Alterations of Placental Sodium in Preeclampsia: Trophoblast Responses.

BACKGROUND: Evidence suggests that increasing salt intake in pregnancy lowers blood pressure, protecting against preeclampsia. We hypothesized that sodium (Na+) evokes beneficial placental signals that are disrupted in preeclampsia. METHODS: Blood and urine were collected from nonpregnant women of reproductive age (n=26) and pregnant women with (n=50) and without (n=55) preeclampsia, along with placental biopsies. Human trophoblast cell lines and primary human trophoblasts were cultured with varying Na+ concentrations. RESULTS: Women with preeclampsia had reduced placental and urinary Na+ concentrations, yet increased urinary angiotensinogen and reduced active renin, aldosterone concentrations, and osmotic response signal TonEBP (tonicity-responsive enhancer binding protein) expression. In trophoblast cell cultures, TonEBP was consistently increased upon augmented Na+ exposure. Mechanistically, inhibiting Na+/K+-ATPase or adding mannitol evoked the TonEBP response, whereas inhibition of cytoskeletal signaling abolished it. CONCLUSIONS: Enhanced Na+ availability induced osmotic gradient-dependent cytoskeletal signals in trophoblasts, resulting in proangiogenic responses. As placental salt availability is compromised in preeclampsia, adverse systemic responses are thus conceivable.

Alterations in Antioxidant Micronutrient Concentrations in Placental Tissue, Maternal Blood and Urine and the Fetal Circulation in Pre-eclampsia.

Trace elements such as selenium and zinc are vital components of many enzymes, including endogenous antioxidants, and can interact with each other. Women with pre-eclampsia, the hypertensive disease of pregnancy, have been reported as having changes in some individual antioxidant trace elements during pregnancy, which are related to maternal and fetal mortality and morbidity. We hypothesised that examination of the three compartments of (a) maternal plasma and urine, (b) placental tissue and (c) fetal plasma in normotensive and hypertensive pregnant women would allow identification of biologically significant changes and interactions in selenium, zinc, manganese and copper. Furthermore, these would be related to changes in the angiogenic markers, placental growth factor (PlGF) and Soluble Fms-Like Tyrosine Kinase-1 (sFlt-1) concentrations. Venous plasma and urine were collected from healthy non-pregnant women (n = 30), normotensive pregnant controls (n = 60) and women with pre-eclampsia (n = 50) in the third trimester. Where possible, matched placental tissue samples and umbilical venous (fetal) plasma were also collected. Antioxidant micronutrient concentrations were measured by inductively coupled plasma mass-spectrometry. Urinary levels were normalised to creatinine concentration. Plasma active PlGF and sFlt-1 concentrations were measured by ELISA. Maternal plasma selenium, zinc and manganese were all lower in women with pre-eclampsia (p < 0.05), as were fetal plasma selenium and manganese (p < 0.05 for all); maternal urinary concentrations were lower for selenium and zinc (p < 0.05). Conversely, maternal and fetal plasma and urinary copper concentrations were higher in women with pre-eclampsia (p < 0.05). Differences in placental concentrations varied, with lower overall levels of selenium and zinc (p < 0.05) in women with pre-eclampsia. Maternal and fetal PlGF were lower and sFlt-1 higher in women with pre-eclampsia; maternal plasma zinc was positively correlated with maternal plasma sFlt-1 (p < 0.05). Because of perceptions that early- and late-onset pre-eclampsia have differing aetiologies, we subdivided maternal and fetal data accordingly. No major differences were observed, but fetal sample sizes were small following early-onset. Disruption in these antioxidant micronutrients may be responsible for some of the manifestations of pre-eclampsia, including contributing to an antiangiogenic state. The potential benefits of mineral supplementation, in women with deficient intakes, during pregnancy to reduce pre-eclampsia remain an important area for experimental and clinical research.

Developing Porous Ortho- and Pyrophosphate-Containing Glass Microspheres; Structural and Cytocompatibility Characterisation.

