Eradication of cervical canal colonization associated with prophylactic cervical cerclage: the look further study.

OBJECTIVES: The perioperative management of the cervical cerclage procedure is not unified. In general population controlling microbiome cervical status does not affect obstetric outcomes, but it might be beneficial in patients with cervical insufficiency. The aim of our study was to present the obstetric, neonatal and pediatric outcomes of patients undergoing the cervical cerclage placement procedure in our obstetric department using a regimen of care that includes control of the microbiological status of the cervix and elimination of the pathogens detected. MATERIAL AND METHODS: Thirty-five patients undergoing cervical cerclage in the 2nd Department of Obstetrics and Gynecology, Medical University of Warsaw, were included in the study. The procedure was performed only after receiving a negative culture from the cervical canal. RESULTS: Thirty-one (88.6%) patients delivered after the 34th and twenty-eight (80.0%) after the 37th week of gestation. The colonization of the genital tract was present in 31% of patients prior to the procedure, in 42% of patients - during the subsequent pregnancy course and in 48% of patients - before delivery. A total of 85% of patients who had miscarriage or delivered prematurely had abnormal cervical cultures. In patients with normal cervical cultures, and 91.7% of women delivered at term. No abnormalities in children's development were found. CONCLUSIONS: Controlling microbiological status of the cervical canal results in better or similar outcomes to those reported by other authors in terms of obstetric and neonatal outcomes. Active eradication of the reproductive tract colonization potentially increases the effectiveness of the cervical cerclage placement.

Varying molecular interactions explain aspects of crowder-dependent enzyme function of a viral protease.

Biochemical processes in cells, including enzyme-catalyzed reactions, occur in crowded conditions with various background macromolecules occupying up to 40% of cytoplasm's volume. Viral enzymes in the host cell also encounter such crowded conditions as they often function at the endoplasmic reticulum membranes. We focus on an enzyme encoded by the hepatitis C virus, the NS3/4A protease, which is crucial for viral replication. We have previously found experimentally that synthetic crowders, polyethylene glycol (PEG) and branched polysucrose (Ficoll), differently affect the kinetic parameters of peptide hydrolysis catalyzed by NS3/4A. To gain understanding of the reasons for such behavior, we perform atomistic molecular dynamics simulations of NS3/4A in the presence of either PEG or Ficoll crowders and with and without the peptide substrates. We find that both crowder types make nanosecond long contacts with the protease and slow down its diffusion. However, they also affect the enzyme structural dynamics; crowders induce functionally relevant helical structures in the disordered parts of the protease cofactor, NS4A, with the PEG effect being more pronounced. Overall, PEG interactions with NS3/4A are slightly stronger but Ficoll forms more hydrogen bonds with NS3. The crowders also interact with substrates; we find that the substrate diffusion is reduced much more in the presence of PEG than Ficoll. However, contrary to NS3, the substrate interacts more strongly with Ficoll than with PEG crowders, with the substrate diffusion being similar to crowder diffusion. Importantly, crowders also affect the substrate-enzyme interactions. We observe that both PEG and Ficoll enhance the presence of substrates near the active site, especially near catalytic H57 but Ficoll crowders increase substrate binding more than PEG molecules.

Insight into the Binding and Hydrolytic Preferences of hNudt16 Based on Nucleotide Diphosphate Substrates.

Nudt16 is a member of the NUDIX family of hydrolases that show specificity towards substrates consisting of a nucleoside diphosphate linked to another moiety X. Several substrates for hNudt16 and various possible biological functions have been reported. However, some of these reports contradict each other and studies comparing the substrate specificity of the hNudt16 protein are limited. Therefore, we quantitatively compared the affinity of hNudt16 towards a set of previously published substrates, as well as identified novel potential substrates. Here, we show that hNudt16 has the highest affinity towards IDP and GppG, with Kd below 100 nM. Other tested ligands exhibited a weaker affinity of several orders of magnitude. Among the investigated compounds, only IDP, GppG, m7GppG, AppA, dpCoA, and NADH were hydrolyzed by hNudt16 with a strong substrate preference for inosine or guanosine containing compounds. A new identified substrate for hNudt16, GppG, which binds the enzyme with an affinity comparable to that of IDP, suggests another potential regulatory role of this protein. Molecular docking of hNudt16-ligand binding inside the hNudt16 pocket revealed two binding modes for representative substrates. Nucleobase stabilization by Π stacking interactions with His24 has been associated with strong binding of hNudt16 substrates.

Crowding affects structural dynamics and contributes to membrane association of the NS3/4A complex.

