Viscoelastic haemostatic point-of-care assays in the management of postpartum haemorrhage: a narrative review.

Viscoelastic haemostatic assays provide rapid testing at the bed-side that identify all phases of haemostasis, from initial fibrin formation to clot lysis. In obstetric patients, altered haemostasis is common as pregnancy is associated with coagulation changes that may contribute to bleeding events such as postpartum haemorrhage, as well as thrombosis events. In this narrative review, we examine the potential clinical utility of viscoelastic haemostatic assays in postpartum haemorrhage and consider the current recommendations for their use in obstetric patients. We discuss the clinical benefits associated with the use of viscoelastic haemostatic assays due to the provision of (near) real-time readouts with a short turnaround, coupled with the identification of coagulation defects such as hypofibrinogenaemia. The use of viscoelastic haemostatic assay-guided algorithms may be beneficial to diagnose coagulopathy, predict postpartum haemorrhage, reduce transfusion requirements and monitor fibrinolysis in women with obstetric haemorrhage. Further studies are required to assess whether viscoelastic haemostatic assay-guided treatment improves clinical outcomes, and to confirm the utility of prepartum viscoelastic haemostatic assay measurements for identifying patients at risk of postpartum haemorrhage.

Post-ruminal supplies of glucose and casein, but not acetate, stimulate milk protein synthesis in dairy cows through differential effects on mammary metabolism.

Amino acids and glucose have been shown to regulate protein synthesis in the mammary gland through their effects on cellular signaling pathways. Acetate might also have an effect on protein synthesis via the AMP-activated kinase signaling pathway, because it is the main energy source for the mammary secretory cell. Thus, the objective of this experiment was to evaluate the effects of casein and energy-yielding nutrients (acetate and glucose), and their combination, on performance and mammary metabolism. Six multiparous Holstein cows, averaging 49 kg of milk/d, were used in a 6 × 6 Latin square design with 14-d periods. Cows were fed to 100% National Research Council requirements for metabolizable protein (MP) and energy (ME) for 9 d, after which they were feed-restricted for 5 d to 85% of their individual ad libitum intake and then abomasally infused with 1 of 6 treatments. Treatments were acetate (A), glucose (G), each at 5% of ad libitum ME intake, casein (C) at 15% of ad libitum MP intake, A + C, G + C, or a saline solution (negative control). Casein infused alone increased milk protein yield numerically, with 25% recovery of the infused casein in milk protein. Glucose infused alone increased milk and milk protein yield and promoted the highest efficiency of nitrogen utilization (37%), with an efficiency of MP use for milk protein of 58%. We discovered no effect of treatment on mammary plasma flow, and the increase in milk protein yield with glucose infusion was brought about by greater mammary AA clearance rate. Infusion of casein and glucose together further increased milk protein yield in an additive fashion, and 47% of the infused casein was recovered in milk protein. Acetate infused alone had no effect on milk protein yield but increased milk fat yield numerically, suggesting that the greater amount of acetate taken up by the mammary gland was used for milk fat synthesis. Infusion of acetate and casein together yielded responses similar to those of casein alone. In conclusion, glucose has a major effect on stimulating milk protein synthesis, and the mammary gland has the ability to increase its supply of nutrients to match its synthetic capacity.

Zooplankton community resilience and aquatic environmental stability on aquaculture practices: a study using net cages.

Fish farming in net cages causes changes in environmental conditions. We evaluated the resilience of zooplankton concerning this activity in Rosana Reservoir (Paranapanema River, PR-SP). Samples were taken near the net cages installed at distances upstream and downstream, before and after net cage installation. The resilience was estimated by the decrease in the groups' abundance after installing the net cages. The zooplankton community was represented by 106 species. The most abundant species were Synchaeta pectinata, S. oblonga, Conochilus coenobasis, Polyarthra dolichoptera and C. unicornis (Rotifera), Ceriodaphnia cornuta, Moina minuta, Bosmina hagmanni and C. silvestrii (Cladocera) and Notodiaptomus amazonicus (Copepoda). The resilience of microcrustaceans was affected in the growing points as this activity left the production environment for longer, delaying the natural ability of community responses. Microcrustaceans groups, mainly calanoid and cyclopoid copepods, had a different return rate. The net cage installation acted as a stress factor on the zooplankton community. Management strategies that cause fewer risks to the organisms and maximize energy flow may help in maintaining system stability.

Targeted cancer immunotherapy with oncolytic adenovirus coding for a fully human monoclonal antibody specific for CTLA-4.