Phosphate-based glasses (PBGs) are promising materials for bone repair and regeneration as they can be formulated to be compositionally similar to the inorganic components of bone. Alterations to the PBG formulation can be used to tailor their degradation rates and subsequent release of biotherapeutic ions to induce cellular responses, such as osteogenesis. In this work, novel invert-PBGs in the series xP2O5·(56 - x)CaO·24MgO·20Na2O (mol%), where x is 40, 35, 32.5 and 30 were formulated to contain pyro (Q1) and orthophosphate (Q0) species. These PBGs were processed into highly porous microspheres (PMS) via flame spheroidisation, with ~68% to 75% porosity levels. Compositional and structural analysis using EDX and 31P-MAS NMR revealed that significant depolymerisation occurred with reducing phosphate content which increased further when PBGs were processed into PMS. A decrease from 50% to 0% in Q2 species and an increase from 6% to 35% in Q0 species was observed for the PMS when the phosphate content decreased from 40 to 30 mol%. Ion release studies also revealed up to a four-fold decrease in cations and an eight-fold decrease in phosphate anions released with decreasing phosphate content. In vitro bioactivity studies revealed that the orthophosphate-rich PMS had favourable bioactivity responses after 28 days of immersion in simulated body fluid (SBF). Indirect and direct cell culture studies confirmed that the PMS were cytocompatible and supported cell growth and proliferation over 7 days of culture. The P30 PMS with ~65% pyro and ~35% ortho phosphate content revealed the most favourable properties and is suggested to be highly suitable for bone repair and regeneration, especially for orthobiologic applications owing to their highly porous morphology.

Increased Placental Cell Senescence and Oxidative Stress in Women with Pre-Eclampsia and Normotensive Post-Term Pregnancies.

Up to 11% of pregnancies extend to post-term with adverse obstetric events linked to pregnancies over 42 weeks. Oxidative stress and senescence (cells stop growing and dividing by irreversibly arresting their cell cycle and gradually ageing) can result in diminished cell function. There are no detailed studies of placental cell senescence markers across a range of gestational ages, although increased levels have been linked to pre-eclampsia before full term. This study aimed to determine placental senescence and oxidative markers across a range of gestational ages in women with uncomplicated pregnancies and those with a diagnosis of pre-eclampsia. Placentae were obtained from 37 women with uncomplicated pregnancies of 37-42 weeks and from 13 cases of pre-eclampsia of 31+2-41+2 weeks. The expression of markers of senescence, oxidative stress, and antioxidant defence (tumour suppressor protein p16INK4a, kinase inhibitor p21, interleukin-6 (IL-6), NADPH oxidase 4 (NOX4), glutathione peroxidases 1, 3, and 4 (GPx1, GPx3, and GPx4), placental growth factor (PlGF), and soluble fms-like tyrosine kinase-1 (sFlt-1)) genes was measured (quantitative real-time PCR). Protein abundance of p16INK4a, IL-6, NOX4, 8-hydroxy-2'-deoxy-guanosine (8-OHdG), and PlGF was assessed by immunocytochemistry. Placental NOX4 protein was higher in post-term than term deliveries and further increased by pre-eclampsia (p < 0.05 for all). P21 expression was higher in post-term placentae (p = 0.012) and in pre-eclampsia (p = 0.04), compared to term. Placental P16INK4a protein expression was increased post-term, compared to term (p = 0.01). In normotensive women, gestational age at delivery was negatively associated with GPx4 and PlGF (mRNA and protein) (p < 0.05 for all), whereas a positive correlation was seen with placental P21, NOX4, and P16INK4a (p < 0.05 for all) expression. Markers of placental oxidative stress and senescence appear to increase as gestational age increases, with antioxidant defences diminishing concomitantly. These observations increase our understanding of placental health and may contribute to assessment of the optimal gestational age for delivery.

IKKγ-Mimetic Peptides Block the Resistance to Apoptosis Associated with Kaposi's Sarcoma-Associated Herpesvirus Infection.

Primary effusion lymphoma (PEL) is a lymphogenic disorder associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Key to the survival and proliferation of PEL is the canonical NF-κB pathway, which becomes constitutively activated following overexpression of the viral oncoprotein KSHV vFLIP (ks-vFLIP). This arises from its capacity to form a complex with the modulatory subunit of the IκB kinase (IKK) kinase, IKKγ (or NEMO), resulting in the overproduction of proteins that promote cellular survival and prevent apoptosis, both of which are important drivers of tumorigenesis. Using a combination of cell-based and biophysical assays together with structural techniques, we showed that the observed resistance to cell death is largely independent of autophagy or major death receptor signaling pathways and demonstrated that direct targeting of the ks-vFLIP-IKKγ interaction both in cells and in vitro can be achieved using IKKγ-mimetic peptides. Our results further reveal that these peptides not only induce cell killing but also potently sensitize PEL to the proapoptotic agents tumor necrosis factor alpha and etoposide and are the first to confirm ks-vFLIP as a tractable target for the treatment of PEL and related disorders.IMPORTANCE KSHV vFLIP (ks-vFLIP) has been shown to have a crucial role in cellular transformation, in which it is vital for the survival and proliferation of primary effusion lymphoma (PEL), an aggressive malignancy associated with infection that is resistant to the majority of chemotherapeutic drugs. It operates via subversion of the canonical NF-κB pathway, which requires a physical interaction between ks-vFLIP and the IKK kinase modulatory subunit IKKγ. While this interaction has been directly linked to protection against apoptosis, it is unclear whether the suppression of other cell death pathways implicated in ks-vFLIP pathogenesis is an additional contributor. We demonstrate that the interaction between ks-vFLIP and IKKγ is pivotal in conferring resistance to apoptosis. Additionally, we show that the ks-vFLIP-IKKγ complex can be disrupted using peptides leading to direct killing and the sensitization of PEL cells to proapoptotic agents. Our studies thus provide a framework for future therapeutic interventions.

