Studies on the Energy Metabolism of Herpetomonads

Herpetomonas ingenoplastis is an intriguing and unusual trypanosomatid with many anaerobic features, including the lack of a complete cytochrome chain, the presence of an acristate mitochondrion and the ability to grow equally well under aerobic and anaerobic conditions. In this study, I compared the glucose catabolism of H. ingenoplastis and the aerobic H. muscarum , with the aim of providing greater insight into the mechanisms whereby H. ingenoplastis manages to meet its energetic requirements under aerobic and anaerobic conditions. The results of this study showed that both H. ingenoplastis and H. muscarum can consume glucose. This correlates with the discovered presence of enzymes of both the Embden-Meyerhof pathway and the pentose-phosphate shunt. The activities of these enzymes are similar in the two organisms. In both species the specific activities of the Embden-Meyerhof pathway enzymes, hexokinase (HK), phosphoglucoseisomerase (PGI) and phosphofructokinase (PFK), were found to be much higher than the activities of two enzymes of the pentose-phosphate shunt, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Glucose consumption varied with gaseous conditions. Anaerobic conditions caused a reduction in glucose consumption, compared with aerobic conditions, in the case of H. muscarum. The addition of CO2 caused an increase in the rate of glucose consumption from that under argon (anaerobic) alone. Similarly for H. ingenoplastis, glucose consumption was reduced under anaerobic conditions compared with aerobic conditions and was stimulated by the presence of CO2. Herpetomonas muscarum was found to produce succinate and acetate as major excretory products of glucose catabolism, and ethanol and propionate as minor excretory products. Compared with aerobic conditions succinate production increased under aerobic conditions in the presence of CO2 by about 2-fold and up to 6-fold in the case of anaerobic conditions in the presence of CO2. Acetate production increased slightly under the two conditions containing high CO2. Herpetomonas ingenoplastis produced propionate as a major end product of glucose catabolism, along with succinate, acetate and ethanol. Ethanol production was found to be greatest under aerobic and aerobic plus CO2 conditions, while succinate and propionate were found to be produced in the greatest quantities under anaerobic plus CO2 conditions. The possibility that propionate production may help supply the energy needs of H. ingenoplastis is discussed. One factor that may be important in the energy metabolism of both herpetomonads are the CO2-fixation pathways. Two CO2-fixing enzymes, 'malic' enzyme (ME) and phosphoenolpyruvate carboxykinase (PEPCK), were found at high activities in both organisms. This correlated with succinate production in the presence of CO2. There were differences between the two species with respect to the tricarboxylic acid (TCA) cycle enzymes. Notable was the presence of fumarate reductase (FR) and the absence of succinate dehydrogenase (SDH), in the case of H. ingenoplastis. Evidence produced suggests that the TCA cycle of H. ingenoplastis operates primarily in the reverse direction. Herpetomomas muscarum was found to have both FR and SDH, suggesting flux through the TCA cycle in both directions. Succinate dehydrogenase/fumarate reductase ratios of 5. 02 and <0. 08 for H. muscarum and H. ingenoplastis, respectively, suggest that H. ingenoplastis is an organism that prefers anaerobic conditions, while H. muscarum is a facultative anaerobe. Differences between H. muscarum and H. ingenoplastis were also found at the subcellular level with respect to studies on the possible presence of glycosomes in the two herpetomonads. Some evidence was obtained for the presence of glycosomes in H. muscarum, in that HK, PGI and malate dehydrogenase (MDH) all were recovered, in part, in the particulate fraction. The distribution of the enzymes of H. ingenoplastis was found to be very different, suggesting that either this organism lacks glycosomes or that the glycosomes are much more labile that those of H. muscarum. Both FR and SDH were found to be particulate in H. muscarum which is consistent with a location in the mitochondrial membrane, perhaps as part of the electron transport chain. The FR activity in H. ingenoplastis seemed to be approximately equally divided between the soluble and particulate fractions. Pyruvate kinase is possibly the major site of glycolytic control in H. ingenoplastis. This enzyme requires MgCI2 to function and is activated by fructose-1,6-bisphosphate (F-1,6-P2) and fructose-2,6-bisphosphate (F-2,6-P2). The enzyme has an apparent Km for ADP of 1.3 mM, much higher than that of other trypanosomatids, and the activity shows sigmoid kinetics with respect to phosphoenolpyruvate (PEP) concentration. (Abstract shortened by ProQuest.)