Promising clinical results have been achieved with monoclonal antibodies (mAbs) such as ipilimumab and tremelimumab that block cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CD152). However, systemic administration of these agents also has the potential for severe immune-related adverse events. Thus, local production might allow higher concentrations at the target while reducing systemic side effects. We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Δ24aCTLA4 expressing complete human mAb specific for CTLA-4 and tested it in vitro, in vivo and in peripheral blood mononuclear cells (PBMCs) of normal donors and patients with advanced solid tumors. mAb expression was confirmed by western blotting and immunohistochemistry. Biological functionality was determined in a T-cell line and in PBMCs from cancer patients. T cells of patients, but not those of healthy donors, were activated by an anti-CTLA4mAb produced by Ad5/3-Δ24aCTLA4. In addition to immunological effects, a direct anti-CTLA-4-mediated pro-apoptotic effect was observed in vitro and in vivo. Local production resulted in 43-fold higher (P<0.05) tumor versus plasma anti-CTLA4mAb concentration. Plasma levels in mice remained below what has been reported safe in humans. Replication-competent Ad5/3-Δ24aCTLA4 resulted in 81-fold higher (P<0.05) tumor mAb levels as compared with a replication-deficient control. This is the first report of an oncolytic adenovirus producing a full-length human mAb. High mAb concentrations were seen at tumors with lower systemic levels. Stimulation of T cells of cancer patients by Ad5/3-Δ24aCTLA4 suggests feasibility of testing the approach in clinical trials.

MHV-68 Open Reading Frame 20 is a nonessential gene delaying lung viral clearance.

Recently, it has been demonstrated that the MHV-68 ORF20-encoded gene product induces cell-cycle arrest at the G2/M phase, followed by apoptosis. To study the role of this conserved gene in vivo, two independent ORF20-deficient MHV-68 viruses and their revertants were constructed. As the replication in vitro of both mutants followed similar kinetics to that of the wild-type and revertant viruses, ORF20 is therefore a nonessential virus gene. No cell cycle arrest could be observed upon infection of cells with wild type MHV-68 or mutant viruses. In addition, no major differences were detected between mock- and virus-infected cells when protein and inactivation levels of the mitotic promoter factor cdc2/cyclinB were analyzed. Following intranasal infection, the recovery of mutant, revertant and wild-type viruses in the lungs was similar. With the ORF20-deficient viruses, however, there was a significant delay of four days in clearance of virus from the lungs. Surprisingly, the magnitude and cell population distribution in the exudates of the lung was essentially similar to mice infected with wild-type, revertant or ORF20-deleted viruses. Subsequent establishment of latency was normal for both mutants, demonstrating that ORF20 does not play a critical role in establishment of a persistent infection. These results indicate that while expression of ORF20 may impact on the pathogenicity of the infection, the observed induction of G2/M arrest in ORF20-expressing cells may not be the primary function of ORF20 in the context of viral infection.

Biodiversity of zooplankton communities in the Upper Paraná River floodplain: interannual variation from long-term studies.

We investigated the effect of interannual variation of hydrosedimentological regime and connectivity on the zooplankton biodiversity in the Upper Paraná River floodplain. Zooplankton samplings were undertaken between 2000 and 2007, in different environments of the floodplain, including connected and isolated floodplain lakes, backwaters, rivers and channels. The zooplankton included 541 species. Rotifers showed the highest species richness and abundance. Among the zooplankton species, 71 represent new occurrence records for the floodplain. The species accumulation curve showed a continuous increase in gamma diversity, demonstrating the importance of long-term research for accurate knowledge of biodiversity in heterogeneous and dynamic ecosystems, such as the floodplains. Interannual beta diversity among studied years indicated a lesser alteration in community composition in 2001, when a long limnophase period was observed. In most of the environments, the highest species richness values were related to the greatest flooding amplitudes. Flooding amplitude, which is associated with connectivity, favors faunal exchange amongst the environments and between the pelagic and littoral zones. This explains the occurrence of both planktonic and non-planktonic species within the community. On the other hand, mean zooplankton abundance values were higher when a long isolation period occurred. Differences between the potamophase and limnophase amplitude associated with connectivity among the environments were the most important factors for the structure and dynamics of the zooplankton community in the Upper Paraná River floodplain.

The conserved UL24 family of human alpha, beta and gamma herpesviruses induces cell cycle arrest and inactivation of the cyclinB/cdc2 complex.

The conserved murine gammaherpesvirus 68 ORF20 has recently been demonstrated to induce G2 cell cycle arrest followed by apoptosis in human and mouse cells. Here, we demonstrate that its homologues UL24 in HSV-1, ORF20 in KSHV and UL76 in HCMV are also inducers of cell cycle arrest followed by apoptosis in both human and mouse cells. The mechanism of action is similar to that reported for MHV-68 ORF20, inactivating the mitotic complex cyclinB/Cdc2.

