Wojciech Witkowski

Age-associated differential microRNA levels in human follicular fluid reveal pathways potentially determining fertility and success of in vitro fertilization.

Abstract Reproductive life span and fertility have been shown to depend on successful early folliculogenesis, which involves cell-to-cell communication and the concerted regulation of gene expression at both the oocyte and granulosa cell levels. Recently, micro RNAs (miRNAs) were identified as fine-tuners of gene expression. Here, we report that miRNAs can readily be detected within membrane-enclosed vesicles of human follicular fluid. MiRNA expression profiling of the follicular fluid of younger (< 31 years) and older (> 38 years) women revealed a set of four differentially expressed miRNAs. The predicted targets of these miRNAs are clearly enriched in genes involved in heparan-sulfate biosynthesis, extracellular matrix–receptor interaction, carbohydrate digestion and absorption, p53 signaling, and cytokine–cytokine-receptor interaction. Several of these pathways have been reported to be determinants of fertility, suggesting that this set of miRNAs and their respective targets should be evaluated in relation to reproductive aging and assisted reproduction.

The Nef-infectivity enigma: mechanisms of enhanced lentiviral infection.

The Nef protein is an essential factor for lentiviral pathogenesis in humans and other simians. Despite a multitude of functions attributed to this protein, the exact role of Nef in disease progression remains unclear. One of its most intriguing functions is the ability of Nef to enhance the infectivity of viral particles. In this review we will discuss current insights in the mechanism of this well-known, yet poorly understood Nef effect. We will elaborate on effects of Nef, on both virion biogenesis and the early stage of the cellular infection, that might be involved in infectivity enhancement. In addition, we provide an overview of different HIV-1 Nef domains important for optimal infectivity and briefly discuss some possible sources of the frequent discrepancies in the field. Hereby we aim to contribute to a better understanding of this highly conserved and therapeutically attractive Nef function.

Contributions of HIV-1 Nef to immune dysregulation in HIV-infected patients: a therapeutic target?

Introduction: HIV accessory protein Nef is a factor responsible for many of the viral pathogenic effects. Progression to AIDS is dramatically delayed and in some well-documented cases completely abolished on infection with naturally occurring HIV strains lacking intact nef sequences in their genomes. The topic of this review is the contribution of Nef to the immune pathology as a possible target in HIV-infected patients. Areas covered: An overview of known Nef functions accounting for its role in pathogenesis is presented, emphasizing interactions with dendritic cells and macrophages, and Nef-induced exosome secretion, all involved in immune dysregulation during the course of HIV infection. Current approaches to Nef inhibition by different classes of compounds are reviewed. Expert opinion: Blocking Nef for therapeutic purposes is a challenging endeavor mainly due to intrinsic properties of this HIV accessory protein. Nef has multiple interfaces to interact with host proteins and lacks a catalytic domain. Potential benefits arising from the development of successful inhibitors could however prove beneficial for reducing gradual deterioration of immune system in chronically infected patients in absence of functional cure.

The Influence of Articular Cartilage Thickness Reduction on Meniscus Biomechanics.

Objective Evaluation of the biomechanical interaction between meniscus and cartilage in medial compartment knee osteoarthritis. Methods The finite element method was used to simulate knee joint contact mechanics. Three knee models were created on the basis of knee geometry from the Open Knee project. We reduced the thickness of medial cartilages in the intact knee model by approximately 50% to obtain a medial knee osteoarthritis (OA) model. Two variants of medial knee OA model with congruent and incongruent contact surfaces were analysed to investigate the influence of congruency. A nonlinear static analysis for one compressive load case was performed. The focus of the study was the influence of cartilage degeneration on meniscal extrusion and the values of the contact forces and contact areas. Results In the model with incongruent contact surfaces, we observed maximal compressive stress on the tibial plateau. In this model, the value of medial meniscus external shift was 95.3% greater, while the contact area between the tibial cartilage and medial meniscus was 50% lower than in the congruent contact surfaces model. After the non-uniform reduction of cartilage thickness, the medial meniscus carried only 48.4% of load in the medial compartment in comparison to 71.2% in the healthy knee model. Conclusions We have shown that the change in articular cartilage geometry may significantly reduce the role of meniscus in load transmission and the contact area between the meniscus and cartilage. Additionally, medial knee OA may increase the risk of meniscal extrusion in the medial compartment of the knee joint.

Replication competent virus as an important source of bias in HIV latency models utilizing single round viral constructs

The central memory T cell (TCM) model forms a unique HIV-1 latency model based on primary cells that closely resemble in vivo TCM. The virus employed in this model is based on an engineered vector incapable of replication after initial infection. We show that despite this strategy, replication competent viral particles are released into the culture medium due to recombination between overlapping sequences of the env deleted HIV genome that is co-transfected with intact env. This finding emphasizes the need for careful data analysis and interpretation if similar constructs are employed and urges for additional caution during laboratory work.