Crystal structure of a KSHV-SOX-DNA complex: insights into the molecular mechanisms underlying DNase activity and host shutoff.

The early lytic phase of Kaposi's sarcoma herpesvirus infection is characterized by viral replication and the global degradation (shutoff) of host mRNA. Key to both activities is the virally encoded alkaline exonuclease KSHV SOX. While the DNase activity of KSHV SOX is required for the resolution of viral genomic DNA as a precursor to encapsidation, its exact involvement in host shutoff remains to be determined. We present the first crystal structure of a KSHV SOX-DNA complex that has illuminated the catalytic mechanism underpinning both its endo and exonuclease activities. We further illustrate that KSHV SOX, similar to its Epstein-Barr virus homologue, has an intrinsic RNase activity in vitro that although an element of host shutoff, cannot solely account for the phenomenon.

Falls assessment and prevention: a multidisciplinary teaching intervention.

BACKGROUND: Falls are a common and important clinical problem, and with ageing populations worldwide it is important for health care professionals to learn about falls management. The multidisciplinary nature of falls teams also provides an ideal opportunity for interprofessional collaboration in teaching. CONTEXT: In this article, we describe a pilot multidisciplinary falls assessment and prevention workshop for second-year medical students at a London medical school. INNOVATION AND IMPLICATIONS: An interprofessional team worked together to design and deliver this workshop. During a 90-minute clinical skills session, students rotated through medical, occupational therapy and physiotherapy areas. They worked in small groups, using brainstorming, discussion and practical exercises to learn about multiple risk factors contributing to falls, and how professionals work together in the management of patients at risk of falling. Evaluation was carried out using a combination of quantitative Likert ratings and qualitative free-text comments. The session was well received, with identified strengths and areas for improvement helping to confirm the importance of this workshop in the curriculum, and leading to improvements in the design for future sessions.

Analysis of nucleotide binding to P97 reveals the properties of a tandem AAA hexameric ATPase.

p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5'-O-(thiotriphosphate) (ATPgammaS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPgammaS can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATPgammaS simultaneously. ATPgammaS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.

Going through the motions: the ATPase cycle of p97.

p97 (VCP, Cdc48), a type II AAA+ ATPase family member, is ubiquitous, essential, highly abundant, and involved in a diverse range of biological functions with roles in membrane fusion, endoplasmic-reticulum associated degradation, transcriptional activation, and cell cycle control. As such, dysfunction of this protein has serious pathological consequences and has been implicated in a variety of cancers and neurodegenerative diseases. p97 has a large number of adaptor proteins through which it transmits energy from ATPase activity to conformational changes which are then exerted onto target proteins. p97 has been studied by a variety of biochemical and structural techniques at various resolutions and stages throughout its ATPase cycle. From these studies, many models have been proposed and consequently a single model for p97's action cannot be suggested. Many questions about the mechanism of p97 still remain, including whether the protomers act in a concerted manner and crucially how the induced changes in p97 are transmitted to its adaptor proteins and target substrates. The elucidation of p97's mechanism is not only important in furthering our knowledge of this intriguing protein and its many functions, but subsequently in the development of potential therapies for diseases associated with p97 dysfunction.

Conformational changes in the AAA ATPase p97-p47 adaptor complex.

The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at approximately 20 Angstroms resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes.

The crystal structure of murine p97/VCP at 3.6A.

p97/VCP is a member of the AAA ATPase family and has roles in both membrane fusion and ubiquitin dependent protein degradation. Here, we present a 3.6A crystal structure of murine p97 in which D2 domain has been modelled as poly-alanine and the remaining approximately 100 residues are absent. The resulting structure illustrates a head-to-tail packing arrangement of the two p97 AAA domains in a natural hexameric state with D1 ADP bound and D2 nucleotide free. The head-to-tail packing arrangement observed in this structure is in contrast to our previously predicted tail-to-tail packing model. The linker between the D1 and D2 domains is partially disordered, suggesting a flexible nature. Normal mode analysis of the crystal structure suggests anti-correlated motions and distinct conformational states of the two AAA domains.