Experiment: Response of Nodularia spumigena to pCO2

The filamentous and diazotrophic cyanobacterium Nodularia spumigena plays a major role in the productivity of the Baltic Sea as it forms extensive blooms regularly. Under phosphorus limiting conditions Nodularia spumigena has a high enzyme affinity for dissolved organic phosphorus (DOP) by production and release of alkaline phosphatase. Additionally, it is able to degrade proteinaceous compounds by expressing the extracellular enzyme leucine aminopeptidase. As atmospheric CO2 concentrations are increasing, we expect marine phytoplankton to experience changes in several environmental parameters including pH, temperature, and nutrient availability. The aim of this study was to investigate the combined effect of CO2-induced changes in seawater carbonate chemistry and of phosphate deficiency on the exudation of organic matter, and its subsequent recycling by extracellular enzymes in a Nodularia spumigena culture. Batch cultures of Nodularia spumigena were grown for 15 days aerated with three different pCO2 levels corresponding to values from glacial periods to future values projected for the year 2100. Extracellular enzyme activities as well as changes in organic and inorganic compound concentrations were monitored. CO2 treatment-related effects were identified for cyanobacterial growth, which in turn was influencing exudation and recycling of organic matter by extracellular enzymes. Biomass production was increased by 56.5% and 90.7% in the medium and high pCO2 treatment, respectively, compared to the low pCO2 treatment and simultaneously increasing exudation. During the growth phase significantly more mucinous substances accumulated in the high pCO2 treatment reaching 363 µg Gum Xanthan eq /l compared to 269 µg Gum Xanthan eq /l in the low pCO2 treatment. However, cell-specific rates did not change. After phosphate depletion, the acquisition of P from DOP by alkaline phosphatase was significantly enhanced. Alkaline phosphatase activities were increased by factor 1.64 and 2.25, respectively, in the medium and high compared to the low pCO2 treatment. In conclusion, our results suggest that Nodularia spumigena can grow faster under elevated pCO2 by enhancing the recycling of organic matter to acquire nutrients.

CRISPR/Cas9-Mediated Gene Editing of ARG1 in Cell Lines and Primary Cells

Arginase 1 deficiency, a urea cycle disorder resulting from an inability of the body to convert arginine into urea, results in hyperargininemia and sporadic episodes of hyperammonemia. Arginase 1 deficiency can lead to a range of developmental disorders and progressive spastic diplegia in children, and current therapeutic options are limited. Clustered regularly interspaced short palindromic repeat (CRISPR) /CRISPR associated protein (Cas) 9 gene editing systems serve as a novel means of treating genetic disorders such as Arginase 1 (ARG1) deficiency, and must be thoroughly examined to determine their curative capabilities. In these experiments numerous guide RNAs and CRISPR/Cas9 systems targeting the ARG1 gene were designed and observed by heteroduplex assay for their targeting capabilities and cleavage efficiencies in multiple cell lines. The CRISPR/Cas9 system utilized in these experiments, along with a panel of guide RNAs targeting various locations in the arginase 1 gene, successfully produced targeted cleavage in HEK293, MCF7, A549, K562, HeLa, and HepG2 cells; however, targeted cleavage in human dermal fibroblasts, blood outgrowth endothelial cells, and induced pluripotent stem cells was not observed. Additionally, a CRISPR/Cas system involving partially inactivated Cas9 was capable of producing targeted DNA cleavage in intron 1 of ARG1, while a Cas protein termed Cpf1 was incapable of producing targeted cleavage. These results indicate a complex set of variables determining the CRISPR/Cas9 systems’ capabilities in the cell lines and primary cells tested. By examining epigenetic factors and alternative CRISPR/Cas9 gene targeting systems, the CRISPR/Cas9 system can be more thoroughly considered in its ability to act as a means towards editing the genome of arginase 1-deficient individuals.