The influence of a change in the meniscus cross-sectional shape on the medio-lateral translation of the knee joint and meniscal extrusion

Objective The purpose of this study was to evaluate the influence of a change in the meniscus cross sectional shape on its position and on the biomechanics of a knee joint. Methods One main finite element model of a left knee joint was created on the basis of MRI images. The model consisted of bones, articular cartilages, menisci and ligaments. Eight variants of this model with an increased or decreased meniscus height were then prepared. Nonlinear static analyses with a fixed flexion/extension movement for a compressive load of 1000 N were performed. The additional analyses for those models with a constrained medio-lateral relative bone translation allowed for an evaluation of the influence of this translation on a meniscus external shift. Results It was observed that a decrease in the meniscus height caused a decrease in the contact area, together with a decrease in the contact force between the flattened meniscus and the cartilage. For the models with an increased meniscus height, a maximal value of force acting on the meniscus in a medio-lateral direction was obtained. The results have shown that the meniscus external shift was approximately proportional to the meniscus slope angle, but that relationship was modified by a medio-lateral relative bone translation. It was found that the translation of the femur relative to the tibia may be dependent on the geometry of the menisci. Conclusions The results have suggested that a change in the meniscus geometry in the cross sectional plane can considerably affect not only the meniscal external shift, but also the medio-lateral translation of the knee joint as well as the congruency of the knee joint.

Curb-to-Barrier Face Distance Variation an a TB51 Bridge Barrier Crash Test Simulation

Abstract This paper addresses the problem of road safety regarding barrier placement as relative to the curb. A short summary of existing regulations is presented. Numerical simulations using the explicit finite element system Ls-Dyna are shown. In the analysis, variable distance between the barrier and the curb is assumed. The obtained result reveals that the distance has little impact on the working width of the barrier.

Vpx-Independent Lentiviral Transduction and shRNA-Mediated Protein Knock-Down in Monocyte-Derived Dendritic Cells

The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting.

Analiza drgań kładki kompozytowej wywołanych działaniem wiatru

in the work, we describe a simplified method for numerical analysis of a FrP composite footbridge in the field of wind induced vibrations. we consider a simply supported structure with a span length of 16 m and U-shape cross-section. Firstly, a two dimensional flow analysis is performed of the fixed bridge cross-section which is subjected to a lateral wind action with 10 m/s velocity. calculations are performed using aNsys FlUeNt 14 software. results of the flow analysis (strouhal’s number) are compared with the results presented in a monograph by a. Flaga, entitled Inzynieria Wiatrowa. Podstawy i zastosowania (Wind Engineering. The bases and applications) (in Polish), arkady, warszawa, 2008, in order to validate calculations. after that, a three dimensional spatial model of the footbridge is built in aBaQUs 6.12-3 finite element method software. a modal dynamics problem is solved, where the loading conditions are adopted on the basis of the flow analysis and applied as an evenly distributed pressure on the bridge deck surface. Finally, the users’ vibration comfort criterion is checked for the considered structure.

Efficient, Vpx independent shRNA transduction of monocyte derived dendritic cells as a tool for studying HIV transmission to primary T-cells

Background: Dendritic cells present at mucosal sites are considered initial targets for HIV transmission. Due to SAMHD1 expression, they are highly resistant to HIV infection themselves, but act as shuttles for viral transmission to T cells. Knock-down of HIV host cellular partners is a powerful method in HIV research. At present, lentiviral shRNA delivery to Monocyte Derived Dendritic Cells (MDDC) requires the use of virus-like particles carrying the Vpx protein in order to alleviate SAMHD1 mediated restriction. Such an approach results in the desired high transduction efficiency, but simultaneously abrogates resistance to HIV thus allowing for viral replication which might obscure the actual outcomes of MDDC-HIV interactions. We present a method for efficient shRNA transduction of MDDC for subsequent studies of HIV transmission to CD4+ T-cells, without the need for Vpx. Methods: MDDC were obtained by culturing primary monocytes for 6 days in presence of lL-4 and GM-CSF. At day 1 after monocyte isolation, cells were transduced with shRNA encoding lentiviral vectors (Sigma MISSION®) at MOI of 1 (titration on HEK293T cell line) by addition of polybrene and using spinoculation. Transduction efficiency was determined by measuring the fraction of eGFP expression (encoded by the lentiviral vector) by flow cytometry. At day 5 post transduction, cells were exposed to NL4.3 HIV virus harboring a marker gene. Twenty four hours later the cells were extensively washed with culture medium to remove unbound virus and autologous, primary CD4+ T cells were added at 1:2 ratio. Infection of T-cells in MDDC-T-cell cocultures was subsequently monitored by flow cytometry to detect marker gene expressing cells in low scatter and/or CD3+ population. Results: Efficient transduction of MDDC (≈95%) as judged by eGFP expression was obtained. This high efficiency avoids puromycin selection and therefore simplifies the protocol while preventing possible side effects. Notably, the transduced MDDC did not show a marked increase in CD80, CD83 and CD86 expression levels. Transduced MDDC are able to capture and transmit the virus to T cells. We have observed a positive correlation between shRNA mediated downregulation of certain MDDC surface markers and transmission of HIV to CD4+ T cells as compared to cells transduced with a control vector encoding a scrambled shRNA sequence not known to target any human gene. Conclusions: We have established a method for lentiviral encoded shRNA transductions allowing for studies of MDDC cellular factors involved in HIV transmission to CD4+ T cells without the need to alleviate SAMHD1 induced restriction. Transduced MDDC remain immature as judged by expression of CD80, CD83 and CD86 cell surface receptors. Preliminary results provide validation of the approach by positively correlating downregulation of MDDC surface markers with trans-infection of CD4+ T cells.