Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers.

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA's life cycle. Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

PinMol: Python application for designing molecular beacons for live cell imaging of endogenous mRNAs.

Molecular beacons are nucleic acid oligomers labeled with a fluorophore and a quencher that fold in a hairpin-shaped structure, which fluoresce only when bound to their target RNA. They are used for the visualization of endogenous mRNAs in live cells. Here, we report a Python program (PinMol) that designs molecular beacons best suited for live cell imaging by using structural information from secondary structures of the target RNA, predicted via energy minimization approaches. PinMol takes into account the accessibility of the targeted regions, as well as the inter- and intramolecular interactions of each selected probe. To demonstrate its applicability, we synthesized an oskar mRNA-specific molecular beacon (osk1236), which is selected by PinMol to target a more accessible region than a manually designed oskar-specific molecular beacon (osk2216). We previously demonstrated osk2216 to be efficient in detecting oskar mRNA in in vivo experiments. Here, we show that osk1236 outperformed osk2216 in live cell imaging experiments.

Return on Investment Analysis of Breast Cancer Screening and Downstaging in Egypt: Implications for Developing Countries.

OBJECTIVE: The aim of this study was to perform a return-on-investment (ROI) analysis of a breast cancer screening program in Egypt by comparing net profit in treatment costs saved to program cost investment. METHODS: The breast cancer downstaging program targeted women living in an Egyptian slum, where residents have low access to health care. Program costs were estimated by using data from interviews with program administrative staff. Screening and treatment costs were estimated by using Ministry of Health medical reimbursement data. Estimates for expected rates of downstaging were modeled on the basis of data from a previous study. ROI, or relative cost savings, was calculated by comparing treatment cost savings to costs for the screening program. A baseline ROI for facility-based screening was calculated, followed by ROIs for different scenarios. RESULTS: Average per-person treatment cost for screened and unscreened patients was estimated to be $28,632 and $58,170, respectively, with a cumulative lifetime risk of 6.36%. Total screening program cost per person was $112.10. The study estimated an expected decrease in late-stage breast cancer diagnosis by 13.7% as a result of the screening program, saving $4,049 in treatment costs per individual diagnosed. The analysis resulted in a positive ROI of 133% for facility-based screening. CONCLUSIONS: Breast cancer represents an increasing burden on health care in Egypt and other developing countries. This study concludes that a community-based downstaging program resulted in a positive ROI, or overall cost-savings. The findings inform that policymakers in low-income and middle-income countries may consider implementing community-based downstaging programs for breast cancer.

Detection of Human Cytomegalovirus in Malignant and Benign Breast Tumors in Egyptian Women.

INTRODUCTION: Previous studies have reported a role for human cytomegalovirus (HCMV) in breast carcinogenesis. We sought to assess the role of HCMV infection in the development and/or progression of breast cancer (BC) among Egyptian patients. PATIENTS AND METHODS: The study included 61 patients with BC cases. Of these 61 patients, 40 had been assessed for HCMV in the blood, BC tissue samples, and adjacent non-neoplastic tissue samples, and 21 had been assessed for HCMV in the tissue only. Tissue samples from 20 patients with fibroadenoma (FA) were also included. As a control group, 41 blood samples obtained from healthy women with no history of cancer were used as a blood control group. HCMV was assessed using enzyme-linked immunosorbent assay, real-time polymerase chain reaction (PCR), and immunohistochemistry (IHC). RESULTS: A significant difference was found in the index value for the anti-CMV IgG antibodies between the BC patients and the control group (P = .001). Using real-time PCR, HCMV DNA was detected in 11 of 61 BC tissues (18%) compared with 1 of 20 FA tissues (5%). HCMV DNA was present in 8 of the 40 plasma samples (20%). Regarding the viral proteins, 21 of 61 samples (34.4%) were positive for early/immediate early (E/IE) and 49 (80.3%) were positive for PP65 expression by IHC. The concordance between the results obtained by the different assays was low. CMVPP65 expression was significantly associated with E/IE protein expression in the malignant and FA groups (P < .001). CONCLUSION: The presence of CMV proteins and DNA in BC tissues suggests a role for this virus. However, the basic criteria to support a causal association of HCMV with BC were not fulfilled.