Thermosensitivity of the reverse transcription process as an inactivation mechanism of lentiviral vectors.

Lentiviral vectors are an important tool for gene transfer research and gene therapy purposes. However, the low stability of these vectors affects their production, storage, and efficacy in preclinical and clinical settings. In the present work the mechanism underlying the thermosensitivity of lentiviral vectors was evaluated. For lentiviral vectors pseudotyped with amphotropic and RDpro envelopes, the capacity to perform reverse transcription was lost rapidly at 37 degrees C, in high correlation with the loss of infectivity. The vector with RDpro envelope presented a higher level of stability than that with amphotropic envelope for both the reverse transcription process and viral infectivity. Reverse transcriptase enzyme inactivation and viral template RNA degradation were not implicated in the loss of the viral capacity to perform reverse transcription. Furthermore, early entry steps in the infection process do not determine the rate of viral inactivation, as the amount of viral RNA and p24 protein entering the cells decreased slowly for both vectors. Taken together, it can be concluded that the reverse transcription process is thermolabile and thus determines the rate of lentiviral inactivation. Strategies to stabilize the reverse transcription process should be pursued to improve the applicability of lentiviral vectors in gene therapy.

Biodiversity of zooplankton communities in the Upper Paraná River floodplain: interannual variation from long-term studies / Biodiversidade de comunidades de zooplâncton na planície de inundação do Alto Rio Paraná: variações interanuais em estudos de longa duração

We investigated the effect of interannual variation of hydrosedimentological regime and connectivity on the zooplankton biodiversity in the Upper Paraná River floodplain. Zooplankton samplings were undertaken between 2000 and 2007, in different environments of the floodplain, including connected and isolated floodplain lakes, backwaters, rivers and channels. The zooplankton included 541 species. Rotifers showed the highest species richness and abundance. Among the zooplankton species, 71 represent new occurrence records for the floodplain. The species accumulation curve showed a continuous increase in gamma diversity, demonstrating the importance of long-term research for accurate knowledge of biodiversity in heterogeneous and dynamic ecosystems, such as the floodplains. Interannual beta diversity among studied years indicated a lesser alteration in community composition in 2001, when a long limnophase period was observed. In most of the environments, the highest species richness values were related to the greatest flooding amplitudes. Flooding amplitude, which is associated with connectivity, favors faunal exchange amongst the environments and between the pelagic and littoral zones. This explains the occurrence of both planktonic and non-planktonic species within the community. On the other hand, mean zooplankton abundance values were higher when a long isolation period occurred. Differences between the potamophase and limnophase amplitude associated with connectivity among the environments were the most important factors for the structure and dynamics of the zooplankton community in the Upper Paraná River floodplain.

O objetivo deste trabalho foi investigar o efeito da variação plurianual do regime hidrosedimentológico e da conectividade sobre a biodiversidade zooplanctônica da planície de inundação do Alto Rio Paraná. As amostragens do zooplâncton foram realizadas entre os anos de 2000 a 2007, em distintos ambientes dessa planície de inundação, incluindo lagoas abertas, lagoas fechadas, ressacos, rios e canais. A comunidade zooplanctônica foi constituída por 541 espécies, sendo os rotíferos o grupo mais especioso e abundante. Dentre essas espécies, 71 representam novas ocorrências para a planície de inundação. A curva de acumulação de espécies mostrou um incremento continuo da diversidade gama evidenciando a importância de estudos de longa duração para o conhecimento da real biodiversidade em ecossistemas heterogêneos e dinâmicos, como são as planícies de inundação. Os resultados da diversidade beta entre os anos estudados mostraram uma menor alteração da composição da comunidade em 2001, quando foi observado um longo período de limnofase. A maior riqueza média de espécies foi associada à grande amplitude de alagamento na maioria dos ambientes. A duração da inundação, associada à conectividade, favorece o intercâmbio de fauna entre os ambientes, bem como entre as regiões pelágica e litorânea, propiciando a ocorrência de espécies planctônicas e não planctônicas na comunidade. Por outro lado, a abundância da comunidade zooplanctônica foi maior quando ocorreu um extenso período de isolamento. Diferenças entre a amplitude de potamofase e a limnofase associada à conectividade entre os ambientes foram fatores preponderantes para estruturação e dinâmica da comunidade zooplanctônica na planície de Inundação do Alto Rio Paraná.

Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo.

