Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

<p>Abstract</p> <p>Background</p> <p>The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings.</p> <p>Results</p> <p>After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2), it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence.</p> <p>Conclusion</p> <p>We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development.</p

Detection of label-free cancer biomarkers using nickel nanoislands and quartz crystal microbalance

We present a technique for the label-free detection and recognition of cancer biomarkers using metal nanoislands intended to be integrated in a novel type of nanobiosensor. His-tagged (scFv)-F7N1N2 is the antibody fragment which is directly immobilized, by coordinative bonds, onto ~5 nm nickel islands, then deposited on the surface of a quartz crystal of a quartz crystal microbalance (QCM) to validate the technique. Biomarker GTPase RhoA was investigated because it has been found to be overexpressed in various tumors and because we have recently isolated and characterized a new conformational scFv which selectively recognizes the active form of RhoA. We implemented a surface chemistry involving an antibiofouling coating of polyethylene glycol silane (PEG-silane) (<2 nm thick) and Ni nanoislands to reach a label-free detection of the active antigen conformation of RhoA, at various concentrations. The methodology proposed here proves the viability of the concept by using Ni nanoislands as an anchoring surface layer enabling the detection of a specific conformation of a protein, identified as a potential cancer biomarker. Hence, this novel methodology can be transferred to a nanobiosensor to detect, at lower time consumption and with high sensitivity, specific biomolecules

Foetal instrumental extractions (IE) at the maternity at Nabil Choucair Health Center (Senegal) from 2005 to 2016: epidemio-clinical and prognostic aspects

Background: The main objective was to take stock of practices on instrumental extractions at the maternity of Nabil Choucair Health Center.Methods: This was a prospective, descriptive study from 1 January 2005 to 31 April 2016. The collection was carried out through the data sheet completed on the basis of an analysis of files, the delivery register and the anaesthesia register of the operating room. The data was entered using the Sphinx software version 5 and the data analysis performed by Epi info version 3.5.Results: 240 instrumental extraction cases were compiled, and their frequency was 0.4%. The mean age of patients was 24.8 years. Obstetric vacuum was the most commonly used instrument (66.4%) and an episiotomy was performed in 73.6%. Maternal complications were dominated by simple perineal tears (6.2%).Conclusions: Instrumental extractions should be rehabilitated in our maternity facilities to combat the outbreak of the caesarean section. Instrumental extractions indications and techniques should be controlled to avoid complication

Practice of diagnostic hysteroscopy after myomectomy in the prevention of intrauterine adhesions: experience of Ouakam military hospital (Dakar, Senegal)

Background: Surgical treatment of myomas may be the cause of some complications, including intrauterine adhesions. The objective of the study was to evaluate the incidence of intrauterine adhesions in patients who underwent myomectomy by laparotomy or hysteroscopic resection.Methods: We carried out a prospective single center study from August 1st 2016 to May 31st 2017 in the gynaecological and obstetrical department of Ouakam the Military Hospital (Senegal). We included patients who underwent myomectomy by laparotomy with opening of the uterine cavity or myomectomy by hysteroscopy. A diagnostic hysteroscopy was performed to search post-operative intrauterine adhesions. For each patient, we studied the socio-demographic aspects, the surgical approach, the delay of diagnostic hysteroscopy, the appearance of uterine cavity and the tolerance of hysteroscopy.Results: 54 patients underwent the diagnostic hysteroscopy. The mean age was 36 years old. The main indication of myomectomy was menorrhagia. 37 patients underwent myomectomy by laparotomy and 17 by hysteroscopy. Each case was followed by a post-operative diagnostic hysteroscopy with a mean delay time of 58 days. We found post-operative intrauterine adhesions in 7 patients (5 in the laparotomy group and 2 in hysteroscopy group).Conclusions: Early diagnostic hysteroscopy after myomectomy should be done in our context to reduce post-operative intrauterine adhesions and preserve the patient’s fertility

Uterine ruptures during labor: a study of 26 cases at Nabil Choucair Medical Center, in the suburbs of Dakar

Background: This study aims to clarify the epidemiological profile of patients and describe the diagnostic, therapeutic and prognostic aspects of uterine ruptures at Nabil Choucair Health Center, in the suburbs of Dakar.Methods: This was a retrospective and prospective, descriptive and analytical study conducted over a period of 112 months, from July 31, 2005 and March 31, 2015; it concerned all diagnosed cases with uterine rupture, managed at Nabil Health Center Choucair Dakar. Data were collected from antenatal notebooks, delivery records, surgical reports and anesthetic records. The following parameters were studied: socio-demographic characteristics (frequency, chracteristics of patient), the course of pregnancy, diagnostic, prognostic, and therapeutic aspects. Data analysis was performed using SPSS software (version 11.0).Results: During the study period, we recorded 26 uterine ruptures from a total of 34,097 deliveries, that is a frequency of 8 out of 10,000 births. The epidemiological profile of the patients was that of a 30-year old woman, on average, few previous deliveries with an average parity of 3, who received on average 3 antenatal and evacuated in 50% of cases. Uterine rupture occurred in 17 patients on a new uterus (65, 4%) and in 9 patients on a scarred uterus (34.6%). Two patients (7%) were in hemorrhagic shock. A laparotomy aimed at specifying the place of rupture and making the treatment was performed in all patients. It was 18 uterine segmento-corporeal ruptures (69.2%), 5 segmental ruptures (19.2%) and 2 corporeal ruptures (7.6%). We recorded 9 uterine ruptures with living fetus (38.5%). Twenty-three patients (88.5%) underwent conservative suture against only 3 hemostasis hysterectomy (11.5%). The postoperative courses were uneventful in 57.7% of cases. Eleven patients (42.3%) had anemia and we recorded maternal deaths, which is a maternal mortality of 3.84%.Conclusions: Uterine rupture is a serious complication that is still responsible for significant maternal and fetal mortality. Prevention is based on careful monitoring of pregnancy and delivery