Quantitative Assessment of Tension Reduction at the Midline Closure During Abdominal Component Separation.

BACKGROUND: Abdominal component separation is used commonly for closure of midline abdominal wounds. The value of each step in reducing tension has not been studied. Our aim was to test whether component separation decreases tension in the midline closure and to quantify the value of each procedural step. STUDY DESIGN: Tension required to bring the rectus muscle to midline was measured using tensiometry after subcutaneous dissection (step 1), external oblique muscle release (step 2), separation of the internal and external oblique muscles (step 3), and internal oblique muscle release (step 4). Measurements were taken in the upper, middle, and lower thirds of the abdominal midline. Distance to midline was also measured after each surgical step. Tension (measured as percent change) and distance were analyzed using Student's t-test with significance set at p < 0.05. RESULTS: In 41 hemi-abdominal defects, tension decreased in middle, upper, and lower thirds of the abdomen by 22.5%, 24.3%, and 34.8% after step 1; 33.4%, 31.8%, and 39.8% after step 2; 26.5%, 22.2%, and 27.4% after step 3; and 33.2%, 28.2%, and 23.5% after step 4. Mean distance change was 0.97 cm, 1.97 cm, 2.22 cm, and 2.59 cm after steps 1 to 4, respectively. CONCLUSIONS: This study shows through a quantitative measure of tension that all steps of the component separation procedure decrease wound tension to variable degrees, with the release of the external and internal oblique muscles being the more effective steps. An internal oblique release is a useful and simple adjunct to the classical component separation procedure.

Chronic Mastitis in Egypt and Morocco: Differentiating between Idiopathic Granulomatous Mastitis and IgG4-Related Disease.

Idiopathic granulomatous mastitis (IGM) is a benign, frequently severe chronic inflammatory lesion of the breast. Its etiology remains unknown and reported cases vary in their presentation and histologic findings with an optimal treatment algorithm yet to be described owing mainly to the disease's heterogeneity. IgG4-related disease (IgG4-RD) is a newly recognized systemic fibroinflammatory condition characterized by a dense lymphoplasmacytic infiltrate with many IgG4-positive plasma cells, storiform fibrosis, and obliterative phlebitis. Immunosuppressive therapy is considered to be an effective first-line therapy for IgG4-RD. We sought to clarify and classify chronic mastitis according to the histologic findings of IgG4-RD mastitis with respect to IGM and to develop a robust diagnostic framework to help select patients for optimal treatment strategies. Using the largest collection to date (43 cases from Egypt and Morocco), we show that despite sharing many features, IGM and IgG4-RD mastitis are separate diseases. To diagnostically separate the diseases, we created a classification schema-termed the Michigan Classification-based upon our large series of cases, the consensus statement on IgG4-RD, and the histologic description of IGM in the literature. Using our classification, we discerned 17 cases of IgG4-RD and 8 cases of IGM among the 43 chronic mastitis cases, with 18 indeterminate cases. Thus, our Michigan Classification can form the basis of rational stratification of chronic mastitis patients between these two clinically and histopathologically heterogeneous diseases.

Clinical utility of certain biomarkers as predictors of breast cancer with or without metastasis among Egyptian females.