THE EFFECT OF PREECLAMPSIA ON RENAL FUNCTION: CROSS-SECTIONAL STUDIES OF POSTPARTUM PREECLAMPTICS AND WOMEN WHO WERE DESTINED TO DEVELOP PREECLAMPSIA

Preeclampsia (PE) is a pregnancy complication that is new-onset of hypertension and proteinuria after 20 weeks of gestation. However, subclinical renal dysfunction may be apparent earlier in gestation prior to the clinical presentation of PE. Although the maternal syndrome of PE resolves early postpartum, women with a history of PE are at higher risk of renal dysfunction later in life. Mineral metabolism, such as phosphate balance is heavily dependent on renal function, yet, phosphate handling in women with a history of PE is largely unknown. To investigate whether women with a history of PE would exhibit changes in phosphate metabolism compared to healthy parous women, phosphate loading test was used. Women with or without a history of PE, who were 6 months to 5 years postpartum, were recruited for this study. Blood and urine samples were collected before and after the oral dosing of 500mg phosphate solution. Biochemical markers of phosphate metabolism and renal function were evaluated. In order to assess the difference in renal function alteration between first trimester women who were or were not destined to develop PE, plasma cystatin C concentration was analysed. After phosphate loading, women with a history of PE had significantly elevated serum phosphate at both 1- and 2-hour, while controls had higher urine phosphate:urine creatinine excretion ratio at 1-hour than women with a history of PE. Women with a history of PE had no changes in intact parathyroid hormone (iPTH) concentration throughout the study period, whereas controls had elevated iPTH at 1-hour from baseline. In terms of renal function in the first trimester, there was no difference in plasma cystatin C concentration between women who were or were not destined to develop PE. The elevation of serum phosphate in women with a history of PE could be due to the delay in phosphate excretion. Prolong elevation of serum phosphate can have serious consequences later in life. Thus, oral phosphate challenge may serve as a useful method of early screening for altered phosphate metabolism and renal function.

THE EFFECT OF PREECLAMPSIA ON RENAL FUNCTION: CROSS-SECTIONAL STUDIES OF POSTPARTUM PREECLAMPTICS AND WOMEN WHO WERE DESTINED TO DEVELOP PREECLAMPSIA

Preeclampsia (PE) is a pregnancy complication that is new-onset of hypertension and proteinuria after 20 weeks of gestation. However, subclinical renal dysfunction may be apparent earlier in gestation prior to the clinical presentation of PE. Although the maternal syndrome of PE resolves early postpartum, women with a history of PE are at higher risk of renal dysfunction later in life. Mineral metabolism, such as phosphate balance is heavily dependent on renal function, yet, phosphate handling in women with a history of PE is largely unknown. To investigate whether women with a history of PE would exhibit changes in phosphate metabolism compared to healthy parous women, phosphate loading test was used. Women with or without a history of PE, who were 6 months to 5 years postpartum, were recruited for this study. Blood and urine samples were collected before and after the oral dosing of 500mg phosphate solution. Biochemical markers of phosphate metabolism and renal function were evaluated. In order to assess the difference in renal function alteration between first trimester women who were or were not destined to develop PE, plasma cystatin C concentration was analysed. After phosphate loading, women with a history of PE had significantly elevated serum phosphate at both 1- and 2-hour, while controls had higher urine phosphate:urine creatinine excretion ratio at 1-hour than women with a history of PE. Women with a history of PE had no changes in intact parathyroid hormone (iPTH) concentration throughout the study period, whereas controls had elevated iPTH at 1-hour from baseline. In terms of renal function in the first trimester, there was no difference in plasma cystatin C concentration between women who were or were not destined to develop PE. The elevation of serum phosphate in women with a history of PE could be due to the delay in phosphate excretion. Prolong elevation of serum phosphate can have serious consequences later in life. Thus, oral phosphate challenge may serve as a useful method of early screening for altered phosphate metabolism and renal function.

Structural and Functional Characterization of the Human Tribbles Homologue 2 Pseudokinase

The Tribbles Homologues are a family of three eukaryotic pseudokinases (Trb1, Trb2, Trb3) that act as allosteric inhibitors and regulatory scaffold sites in pathways governing adipogenesis, cell proliferation and insulin signaling. The Tribbles Homologues have the same overall tertiary structure of the eukaryotic protein kinase domain, but lack multiple residues necessary to catalysis in the nucleotide-binding P-loop and the Mg2+-coordinating DFG motif. Trb1 has been shown conclusively to be incapable of binding ATP, whereas a recent study presents evidence that Trb2 autophosphorylates independently of Mg2+ in vitro. This finding is surprising given the high degree of sequence similarity between the two proteins (71%), and suggests unique nucleotide binding and phosphotransfer mechanisms. The goal of this project was to investigate whether Trb2 possesses kinase activity or not and determine its structural basis. A method for the high-yield recombinant expression and purification of stable Trb2 was developed. Trb2 nucleotide binding and autophosphorylation could not be detected across multiple experimental approaches, including thermal shift assays, MANT-ATP fluorescence, radiolabeled phosphate incorporation, and nonspecific ATPase activity assays. Further characterization also revealed that Trb2 forms homomultimers with possible functional consequences, and extensive crystallization screening has yielded multiple promising conditions that could produce diffraction-quality crystals with further optimization. This project explores the difficulties in functionally characterizing putatively active pseudokinases, and proposes a structural basis for the conserved pseudokinase features of the Tribbles homologues.

Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation.

METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1β response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover.

RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1β release from macrophages, through lysosomal destabilization. IL-1β secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1β release was significantly reduced in primary macrophages from ctss(-/-) mice.

CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1β release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.

Supporting Cubane’s Renaissance: Metathesis reactions on 4-iodo-1-vinylcubane and Stetter reaction on 1-iodocubane-4-carboxaldehyde

Cubane is a peculiar cube-shaped alkane molecule with a rigid, regular structure. This makes it a good scaffold, i.e. a molecular platform to which the substituents are arranged in a specific and fixed orientation. Moreover, cubane has a body diagonal of 2.72 Å, very similar to the distance across the benzene ring, i.e. 2.79 Å. Thus, it would be possible to use cubane as a scaffold in medicinal and material chemistry as a benzene isostere 1,2. This could lead to advantages in terms of solubility and toxicity and could provide novel properties. For this purpose, the possibility of performing “modern organic chemistry” on the cubane scaffold has to be studied. This project was entirely carried out in the framework of the Erasmus+ mobility programme at the Trinity College (Dublin, IRL) under the supervision of prof. M. O. Senge. The main goal of this project was to widen the knowledge on cubane chemistry. In particular, it was decided to test reactions that were never applied to the scaffold before, such as metathesis of 4-iodo-1-vinylcubane and Stetter reaction of 1-iodocubane-4-carboxaldehyde. These two molecules were synthesized in 10 and 9 steps respectively from commercially available cyclopentanone, following a known procedure. Unfortunately, metathesis with different olefins, such as styrene, α,β unsaturated compounds and linear α-olefins failed under different conditions, highlighting cubane behaves as a Type IV, challenging olefin under metathesis conditions. Even the employment of a specific catalyst for hindered olefins failed in the cross-coupling with linear α-olefins. On the other hand, two new molecules were synthesized via Stetter reaction and benzoin condensation respectively. Even if the majority of the reactions were not successful, this work can be seen as an inspiration for further investigation on cubane chemistry, as new questions were raised and new opportunities were envisioned.

Renal Drug Transporters – Clinical Significance and Role in Drug-Drug Interactions

Thesis (Ph.D.)--University of Washington, 2016-07

Congenital adrenal hyperplasia: focus on the molecular basis of 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by defects in one of several steroidogenic enzymes involved in the synthesis of cortisol from cholesterol in the adrenal glands. More than 90% of cases are caused by 21-hydroxylase deficiency, and the severity of the resulting clinical symptoms varies according to the level of 21-hydroxylase activity. 21-Hydroxylase deficiency is usually caused by mutations in the CYP21A2 gene, which is located on the RCCX module, a chromosomal region highly prone to genetic recombination events that can result in a wide variety of complex rearrangements, such as gene duplications, gross deletions and gene conversions of variable extensions. Molecular genotyping of CYP21A2 and the RCCX module has proved useful for a more accurate diagnosis of the disease, and prenatal diagnosis. This article summarises the clinical features of 21-hydroxylase deficiency, explains current understanding of the disease at the molecular level, and highlights recent developments, particularly in diagnosis.