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Maternal Circulating Levels of Activin A, Inhibin A, sFlt-1 and Endoglin at Parturition in Normal Pregnancy and Pre-Eclampsia

Background: Maternal circulating levels of anti-angiogenic factors such as soluble fms-like tyrosine kinase-1 (sFlt-1), endoglin (sEng) and placental proteins like activin A and inhibin A are increased before the onset of pre-eclampsia. There is evidence for oxidative stress in pre eclampsia. Recently it was shown that placental oxygen concentration is related to sFlt-1 and inhibin A. In addition it is reported that oxidative stress markers are increased in placental tissue delivered after labour. Therefore, the objective of this study is to investigate if these proteins are altered in maternal circulation of labouring preeclampsia and normal pregnancies. Methodology: To assess the effects of labour, samples were taken from 10 normal pregnant (NP) and 10 pre-eclamptic (PE) women pre-labour, full dilation, placental delivery and 24 h. To assess the effects of placental delivery, plasma samples were taken from 10NP and 10PE women undergoing elective Caesarean section, pre-delivery, placental delivery and 10 min, 60 min and 24 h post delivery. SFlt-1 and sEng and activin A and inhibin A were measured using commercial and in house ELISA’s respectively. Results: The levels of sFlt-1 and sEng were significantly higher in PE compared to NP women in both groups. In labour, sFlt- 1 levels increased significantly at full dilatation in PE women, before declining by 24 hr. However there was no significant rise in sEng levels in labour. Activin A and inhibin A levels declined rapidly with placental delivery in NP and PE pregnancies. There was a significant rise in activin A levels during labour in PE compared to pre labour, but inhibin levels did not increase. Conclusion: Labour in pre-eclamptic women increases the levels of sFlt-1 and activin A. This pilot data suggests that increase in the maternal levels of these factors in labour could predict and/or contribute to the maternal syndrome postpartum.Citation: Reddy, A. et al. (2009). 'Maternal circulating levels of activin A, inhibin A, sFlt-1 and endoglin at parturition in normal pregnancy and pre-eclampsia', PLoS ONE 4(2): e4453. [Available at http://www.plosone.org]. © 2009 Reddy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Measurement of refractive index by nanoparticle tracking analysis reveals heterogeneity in extracellular vesicles

Introduction: Optical techniques are routinely used to size and count extracellular vesicles (EV). For comparison of data from different methods and laboratories, suitable calibrators are essential. A suitable calibrator must have a refractive index (RI) as close to that of EV as possible but the RI of EV is currently unknown. To measure EV, RI requires accurate knowledge of size and light scattering. These are difficult to measure as most EVs cannot be resolved by light microscopy and their diameter is smaller than the wavelength of visible light. However, nanoparticle tracking analysis (NTA) provides both size and relative light scattering intensity (rLSI) values. We therefore sought to determine whether it was possible to use NTA to measure the RI of individual EVs. Methods: NTA was used to measure the rLSI and size of polystyrene and silica microspheres of known size and RI (1.470 and 1.633, respectively) and of EV isolated from a wide range of cells. We developed software, based on Mie scattering code, to calculate particle RI from the rLSI data. This modelled theoretical scattering intensities for polystyrene and silica microspheres of known size (100 and 200 nm) and RI. The model was verified using data from the polystyrene and silica microspheres. Size and rLSI data for each vesicle were processed by the software to generate RI values. Results: The following modal RI measurements were obtained: fresh urinary EV 1.374, lyophilised urinary EV 1.367, neuroblastoma EV 1.393, blood EV 1.398, EV from activated platelets 1.390, small placental EV 1.364–1.375 and 1.398–1.414 for large placental EV (>200 nm). Large placental EV had a significantly higher RI than small placental EV (p<0.0001). The spread of RI values was narrower for small EV than for the more heterogeneous large EV. Discussion: Using NTA and Mie scattering theory, we have demonstrated that it is possible to estimate the RI of sub-micron EV using NTA data. EV typically had a modal RI of 1.37–1.39, whereas values of >1.40 were observed for some large (>200 nm) microvesicles. Conclusion: This method for measuring EV RI will be useful for developing appropriate calibrators for EV measurement

Combinations of Maternal KIR and Fetal HLA-C Genes Influence the Risk of Preeclampsia and Reproductive Success

Preeclampsia is a serious complication of pregnancy in which the fetus receives an inadequate supply of blood due to failure of trophoblast invasion. There is evidence that the condition has an immunological basis. The only known polymorphic histocompatibility antigens on the fetal trophoblast are HLA-C molecules. We tested the idea that recognition of these molecules by killer immunoglobulin receptors (KIRs) on maternal decidual NK cells is a key factor in the development of preeclampsia. Striking differences were observed when these polymorphic ligand: receptor pairs were considered in combination. Mothers lacking most or all activating KIR (AA genotype) when the fetus possessed HLA-C belonging to the HLA-C2 group were at a greatly increased risk of preeclampsia. This was true even if the mother herself also had HLA-C2, indicating that neither nonself nor missing-self discrimination was operative. Thus, this interaction between maternal KIR and trophoblast appears not to have an immune function, but instead plays a physiological role related to placental development. Different human populations have a reciprocal relationship between AA frequency and HLA-C2 frequency, suggesting selection against this combination. In light of our findings, reproductive success may have been a factor in the evolution and maintenance of human HLA-C and KIR polymorphisms

State legislators are less likely to respond to constituents who are not white

The election of Donald Trump in 2016 has reignited debates over immigration and race in America. In new research, Micah Gell-Redman, Neil Visalvanich, Charles Crabtree, and Christopher Fariss examine how state legislators respond to minority and immigrant constituents by sending emails to more than 5,000 elected officials in 42 states. They find that Black and Hispanic senders were less likely to receive responses compared to whites, and were especially less likely from Republican legislators. Asians, they write, were even less likely to receive responses, but this was regardless of whether the legislator was a Republican or Democrat

