Carbon sources and pathways for citrate secreted by human prostate cancer cells determined by NMR tracing and metabolic modeling.

Prostate epithelial cells have the unique capacity to secrete large amounts of citrate, but the carbon sources and metabolic pathways that maintain this production are not well known. We mapped potential pathways for citrate carbons in the human prostate cancer metastasis cell lines LNCaP and VCaP, for which we first established that they secrete citrate (For LNCaP 5.6 ± 0.9 nmol/h per 106 cells). Using 13C-labeled substrates, we traced the incorporation of 13C into citrate by NMR of extracellular fluid. Our results provide direct evidence that glucose is a main carbon source for secreted citrate. We also demonstrate that carbons from supplied glutamine flow via oxidative Krebs cycle and reductive carboxylation routes to positions in secreted citrate but likely do not contribute to its net synthesis. The potential anaplerotic carbon sources aspartate and asparagine did not contribute to citrate carbons. We developed a quantitative metabolic model employing the 13C distribution in extracellular citrate after 13C glucose and pyruvate application to assess intracellular pathways of carbons for secreted citrate. From this model, it was estimated that in LNCaP about 21% of pyruvate entering the Krebs cycle is converted via pyruvate carboxylase as an anaplerotic route at a rate more than sufficient to compensate carbon loss of this cycle by citrate secretion. This model provides an estimation of the fraction of molecules, including citrate, leaving the Krebs cycle at every turn. The measured ratios of 13C atoms at different positions in extracellular citrate may serve as biomarkers for (malignant) epithelial cell metabolism.

Transformation of coffee-growing landscapes across Latin America. A review.

In Latin America, the cultivation of Arabica coffee (Coffea arabica) plays a critical role in rural livelihoods, biodiversity conservation, and sustainable development. Over the last 20 years, coffee farms and landscapes across the region have undergone rapid and profound biophysical changes in response to low coffee prices, changing climatic conditions, severe plant pathogen outbreaks, and other drivers. Although these biophysical transformations are pervasive and affect millions of rural livelihoods, there is limited information on the types, location, and extent of landscape changes and their socioeconomic and ecological consequences. Here we review the state of knowledge on the ongoing biophysical changes in coffee-growing regions, explore the potential socioeconomic and ecological impacts of these changes, and highlight key research gaps. We identify seven major land-use trends which are affecting the sustainability of coffee-growing regions across Latin America in different ways. These trends include (1) the widespread shift to disease-resistant cultivars, (2) the conventional intensification of coffee management with greater planting densities, greater use of agrochemicals and less shade, (3) the conversion of coffee to other agricultural land uses, (4) the introduction of Robusta coffee (Coffea canephora) into areas not previously cultivated with coffee, (5) the expansion of coffee into forested areas, (6) the urbanization of coffee landscapes, and (7) the increase in the area of coffee produced under voluntary sustainability standards. Our review highlights the incomplete and scattered information on the drivers, patterns, and outcomes of biophysical changes in coffee landscapes, and lays out a detailed research agenda to address these research gaps and elucidate the effects of different landscape trajectories on rural livelihoods, biodiversity conservation, and other aspects of sustainable development. A better understanding of the drivers, patterns, and consequences of changes in coffee landscapes is vital for informing the design of policies, programs, and incentives for sustainable coffee production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13593-021-00712-0.

Pesticide risk perceptions among bystanders of aerial spraying on bananas in Costa Rica.

Little is known about how bystanders perceive risks from pesticide use in areas with frequent aerial spraying of pesticides. This research aims to better understand how bystanders (school workers) from three counties of the Limón province in Costa Rica, who did not have a contractual relationship with agricultural production, perceive risks of pesticides in the areas where they work and live. A face-to-face survey was carried out among 475 school workers, of whom 455 completed all 33 questions on pesticide risk perception. An exploratory factor analysis characterized underlying perceptions of pesticide exposure. Nine factors explained 40% of total variance and concerned severity and magnitude of perceived risk, manageability, benefits and support of pesticide use, amongst others. We subsequently analyzed what variables explained the five factors with satisfactory internal consistency, using separate multivariable linear regression models. Older school workers, (male) elementary teachers, and women school workers (particularly from schools situated near agricultural fields with aerial spraying of pesticides), felt greater severity and/or magnitude of risk from pesticide use. This study shows that bystanders are concerned about health risks from pesticide use. Their risk perceptions are not only shaped by gender and age like previously reported in the literature, but also by job title and geographical context. Understanding of what hazards people care about and how they deal with them is essential for successful risk management, bystanders should therefore be considered as a relevant actor in debates around pesticide issues and for informing the development of regulations and risk reduction strategies.

