Prokaryotic expression, purification and immunogenicity in rabbits of the small antigen of hepatitis delta virus

Funding Information: Expression and purification of HDV antigen was supported by Russian Foundation for Basic Research (grant 16-04-01490a). Evaluation of serum by Western blot and confocal microscopy was supported by Russian Science Foundation (grant 14-14-01021). Experiments in rabbits were supported by the Swedish Institute grants 09272_2013 and 19806_2016. Cross-border collaboration of the partners, exchange of the materials and standard operation procedures used in the study, and dissemination of the data were supported by the EU Twinning project VACTRAIN, contract nr 692293. Publisher Copyright: © 2016 by the authors; licensee MDPI, Basel, Switzerland.Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 µg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 µg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.publishersversionPeer reviewe

Clinical evaluation of early postpartum pain and healing outcomes after mediolateral versus lateral episiotomy

a b s t r a c t a r t i c l e i n f o Objective: To evaluate short-term perineal pain among primiparous women after mediolateral episiotomy (MLE) and lateral episiotomy (LE). Methods: The prospective randomized study was conducted in the Czech Republic during 2010-2012. Consecutive primiparous women who gave birth at or after 37 weeks of pregnancy and had indications for an episiotomy were enrolled and randomly assigned to undergo MLE or LE. Patients were unaware of the episiotomy type performed. The primary outcomes were pain at 24 hours, 72 hours, and 10 days post partum, measured by a visual analog scale, verbal rating scale, interference with activities of daily living, and amount of analgesic use. Results: The analysis included 266 women who underwent MLE and 297 women who underwent LE. Complete relief of pain was observed in 6 (2.3%) of 266 women after 24 hours, 21 (8.0%) of 264 after 72 hours, and 77 (29.1%) of 265 after 10 days in the MLE group, and in 11 (3.9%) of 285, 23 (7.7%) of 297, and 78 (26.4%) of 295 in the LE group, respectively (P = 0.36). There were no significant differences in overall pain scores from any rating system or in the amount of analgesics used. Conclusion: Incidence and extent of pain in the first 10 days after LE correspond to those after adequately performed MLE

Initial placement of fingers and vectors of subsequent coordinated movements in evaluated modifications of MPP.

<p>Initial placement of fingers and vectors of subsequent coordinated movements in evaluated modifications of MPP.</p

The order of effectiveness of VMPP modifications (in brackets), relative perineal tension at the fourchette during expulsion of small, normal and large fetal head.

<p>The relative perineal tissue tension provided in percentage with the pre-set maximum tension in the "hands-off" model at 100% and the pre-set tension at rest at 0%.</p

The most effective modification of VMPP calculated from numerical model during the expulsion of an average-sized fetal head.

<p>NB The initial position of fingers is 12 cm apart and 2 cm anteriorly from the posterior fourchette (Fig 1A). The fingers, still in contact with the perineal skin, are subsequently moved from each side 1 cm towards the midline. No movement in an antero-posterior dimension is performed (Fig 1B).</p

Direct comparison between perineal tensions of a variety of MPP simulations with respect to different sizes of the fetal head and between normal fetal head expulsion without any intervention.

<p>The maximum tension during the "hands-off" simulation with normal fetal head is the referrential tension, hence the proportion for this simulation is 1.00. The lower the number the higher the efficiency of the simulated intervention.</p

Mid-sagittal plane of the segment of the perineum during the "hands-off" simulation and stress distribution in the tissue at the moment of fetal head expulsion with areas where the tension exceeded 20%, 40% and 60% of the maximum tension (i.e. 100%).

<p>Mid-sagittal plane of the segment of the perineum during the "hands-off" simulation and stress distribution in the tissue at the moment of fetal head expulsion with areas where the tension exceeded 20%, 40% and 60% of the maximum tension (i.e. 100%).</p

European guidelines on perinatal care- Peripartum care Episiotomy

1. Episiotomy should be performed by indication only, and not routinely (Moderate quality evidence +++-; Strong recommendation). Accepted indications for episiotomy are to shorten the second stage of labor when there is suspected fetal hypoxia (Low quality evidence ++–; Weak recommendation); to prevent obstetric anal sphincter injury in vaginal operative deliveries, or when obstetric sphincter injury occurred in previous deliveries (Moderate quality evidence +++-; Strong recommendation) 2. Mediolateral or lateral episiotomy technique should be used (Moderate quality evidence +++-; Strong recommendation). Labor ward staff should be offered regular training in correct episiotomy techniques (Moderate quality evidence +++-; Strong recommendation). 3. Pain relief needs to be considered before episiotomy is performed, and epidural analgesia may be insufficient. The perineal skin needs to be tested for pain before an episiotomy is performed, even when an epidural is in place. Local anesthetics or pudendal block need to be considered as isolated or additional pain relief methods (Low quality evidence ++–; Strong recommendation). 4. After childbirth the perineum should be carefully inspected, and the anal sphincter palpated to identify possible injury (Moderate quality evidence +++-; Strong recommendation). Primary suturing immediately after childbirth should be offered and a continuous suturing technique should be used when repairing an uncomplicated episiotomy (High quality evidence ++++; Strong recommendation

Fetal head size and effect of manual perineal protection

Cílem studie je vyhodnotit zda dříve identifikovaná modifikace Vídeňské metody manuální perineální protekce je také nejefektivnější pro redukci perineálního napětí v případech s výrazně menší nebo větší hlavičkou plodu.The aim of this study was to evaluate whether a previously identified modification of Viennese method of perineal protection remains most effective for reduction of perineal tension in cases with substantially smaller or larger fetal heads