Assessing Peracetic Acid Application Methodology and Impacts on Fluidized Sand Biofilter Performance

Nitrifying biofilters oxidize harmful ammonia excreted by fish into less toxic nitrate within recirculating aquaculture systems (RAS). Biofilter performance and resulting RAS water quality largely depend on a robust microbiome that effectively converts nitrogenous wastes; however, occasional use of water disinfectants may also be necessary to reduce or eliminate specific fish pathogens. Disinfectants and sanitizers such as peracetic acid (PAA) work by disrupting microbial activity and could unintentionally alter the microbially-driven nitrification biofiltration process if allowed to circulate within an RAS. Furthermore, the target concentration and application method of PAA may influence the level of biofilter disruption. For this study, 12 replicated experimental-scale fluidized sand biofilters were dosed with PAA to achieve target concentrations ranging from 1.0-–2.5 mg/L, a typical low-dose treatment range to reduce or eliminate opportunistic pathogens. Two application methods were compared, including (i) a single pulse of PAA added every other day for five days, and (ii) smaller doses of PAA added every five minutes over four hours. The PAA decay was monitored and predosing and postdosing water quality parameters were assessed. Regardless of the target concentration or application method, PAA addition within the tested range did not cause significant disruption to the biofilters’ nitrification processes. This research demonstrates that PAA may be a viable water sanitizer for the RAS industry, although further research to refine safe application protocols is necessary.Assessing Peracetic Acid Application Methodology and Impacts on Fluidized Sand Biofilter PerformancepublishedVersio

CD8+ T-cell recognition of a synthetic epitope formed by t-butyl modification

We set out to clone Bax-specific CD8+ T cells from peripheral blood sam- ples of patients with primary chronic lymphocytic leukaemia. A number of clones were generated using a Bax peptide pool and their T-cell epitope was mapped to two peptides sharing a common 9-amino-acid sequence (LLSYFGTPT), restricted by HLA-A*0201. However, when these T-cell clones were tested against highly purified syntheses (> 95%) of the same peptide sequence, there was no functional response. Subsequent mass spectrometric analysis and HPLC fractionation suggested that the active component in the original crude peptide preparations (77% pure) was a peptide with a tert-butyl (tBu) modification of the tyrosine residue. This was confirmed by modification of the inactive wild-type sequence to gen- erate functionally active peptides. Computer modelling of peptide:HLA- A*0201 structures predicted that the tBu modification would not affect interactions between peptide residues and the HLA binding site. However, these models did predict that the tBu modification of tyrosine would result in an extension of the side chain out of the peptide-binding groove up towards the T-cell receptor. This modified product formed < 1% of the original P603 crude peptide preparation and < 0.05% of the origi- nal 23-peptide mixture used for T-cell stimulation. The data presented here, illustrate the potential for chemical modifications to change the immunogenicity of synthetic peptides, and highlight the exquisite capacity of T-cell receptors to discriminate between structurally similar peptide sequences. Furthermore, this study highlights potential pitfalls associated with the use of synthetic peptides for the monitoring and modulating of human immune responses

Concurrent whole brain radiotherapy and bortezomib for brain metastasis

Abstract Background Survival of patients with brain metastasis particularly from historically more radio-resistant malignancies remains dismal. A phase I study of concurrent bortezomib and whole brain radiotherapy was conducted to determine the tolerance and safety of this approach in patients with previously untreated brain metastasis. Methods A phase I dose escalation study evaluated the safety of bortezomib (0.9, 1.1, 1.3, 1.5, and 1.7 mg/m2) given on days 1, 4, 8 and 11 of whole brain radiotherapy. Patients with confirmed brain metastasis were recruited for participation. The primary endpoint was the dose-limiting toxicity, defined as any ≥ grade 3 non-hematologic toxicity or grade ≥ 4 hematologic toxicity from the start of treatment to one month post irradiation. Time-to-Event Continual Reassessment Method (TITE-CRM) was used to determine dose escalation. A companion study of brain diffusion tensor imaging MRI was conducted on a subset of patients to assess changes in the brain that might predict delayed cognitive effects. Results Twenty-four patients were recruited and completed the planned therapy. Patients with melanoma accounted for 83% of all participants. The bortezomib dose was escalated as planned to the highest dose of 1.7 mg/m2/dose. No grade 4/5 toxicities related to treatment were observed. Two patients had grade 3 dose-limiting toxicities (hyponatremia and encephalopathy). A partial or minor response was observed in 38% of patients. Bortezomib showed greater demyelination in hippocampus-associated white matter structures on MRI one month after radiotherapy compared to patients not treated with bortezomib (increase in radial diffusivity +16.8% versus 4.8%; p = 0.0023). Conclusions Concurrent bortezomib and whole brain irradiation for brain metastasis is well tolerated at one month follow-up, but MRI changes that have been shown to predict delayed cognitive function can be detected within one month of treatment.http://deepblue.lib.umich.edu/bitstream/2027.42/112849/1/13014_2013_Article_928.pd