Proton-pump inhibitors adverse effects: a review of the evidence and position statement by the Sociedad Española de Patología Digestiva

Introduction: In the last few years a significant number of papers have related the use of proton-pump inhibitors (PPIs) to potential serious adverse effects that have resulted in social unrest. Objective: The goal of this paper was to provide a literature review for the development of an institutional position statement by Sociedad Española de Patología Digestiva (SEPD) regarding the safety of long-term PPI use. Material and methods: A comprehensive review of the literature was performed to draw conclusions based on a critical assessment of the following: a) current PPI indications; b) vitamin B12 deficiency and neurological disorders; c) magnesium deficiency; d) bone fractures; e) enteric infection and pneumonia; f) interactions with thienopyridine derivatives; e) complications in cirrhotic patients. Results: Current PPI indications have remained unchanged for years now, and are well established. A general screening of vitamin B12 levels is not recommended for all patients on a PPI; however, it does seem necessary that magnesium levels be measured at therapy onset, and then monitored in subjects on other drugs that may induce hypomagnesemia. A higher risk for bone fractures is present, even though causality cannot be concluded for this association. The association between PPIs and infection with Clostridium difficile is mild to moderate, and the risk for pneumonia is low. In patients with cardiovascular risk receiving thienopyridines derivatives it is prudent to adequately consider gastrointestinal and cardiovascular risks, given the absence of definitive evidence regardin potential drug-drug interactions; if gastrointestinal risk is found to be moderate or high, effective prevention should be in place with a PPI. PPIs should be cautiously indicated in patients with decompensated cirrhosis. Conclusions: PPIs are safe drugs whose benefits outweigh their potential side effects both short-term and long-term, provided their indication, dosage, and duration are appropriate.

Iron deficiency anaemia among apparently healthy pre-school children in Lagos, Nigeria.

Background: Iron deficiency, and specifically iron deficiency anaemia, remains one of the most severe and important nutritional deficiencies in the world today. Objective: To estimate the prevalence and associated factors for iron deficiency anaemia among pre-school children in Lagos. Methodology: The study was conducted from December 2009 to February 2010 at the outpatient clinics of Lagos State University Teaching Hospital, Lagos. Serum iron, total iron binding capacity, transferrin saturation and serum ferritin were assayed in subjects. The primary outcome measured was iron deficiency anaemia established based on the following criteria: hemoglobin <11.0 g/dl1 plus 2 or more of the following: MCV <70fl, transferrin saturation <10% or serum ferritin <15ng/ dL. Statistical analysis included Pearson Chi square analysis and logistic regression analysis. Results: A total of 87 apparently healthy subjects were recruited. Only one subject had iron depletion and this child belonged to the ≤ 2 years age category. None of the recruited subjects had iron deficiency without anaemia. Nine of the study subjects (10.11%) had iron deficiency anaemia. The prevalence of iron deficiency anaemia was significantly higher among younger age group than in the older age group (19.1% Vs 2.1%, p = 0.022). The prevalence of iron deficiency anaemia was significantly higher among subjects with weight-for-age, and weight-for-height Z scores below two standard scores (83.3% and 75.0% respectively, p = <0.001 and 0.001 respectively). Conclusion: The overall prevalence of iron deficiency anaemia among study subjects was 10.11%. Iron deficiency anaemia was more common in children aged two years and below. Weight-for-age and weight-for-height Z scores below minus two standard scores were strongly associated with iron deficiency anaemia.

Cadmium availability and accumulation by lettuce and rice

Among the toxic elements, Cd has received considerable attention in view of its association with a number of human health problems. The objectives of this study were to evaluate the Cd availability and accumulation in soil, transfer rate and toxicity in lettuce and rice plants grown in a Cd-contaminated Typic Hapludox. Two simultaneous greenhouse experiments with lettuce and rice test plants were conducted in a randomized complete block design with four replications. The treatments consisted of four Cd rates (CdCl2), 0.0; 1.3; 3.0 and 6.0 mg kg(-1), based on the guidelines recommended by the Environmental Agency of the State of São Paulo, Brazil (Cetesb). Higher Cd rates increased extractable Cd (using Mehlich-3, Mehlich-1 and DTPA chemical extractants) and decreased lettuce and rice dry matter yields. However, no visual toxicity symptoms were observed in plants. Mehlich-1, Mehlich-3 and DTPA extractants were effective in predicting soil Cd availability as well as the Cd concentration and accumulation in plant parts. Cadmium concentration in rice remained below the threshold for human consumption established by Brazilian legislation. on the other hand, lettuce Cd concentration in edible parts exceeded the acceptable limit.