Pyruvate-lactate exchange and glucose uptake in human prostate cancer cell models. A study in xenografts and suspensions by hyperpolarized [1-13 C]pyruvate MRS and [18 F]FDG-PET.

Reprogramming of energy metabolism in the development of prostate cancer can be exploited for a better diagnosis and treatment of the disease. The goal of this study was to determine whether differences in glucose and pyruvate metabolism of human prostate cancer cells with dissimilar aggressivenesses can be detected using hyperpolarized [1-13 C]pyruvate MRS and [18 F]FDG-PET imaging, and to evaluate whether these measures correlate. For this purpose, we compared murine xenografts of human prostate cancer LNCaP cells with those of more aggressive PC3 cells. [1-13 C]pyruvate was hyperpolarized by dissolution dynamic nuclear polarization (dDNP) and [1-13 C]pyruvate to lactate conversion was followed by 13 C MRS. Subsequently [18 F]FDG uptake was investigated by static and dynamic PET measurements. Standard uptake values (SUVs) for [18 F]FDG were significantly higher for xenografts of PC3 compared with those of LNCaP. However, we did not observe a difference in the average apparent rate constant kpl of 13 C label exchange from pyruvate to lactate between the tumor variants. A significant negative correlation was found between SUVs from [18 F]FDG PET measurements and kpl values for the xenografts of both tumor types. The kpl rate constant may be influenced by various factors, and studies with a range of prostate cancer cells in suspension suggest that LDH inhibition by pyruvate may be one of these. Our results indicate that glucose and pyruvate metabolism in the prostate cancer cell models differs from that in other tumor models and that [18 F]FDG-PET can serve as a valuable complementary tool in dDNP studies of aggressive prostate cancer with [1-13 C]pyruvate.

Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with (13)C hyperpolarised NMR.

Hyperpolarised (HP) (13)C NMR allows enzymatic activity to be probed in real time in live biological systems. The use of in vitro models gives excellent control of the cellular environment, crucial in the understanding of enzyme kinetics. The increased conversion of pyruvate to lactate in cancer cells has been well studied with HP (13)C NMR. Unfortunately, the equally important metabolic step of lactate transport out of the cell remains undetected, because intracellular and extracellular lactate are measured as a single resonance. Furthermore, typical experiments must be performed using tens of millions of cells, a large amount which can lead to a costly and sometimes highly challenging growing procedure. We present a relatively simple set-up that requires as little as two million cells with the spectral resolution to separate the intracellular and extracellular lactate resonances. The set-up is tested with suspensions of prostate cancer carcinoma cells (PC3) in combination with HP [1-(13)C]pyruvate. We obtained reproducible pyruvate to lactate label fluxes of 1.2 and 1.7 nmol/s per million cells at 2.5 and 5.0 mM pyruvate concentrations. The existence of a 3-Hz chemical shift difference between intracellular and extracellular lactate enabled us to determine the lactate transport rates in PC3. We deduced a lactate export rate of 0.3 s(-1) and observed a decrease in lactate transport on addition of the lactate transport inhibitor α-cyano-4-hydroxycinnamic acid.

Social movements and risk perception: unions, churches, pesticides and bananas in Costa Rica.

BACKGROUND: Between 1992 and 2010 in the Costa Rican Caribbean, a social movement coalition called Foro Emaús sought to change people's view on problems of high pesticide use in banana production. OBJECTIVE: To understand the formation and membership of Foro Emaús, its success period, and its decline. METHODS: Semi-structured interviews of 28 key actors; a questionnaire survey among school personnel (n = 475) in Siquirres, Matina, and Talamanca counties; and secondary data from newspapers, leaflets, and movement documents were used. RESULTS: Foro Emaús developed activism around pesticide issues and put pressure on governmental agencies and banana companies and shaped people's perception of pesticide risks. The success of the Foro Emaús movement led to the reinforcement of a counteracting social movement (Solidarismo) by conservative sectors of the Catholic Church and the banana companies. We found that the participation of unions in Foro Emaús is an early example of social movement unionism. CONCLUSIONS: Scientific pesticide risk analysis is not the only force that shapes emerging societal perceptions of pesticide risk. Social movements influence the priority given to particular risks and can be crucial in putting health and environmental risk issues on the political and research agenda.

Pesticide risk perceptions and the differences between farmers and extensionists: towards a knowledge-in-context model.

