human beta- or gamma-globin genes into the hematopoietic stem cells of patients with homozygous beta-thalassemia, is a promising therapy for the future. High-level lineage-specific stable globin expression in transduced cells reinfused into patients in an autologous transplantation setting could be curative, if successful. Previous studies have shown high-level donor chimerism in nonmyeloablated non-thalassemic hosts. We have now studied the conditions for stable long-term engraftment of normal cells into a thalassemia mouse model that lead to high-level donor chimerism and correction of the abnormal phenotype. Thalassemic female mice treated with 0 to 300 cGy whole-body irradiation received transplantations of donor cells harvested from wild-type males. Engraftment of male cells was quantitated by Y-chromosome polymerase chain reaction analysis of blood and marrow progenitors, and changes in hemoglobin levels, red cell morphology, and spleen size were measured at various times posttransplantation. High-level stable donor cell engraftment was achieved in mice given 200 cGy and receiving transplants of 2 x 10(7) or more donor cells. The anemia, abnormal peripheral blood smears, and splenomegaly improved in the thalassemic mice that had successful engraftment. These studies demonstrate that stable and successful levels of engraftment of normal cells can correct the thalassemic phenotype without fully myeloablating the host. This animal model should allow us to test the amount of cytoreduction required and the level of engraftment and beta-globin expression needed in autologous transplantation of beta-globin gene-transduced cells to correct the abnormal phenotype in thalassemic mice, and it may be relevant to human clinical trials, as well.     

Validation of the EntericBio Panel II® multiplex polymerase chain reaction system for detection of Campylobacter spp., Salmonella spp., Shigella spp., and verotoxigenic E. coli for use in a clinical diagnostic setting

A total of 717 faeces samples were tested prospectively using the EntericBio Panel II® detection system (Serosep, Limerick, Ireland), in parallel with routine laboratory testing, which combines the EntericBio® system with retrospective culture of each specimen where a target is detected. Discrepancy analysis was conducted using molecular methods. The EntericBio Panel II® assay produced 585 negative and 132 positive results, namely, Campylobacter spp. (n = 66); SLT 1 and/or SLT 2 (n = 64); Salmonella spp. (n = 5); and Shigella spp. (n = 0). Three samples were positive for more than 1 target. Of these results, 4 Campylobacter spp. detections and 4 SLT 1/ SLT 2 detections remained unconfirmed, and the system failed to detect 2 Campylobacter spp. targets detected by routine laboratory detection. The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency were calculated to be 98.4%, 98.7%, 93.9%, 99.7%, and 98.6%, respectively.     

The dosimetric impact of supraclavicular nodal irradiation on the thyroid gland in patients with breast cancer

PurposeThe thyroid is not routinely considered an organ at risk in supraclavicular (SC) nodal radiation therapy (RT) for breast cancer. We compared the dosimetric impact of the following 2 RT planning techniques on the thyroid: (1) conventional single anterior field to encompass the SC nodal volume defined clinically; and (2) 3-dimensional conformal radiation therapy (3DCRT) planning to encompass the computed tomography (CT)-contoured SC nodal volume.Methods and MaterialsThe thyroid, SC nodal volumes, and organs at risk were contoured on the planning CT of 20 patients who received 50 Gy in 2-Gy daily fractions to the breast or chest wall, and SC nodes. Comparisons of dosimetric parameters between the techniques were performed: thyroid, mean and maximum dose, V5, V30, and V50 (percentage of thyroid receiving ≥ 5 Gy, ≥ 30 Gy, and ≥ 50 Gy, respectively); SC nodal volume, homogeneity index (HI, percentage volume receiving 95%-107% of prescribed dose); and maximum doses of spinal cord and brachial plexus. Anatomic characteristics that influenced the dose distributions were investigated.ResultsThe 3DCRT planning technique significantly increased all thyroid dosimetric measures (mean dose 17.2 Gy vs 26.7 Gy; maximum dose 48.5 Gy vs 51.9 Gy; V5 45.7% vs 64.9%; V30 33.7% vs 48%; and V50 0.6% vs 26.7%; P < .001). It improved HI for the SC nodal volumes (P < .001) but resulted in higher maximum doses to the spinal cord (6.1 Gy vs 30 Gy) and brachial plexus (43.2 Gy vs 51.4 Gy). The thyroid volume and depth of SC nodes did not influence the thyroid dose distribution. The depth of SC nodes impacted on the HI of SC nodal volumes in the conventional technique (P = .004).ConclusionsThe 3DCRT planning improved dosimetric coverage of the SC nodal volume but increased thyroid radiation doses. The potential adverse effects of incidental thyroid irradiation should be considered while improving dosimetric coverage in SC nodal irradiation for breast cancer.