Distribution of Ra-226, Th-232 and K-40 in soils and sugar cane crops at Corumbatai river basin, São Paulo State, Brazil

The common use of phosphate fertilizers NPK and amendments in sugar cane crops in Brazilian agriculture may increase the Ra-226, Th-232 and K-40 activity concentrations in soils and their availability for plants and human food chain. Thus, the main aim of this study was to evaluate the distribution of Ra-226, Th-232 and K-40 in soils and sugar cane crops in the Corumbatai river basin, São Paulo State, Brazil. The gamma spectrometry was utilized to measure the Ra-226, Th-232 and K-40 activity concentration in all samples. The soil-to-sugar cane transfer factors (TF) were quantified using the ratio between the radionuclide activity concentration in sugar cane and its activity concentration in soil. The results show that, although radionuclides incorporated in phosphate fertilizers and amendments are annually added in the sugar cane crops, if utilized in accordance with the recommended rates, their use does not lead to hazards levels in soils. The soil-to-sugar cane transfer of radionuclides occurred in the following order K-40 > Ra-226 > Th-232. Therefore, under these conditions, radionuclides intake through consumption of sugar is not hazardous to human health. (C) 2009 Elsevier Ltd. All rights reserved.

Sociodemographic factors influencing adherence to antenatal iron supplementation recommendations among pregnant women in Malawi: Analysis of data from the 2010 Malawi Demographic and Health Survey

Background and Aim: Maternal morbidity and mortality statistics remain unacceptably high in Malawi. Prominent among the risk factors in the country is anaemia in pregnancy, which generally results from nutritional inadequacy (particularly iron deficiency) and malaria, among other factors. This warrants concerted efforts to increase iron intake among reproductive-age women. This study, among women in Malawi, examined factors determining intake of supplemental iron for at least 90 days during pregnancy. Methods: A weighted sample of 10,750 women (46.7%), from the 23,020 respondents of the 2010 Malawi Demographic and Health Survey (MDHS), were utilized for the study. Univariate, bivariate, and regression techniques were employed. While univariate analysis revealed the percent distributions of all variables, bivariate analysis was used to examine the relationships between individual independent variables and adherence to iron supplementation. Chi-square tests of independence were conducted for categorical variables, with the significance level set at P < 0.05. Two binary logistic regression models were used to evaluate the net effect of independent variables on iron supplementation adherence. Results: Thirty-seven percent of the women adhered to the iron supplementation recommendations during pregnancy. Multivariate analysis indicated that younger age, urban residence, higher education, higher wealth status, and attending antenatal care during the first trimester were significantly associated with increased odds of taking iron supplementation for 90 days or more during pregnancy (P < 0.01). Conclusions: The results indicate low adherence to the World Health Organization’s iron supplementation recommendations among pregnant women in Malawi, and this contributes to negative health outcomes for both mothers and children. Focusing on education interventions that target populations with low rates of iron supplement intake, including campaigns to increase the number of women who attend antenatal care clinics in the first trimester, are recommended to increase adherence to iron supplementation recommendations.

Ecotoxicological assessment of sediments from the Santos and São Vicente estuarine system- Brazil

O sedimento representa um importante depósito de contaminantes e uma fonte de contaminação para a cadeia alimentar aquática. Testes de toxicidade usando anfípodos como organismos-teste são empregados para avaliar sedimentos marinhos e estuarinos, juntamente com análises químicas. O presente trabalho tem como objetivo avaliar a qualidade de sedimentos de seis estações situadas no Sistema Estuarino e Portuário de Santos e São Vicente (São Paulo-Brasil), usando testes de toxicidade aguda com sedimento com anfípodos (Tiburonella viscana) e análises químicas de metais, PCB, e PAH. Outros parâmetros do sedimento foram analisados, como carbono orgânico e granulometria. Foram observados níveis de contaminação mais altos na porção interna do estuário onde se localiza o Porto de Santos e a zona industrial. Os testes de toxicidade mostraram resultados adversos significantes para a maioria das amostras testadas, e os sedimentos da porção interna do estuário apresentaram toxicidade mais alta. As análises de componentes principais indicaram uma relação forte entre contaminação do sedimento e toxicidade. As correlações positivas destes fatores nas amostras estudadas foram usadas para estabelecer os pesos das concentrações químicas que estão associadas com os efeitos adversos. Tais análises permitiram estimar valores limiares de efeito para a contaminação de sedimento através de análises multivariadas, identificando os contaminantes associados com o efeito biológico. Estes valores sugeridos são: Cu, 69.0; Pb, 17.4; Zn, 73.3(mg.kg-1); PAHs, 0.5 (mg.kg-1) e PCBs, 0.1 (µg.kg-1).