Pseudo tumor tuberculosis of the uterine cervix: about a case at the colposcopy and cervico vaginal pathologies unit of the University hospital Le Dantec, Dakar, Senegal

Tuberculosis is still common in developing countries and particularly in sub-Saharan Africa since the advent of the HIV/AIDS. Genital tuberculosis usually affects young women in genital activity period. The most frequent locations are tubal, endometrial and ovarian. The cervical location is rare. We report on the case of a 36 years old patient with eight pregnancies and eight deliveries who lives in a rural area and has got in her history 8 vaginal deliveries with four living children and 4 dead children and who was referred by a colleague for a "tumor of the uterine cervix". In her medical history, there was a BCG vaccination during childhood and she had never received Pap smear. The colposcopy revealed an ulcerating budding tumor of the cervix with necrotic areas. The colposcopy biopsy revealed fibrocaseous tuberculosis of the uterine cervix. Tuberculosis is still a common disease in developing countries. The cervical localization is rare but should be considered in case of an ulcerating tumor budding of the cervix

Preferential Transfer of Certain Plasma Membrane Proteins onto T and B Cells by Trogocytosis

T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRγ found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis

Assessment of the quality of the management of childbirth by vaginal delivery in 5 reference maternity clinics in Dakar, Senegal

Background: The current strategy to fight maternal and perinatal mortalities is mainly based on three pillars: family planning, skilled delivery and emergency obstetric and neonatal cares. The objective of the study was to assess the quality of the management of low-risk deliveries in 5 reference maternity clinics in the region of Dakar.             Methods: This is an observation-based multicentric, prospective and descriptive study, carried out over a four-month period, from September 1st to December 31st, 2012, in 5 reference maternity hospitals in the region of Dakar. For each delivery, the focus was put on parturient socio-demographic features, the surveillance techniques of the labour, the handling of the exit and delivery phases, materno-fetal complications but also on the new-born care. To assess the quality of childbirth management, a comparison between the practices was made and observed on the field and the World Health Organization's recommendations which fall into 4 categories (A, B, C and D) depending on the appropriateness or not of their use. For data input and analysis, we used the 13.0 version of the SPSS software. Results: During the study period, 200 childbirths were observed. The epidemiologic profile used for this study was that of a literate woman with a low record of childbirth who was 27 on average with a mean parity of 3. Forty seven percent of childbirth deliveries were observed in Roi Baudouin hospital centre. The A-category recommendations of the World Health Organization, such as the consumption of drinks, mobility during labour, the use of a partograph, the active management of the third stage of labour (AMTSL) and the examination of the placenta were applied in 22.5%, 86.2%, 23.4%, 100% and 23.4% of cases respectively. As for the B-category recommendations, which recommend the removal of those practices deemed harmful such as the back position during labour, the placement of an intravenous line and the oxytocin infusion, they were carried out in 91.84%, 98.08% and 7.28% of cases respectively. C and D categories which deal with non-recommended practices such as the use of abdominal expressions during labour and episiotomy were used in 47.6% and 39.7% of cases respectively.Conclusions: In our study, the most followed A-category recommendations from the WHO are: encouraged mobility, the use of single-use equipment, AMTSL and skin-on-skin contact. However, some B, C and D-category practices are still widely used in our maternity hospitals. The health authorities should make sure that the WHO recommendations are followed

Active management of third stage of labour with low doses of oral misoprostol and oxytocin on low: risk parturient in a Sub-Saharan hospital, Dakar, Sénégal

Background: Assess the effectiveness of oral misoprostol as an alternative to oxytocin in the active management of the third stage of labour in Dakar/Senegal.Methods: Randomized controlled clinical trial conducted in the maternity ward of a university hospital on 304 women who had vaginal delivery. These women were randomly assigned into 2 groups based on active delivery conditions: the first group received an oral administration of misoprostol (400 mcg) and the second group 5 IU oxytocin through intravenous route.Results: The average volume of blood loss was 196.55 ml in the misoprostol group and 208.39 ml in the oxytocin group (p=0.63). The incidence of postpartum haemorrhage (>500 cc) was 6.49% in the misoprostol group and 9.33% in the oxytocin group (p=0.358). The average rate of haemo globin decline was 0.38 g/dl in the misoprostol group and 0.29 g/dl in the oxytocin group (p=0.99). The proportion of hyperthermia, shivering, and nausea in the misoprostol and oxytocin groups were respectively: 2.59% against 0.6% (p=0.123), 7.14% against 2% (p=0.001) and 2.59% against 0.6% (p=0.498).Conclusions: In Senegal, Misoprostol despite its side effects, is an effective alternative to oxytocin in the active management of the third stage of labour for low-risk parturient women to reduce the risk of maternal deaths due to post-partum hemorrhage