Using molecular dynamics simulations, we describe how crowded environments affect the internal dynamics and diffusion of the hepatitis C virus proteases NS3/4A. This protease plays a key role in viral replication and is successfully used as a target for antiviral treatment. The NS3 enzyme requires a peptide cofactor, called NS4A, with its central part interacting with the NS3 ß-sheet, and flexible, protruding terminal tails that are unstructured in water solution. The simulations describe the enzyme and water molecules at atomistic resolution, whereas crowders are modeled via either all-atom or coarse-grained models to emphasize different aspects of crowding. Crowders reflect the polyethylene glycol (PEG) molecules used in the experiments to mimic the crowded surrounding. A bead-shell model of folded coarse-grained PEG molecules considers mainly the excluded volume effect, whereas all-atom PEG models afford more protein-like crowder interactions. Circular dichroism spectroscopy experiments of the NS4A N-terminal tail show that a helical structure is formed in the presence of PEG crowders. The simulations suggest that crowding may assist in the formation of an NS4A helical fragment, positioned exactly where a transmembrane helix would fold upon the NS4A contact with the membrane. In addition, partially interactive PEGs help the NS4A N-tail to detach from the protease surface, thus enabling the process of helix insertion and potentially helping the virus establish a replication machinery needed to produce new viruses. Results point to an active role of crowding in assisting structural changes in disordered protein fragments that are necessary for their biological function.

Crowded environment affects the activity and inhibition of the NS3/4A protease.

Kinetic parameters characterizing the catalytic activities of enzymes are typically investigated in dilute solutions. However, in reality, these reactions occur in cells that, in addition to water and ions, are full of other macromolecules including proteins, nucleic acids, lipids, and metabolites. Such a crowded environment might affect enzyme-catalyzed reaction rates, so it is necessary to mimic the crowd in laboratory settings. We determined the effect of macromolecular crowders on the activity of the hepatitis C virus protease NS3/4A. As crowders we used polyethylene glycol (PEG), Ficoll, and bovine serum albumin. Using the fluorescence assay with a labeled peptide substrate, we found that the crowders affected the kinetics of the NS3/4A-catalyzed reaction differently. The Ficoll crowders increased and PEG decreased the initial and maximum reaction velocities. To explain the opposite effects exerted by PEG as compared to Ficoll, we performed molecular dynamics simulations of NS3/4A in explicit solvent and surrounded by its peptide substrates and PEG molecules. The simulations suggest both hydrophobic and polar/electrostatic interactions between PEG and NS3/4A with hydrogen bonds formed between PEG oxygens and NS3/4A amino acids rich in hydrogen bonds donors. The NS3/4A protease is a known target for telaprevir, an anti-viral drug. We found that Ficoll changes the inhibition constant for telaprevir suggesting that the effect of crowders should also be considered in inhibitor design.

Modeling Crowded Environment in Molecular Simulations.

Biomolecules perform their various functions in living cells, namely in an environment that is crowded by many macromolecules. Thus, simulating the dynamics and interactions of biomolecules should take into account not only water and ions but also other binding partners, metabolites, lipids and macromolecules found in cells. In the last decade, research on how to model macromolecular crowders around proteins in order to simulate their dynamics in models of cellular environments has gained a lot of attention. In this mini-review we focus on the models of crowding agents that have been used in computer modeling studies of proteins and peptides, especially via molecular dynamics simulations.

Association between antenatal classes attendance and perceived fear and pain during labour.

OBJECTIVES: Antenatal classes are a common method of preparation for birth with proven efficiency in improving perinatal outcomes. Yet, their impact on fear perception during labour has not been identified. The aim of the study was to analyse whether preparation for labour by means of antenatal classes attendance could be associated with decrease in level of experienced fear and pain during birth. MATERIALS AND METHODS: It was a cross-sectional study of 147 women who had given vaginal births. Data was collected from mothers between 24 and 72 h postpartum. Patients answered self-reported questionnaires concerning subjective perception of birth including Delivery Fear Scale (DFS) and Numeric Rating Scale (NRS) for fear and pain assessment. The study group was divided into subgroups depending on parity and antenatal classes attendance. RESULTS: Patients in the primiparas subgroup who attended antenatal classes scored lower in the DFS (48.7 ± 23.5 vs. 60.2 ± 16.5, p < .03). There was no difference in the DFS score in the multiparas subgroup (p < .90). No significant differences in the NRS score depending on antenatal classes attendance in any subgroup were observed. CONCLUSION: Participation in antenatal classes should be advised to every pregnant primiparous woman as this type of non-invasive preparation lowers level of fear experienced during childbirth.