The objective of this study is to explore and correlate the value of certain biomarkers in breast cancer (BC) females with and without metastasis after undergoing the surgical treatment protocol in the National Cancer Institute in Egypt. Thirty females (33-69 years), diagnosed as early breast cancer patients with or without metastasis, and 20 healthy individuals were selected for this study. The biomarkers under investigation were vascular endothelial growth factor (VEGF), C-reactive protein (CRP), interleukin-6 (IL-6), and interleukin-8 (IL-8). The correlation between these markers and the tumor grade was also evaluated. The results revealed a significant increase (p < 0.0001) in VEGF, CRP, IL-6, and IL-8 in breast cancer patients with or without metastasis as compared to the healthy group. Surgical treatment of metastatic BC females showed a significant reduction of those parameters by variable degrees, whereas BC females without metastasis recorded the most inhibition levels. Also, there was positive correlation (p < 0.0001) between those biomarkers and the tumor grades. We also noticed an association between VEGF and IL-8 as well as CRP and IL-6. In conclusion, the selected biomarkers may be beneficial for the prognosis of breast cancer and seem to be a diagnostic tool to differentiate between BC with or without metastasis. The descried surgical treatment protocol succeeded to attenuate the elevated biomarker levels and improve patient survival which deserves more extensive studies.

Risk factors for chronic mastitis in morocco and egypt.

Chronic mastitis is a prolonged inflammatory breast disease, and little is known about its etiology. We identified 85 cases and 112 controls from 5 hospitals in Morocco and Egypt. Cases were women with chronic mastitis (including periductal, lobular, granulomatous, lymphocytic, and duct ectasia with mastitis). Controls had benign breast disease, including fibroadenoma, benign phyllodes, and adenosis. Both groups were identified from histopathologically diagnosed patients from 2008 to 2011, frequency-matched on age. Patient interviews elicited demographic, reproductive, breastfeeding, and clinical histories. Cases had higher parity than controls (OR = 1.75, 1.62-1.90) and more reported history of contraception use (OR = 2.73, 2.07-3.61). Cases were less likely to report wearing a bra (OR = 0.56, 0.47-0.67) and less often used both breasts for breastfeeding (OR = 4.40, 3.39-5.72). Chronic mastitis cases were significantly less likely to be employed outside home (OR = 0.71, 0.60-0.84) and more likely to report mice in their households (OR = 1.63, 1.36-1.97). This is the largest case-control study reported to date on risk factors for chronic mastitis. Our study highlights distinct reproductive risk factors for the disease. Future studies should further explore these factors and the possible immunological and susceptibility predisposing conditions.

Pseudorabies virus and herpes simplex virus type 1 utilize different tegument-glycoprotein interactions to mediate the process of envelopment.

BACKGROUND AND OBJECTIVE: During herpesvirus envelopment capsids, tegument polypeptides and membrane proteins assemble at the site of budding, and a cellular lipid bilayer becomes refashioned into a spherical envelope. A web of interactions between tegument proteins and the cytoplasmic tails of viral glycoproteins play a critical role in this process. We have previously demonstrated that for herpes simplex virus (HSV)-1 the cytoplasmic tail of glycoprotein H (gH) binds the tegument protein VP16. The HSV and pseudorabies virus (PRV) genomes are essentially collinear, and individual gene products show significant sequence homology. However, the demarcation of function often differs between PRV and HSV proteins. The goal of this study was to determine whether PRV gH and VP16 interact in a manner similar to their homologs in HSV. METHODS: A fusion protein pull-down assay was performed in which a PRV gH cytoplasmic tail-glutathione S-transferase fusion protein, bound to glutathione-Sepharose beads, was incubated with PRV-infected cell cytosol, washed and subjected to Western blot analysis using anti-PRV VP16 antisera. RESULTS: Western blots indicate that PRV VP16 does not specifically bind to the PRV gH tail. CONCLUSION: Our results highlight that, despite the relatively close evolutionary relationship between HSV and PRV, there are significant differences in their protein interactions that drive envelopment.

Elastography ultrasound and questionable breast lesions: does it count?