Oncolytic viruses kill cancer cells by tumor-selective replication. Clinical data have established the safety of the approach but also the need of improvements in potency. Efficacy of oncolysis is linked to effective infection of target cells and subsequent productive replication. Other variables include intratumoral barriers, access to target cells, uptake by non-target organs and immune response. Each of these aspects relates to the location and degree of virus replication. Unfortunately, detection of in vivo replication has been difficult, labor intensive and costly and therefore not much studied. We hypothesized that by coinfection of a luciferase expressing E1-deleted virus with an oncolytic virus, both viruses would replicate when present in the same cell. Photon emission due to conversion of D-Luciferin is sensitive and penetrates tissues well. Importantly, killing of animals is not required and each animal can be imaged repeatedly. Two different murine xenograft models were used and intratumoral coinjections of luciferase encoding virus were performed with eight different oncolytic adenoviruses. In both models, we found significant correlation between photon emission and infectious virus production. This suggests that the system can be used for non-invasive quantitation of the amplitude, persistence and dynamics of oncolytic virus replication in vivo, which could be helpful for the development of more effective and safe agents.

Platelet activation is increased in cyclosporin A-induced hypertensive rats.

One of the most severe side effects of the immunosuppressive agent, cyclosporin A (CsA), is increased risk of thromboembolic complications and drug-related hypertension. Because platelets might be involved in these processes, we tested the possibility of CsA affecting platelet activation, which might contribute to these adverse drug reactions. The experiments were done using Wistar rats, treated or not (control) with CsA (Sandimmun Neoral), 5 and 30 mg/kg/day, for 7 weeks. Systolic, diastolic, and mean blood pressures, intracellular free calcium concentration ([Ca2+]i), platelet serotonin (5-HT) contents, and aggregation were determined, at weeks 0, 2, and 7 of treatment. Inositol phosphates (InsP) production, platelet thromboxane A2 (TXA2) generation, and morphology of platelets, through electron microscopy studies, also were compared. It was demonstrated that blood pressures increased in the CsA-treated groups, when compared with the control group, after 2 and 7 weeks of administration. CsA at both "attack" and "maintenance" doses increased basal, 5-HT, and thrombin-evoked [Ca2+]i after 2 and 7 weeks versus the control group. However, basal and evoked InsP production was stimulated by 5 mg/kg of CsA, but inhibited by 30 mg/kg, when compared with the control. Platelet 5-HT contents decreased significantly after 2 and 7 weeks in the CsA-treated groups, when compared with the control group. Collagen-induced whole blood platelet aggregation increased drastically in the "attack" CsA-treated group, whereas adenosine diphosphate (ADP)-induced platelet aggregation did not reach statistical significance. Finally, in vitro basal, collagen-, and ADP-evoked platelet TXA2 generation increased in both CsA concentrations, versus the control. In conclusion, our study demonstrates that both CsA doses alter platelet calcium homeostasis (even affecting the calcium fluxes differently), 5-HT and TXA2 contents and aggregation, which might contribute to the development and/or maintenance of high blood pressures and increased risk of thromboembolic complications.

Ultrastructure of the mature pollen of Michelia figo (Lour.) Spreng. (Magnoliaceae).

The structural organization of the Michelia figo mature pollen was investigated. The pollen wall consisted of an outer exine and an inner intine, the former being coated by a thin polysaccharide pellicle. The intine comprised three structurally distinct layers that were equally thick throughout the pollen surface. The generative cell (GC) was closely associated with the vegetative cell (VC) nucleus and its periplasm was found to maintain communication with the sporoderm through a complex plasmalemmic cord. In the freeze-fixed pollen a fluffy coat was detected on the cytoplasmic face of the VC plasmalemma bordering the GC. The plastids were present in only the VC and usually contained abundant small starch grains. In a few pollen grains, however, little or no starch existed and in this case one or more electron dense inclusions appeared in the plastids. Microbodies were found in both the VC and GC. In the VC they presumably have a glyoxysomal function as indicated by the numerous lipid droplets in the cytoplasm and the spatial relationship of microbodies with lipid droplets and/or mitochondria. In the GC the function of the microbodies is unclear once this cell had no abundant lipid reserves and the microbodies did not show any preferential relationship with other organelles. The most conspicuous feature of the VC cytoplasm was the high amount of storage vacuoles which displayed a striking different appearance after one and the other of the fixation techniques used. In contrast to the chemically fixed pollen they were quite polymorphic in the freeze-fixed pollen, and appeared uniformly filled with fibrillar material. Enzymatic digestion with protease has revealed most of this material to be proteinaceous in nature. The existence of phytin reserves is, however, also probable. These protein storage vacuoles closely resemble those in storage tissues of seeds and fruits.