OBJECTIVE: To check possible additional value of using elastography ultrasound in the specification of questionable breast lesions. SUBJECTS AND METHODS: Questionable breast lesions on gray scale ultrasound examination had been further evaluated by elastography ultrasound in 97 cases with median age of 42 years. The studied lesions were pathologically proven (58 benign and 39 malignant) using true cut tissue/surgical excision biopsy that was considered the gold standard of reference. RESULTS: Conventional ultrasound categorization before biopsy included: category 3 (probably benign) in 42.3% (n=41), category 4a (low suspicion of malignancy) in 13.4% (n=13), category 4b (intermediate suspicion of malignancy) in 16.5% (n=16) and category 4c (moderate suspicion of malignancy) in 27.8% (n=27). We had evaluated elastography ultrasound regarding elastography strain scoring and quantitative strain ratio. Sensitivity, specificity and accuracy were 89.7%, 86.2% and 87.6% for conventional ultrasound, 92.3%, 74.1% and 81.4% for elastogram 5-point scoring method and 87.1%, 89.6% and 88.6% for the calculated strain ratios respectively in the assessment of the examined breast lesions. CONCLUSION: Ultrasound elastography, using both qualitative and quantitative methods can improve the performance of conventional B-mode ultrasound and enhance its specificity and accuracy in the diagnosis of questionable (BI-RADS categories 3 and 4) breast lesions.

Attitudes of Cairo University medical students toward smoking: the need for tobacco control programs in medical education.

BACKGROUND: Tobacco smoking rates are increasing in developing countries and so are tobacco-related chronic diseases. Reported figures from the WHO show rates of smoking in Egypt as high as 20% but limited information is available about smoking specifically among physicians and medical students. MATERIALS AND METHODS: Final-year medical students of Cairo University were surveyed regarding their tobacco behavior and attitudes using a modified Global Health Professions Student Survey. We approached 220 students by randomly selecting clinical units into which they were assigned and requested completion of the survey. RESULTS: Ever users of some form of tobacco comprised 46.7% of students sampled, current users of cigarettes comprised 17.4%, and current users of water pipe 'sheesha' comprised 17.6%. The vast majority (87.7%) of students believed that smoking is a public health problem in Cairo and supported restriction of tobacco. Yet, only 58.5% stated that they were taught it is important for physicians to provide tobacco education materials to patients. Among ever users of cigarettes, 54.4% believed health professionals do not serve as health role models for patients, and only a small percentage of all students (34.2%) stated that they had received some form of training on smoking cessation in their medical curriculum to be able to instruct patients. CONCLUSION AND RECOMMENDATIONS: A high rate of smoking was revealed among medical students in Cairo. Overall, approximately 23.4% of students were currently smoking cigarettes and/or sheesha, and 46.7% were ever users of some form of tobacco. A formal antitobacco program for medical students should be incorporated into their medical curriculum to change the attitudes of medical students and overcome the anticipated increase in chronic diseases in Egypt.

Split liver transplantation for hepatocellular carcinoma.

BACKGROUND/AIMS: Liver transplantation (LTx) is recognized as the treatment of choice for small hepatocellular carcinomas (HCC) in patients with end-stage liver failure. However, because of limited organ availability, not all those who qualify can benefit from full-size LTx. METHODOLOGY: Over a 28-month period, we transplanted 6 deceased donor split liver allografts in stable recipients with HCC and cirrhosis. RESULTS: There were 5 men and one woman with a median age of 60 years. Median waiting time to LTx was 82 days. Extended right split grafts (segments I, IV-VIII) were transplanted in 5 patients. One patient received a right split graft. In situ harvesting was performed in 4 instances and ex situ in the remaining 2. Median cold ischemia time was 10 hours. Primary non-function was not observed. Median intensive care unit stay was 4 days. There were neither vascular nor biliary complications. The postoperative course was uneventful in half of the patients. Two patients were re-operated because of an abscess caused by a liver necrosis in the marginal zone of segment IV. One patient died for reasons unrelated to liver function or to the surgical intervention. The remaining 5 patients are alive after a median follow-up of 20 months. CONCLUSIONS: Deceased donor split LTx constitutes an additional option for patients with HCC and cirrhosis. The potential risks of using "split livers" as well as the potential benefits of transplanting patients unlikely to survive the waiting list period must be evaluated on an individual basis.