UNLABELLED: A growing body of literature analyzes farmer perceptions of pesticide risk, but much less attention has been given to differences in risk perception between farmers and technical experts. Furthermore, inconsistencies in knowledge have too easily been explained in terms of lack of knowledge rather than exploring the underlying reasons for particular forms of thinking about pesticide risks. By doing this, the division between expert and lay knowledge has been deepened rather than transcended. OBJECTIVE: This study aims to understand differences and similarities among the perceptions of pesticide risks of farmers, farm workers, and technical experts such as extensionists, by applying a social science approach towards knowledge and risk attitudes. METHODS: Semi-structured interviews and field observations were conducted to smallholders, farm workers, extensionists, health professionals and scientists involved in the use and handling of pesticides. Subsequently, a survey was carried out to quantify the farmers and extensionists' acceptance or rejection of typical assertions expressed previously in the semi-structured interviews. RESULTS: Smallholders showed to gain knowledge from their own experiences and to adapt pesticides practices, which is a potential basis for transforming notions of pesticide safety and risk reduction strategies. Though extensionists have received formal education, they sometimes develop ideas deviating from the technical perspective. The risk perception of the studied actors appeared to vary according to their role in the agricultural labor process; they varied much less than expected according to their schooling level. CONCLUSIONS: Commitment to the technical perspective is not dramatically different for extensionists on the one hand and farmers as well as farm workers on the other hand. Ideas about a supposed lack of knowledge by farmers and the need of formal training are too much driven by a deficit model of knowledge. Further research on risk perceptions of pesticides and training of rural people will benefit from the development of a knowledge-in-context model.

The distribution and function of chondroitin sulfate and other sulfated glycosaminoglycans in the human bladder and their contribution to the protective bladder barrier.

PURPOSE: Glycosaminoglycan replenishment therapies are commonly applied to treat bladder inflammatory conditions such as bladder pain syndrome/interstitial cystitis. Although there is evidence that these therapies are clinically effective, much is still unknown about the location and function of different types of glycosaminoglycans in the bladder. We investigated the location of sulfated glycosaminoglycans in the bladder and evaluated their contribution to the urothelial barrier. MATERIALS AND METHODS: The location of different glycosaminoglycans (heparan sulfate, chondroitin sulfate and dermatan sulfate) in human and porcine bladders was investigated with immunofluorescence staining and isolating glycosaminoglycans using selective urothelial sampling techniques. Barrier function was evaluated with transepithelial electrical resistance measurements (Ω.cm(2)) on primary porcine urothelial cell cultures. The contribution of different glycosaminoglycans to the bladder barrier was investigated with specific glycosaminoglycan digesting enzymes and protamine. RESULTS: High glycosaminoglycan concentrations are located around the urothelial basal membrane and at the urothelial luminal surface. After removing the glycosaminoglycan layer, urothelial permeability increased. Natural recovery of the glycosaminoglycan layer takes less than 24 hours. Chondroitin sulfate was the only sulfated glycosaminoglycan that was located on the urothelial luminal surface and that contributed to urothelial barrier function. CONCLUSIONS: This study reveals an important role for chondroitin sulfate in bladder barrier function. Therapies aiming at restoring the luminal glycosaminoglycan layer in pathological conditions such as bladder pain syndrome/interstitial cystitis are based on a sound principle.

The mechanoreceptor TRPV4 is localized in adherence junctions of the human bladder urothelium: a morphological study.

PURPOSE: TRPV4 (transient receptor potential vanilloid 4 channel) is a nonselective cation channel involved in different sensory functions that was recently implicated in bladder mechanosensation. We investigated the cellular site of TRPV4 in bladder urothelium and explored a molecular connection between TRPV4 and urothelial adherence junctions. MATERIALS AND METHODS: We obtained healthy tissues sections from cystectomy in humans due to cancer in 3 and noncancerous conditions in 2. Besides human biopsies tissues from 7 normal and 7 TRPV4-/-mice, and the urothelial cell line RT4 were also used. Experiments were done with polyclonal antibody against TRPV4 (against the N-terminus of rat TRPV4). A molecular connection between TRPV4 and different adherence junction components was investigated using immunofluorescence, Western blot and immunoprecipitation. RESULTS: Results revealed TRPV4 on urothelial cell membranes near adherence junctions. Results were comparable in the urothelial cell line, human bladders and mouse bladders. Subsequent immunoprecipitation experiments established a molecular connection of TRPV4 to α-catenin, an integral part of the adherence junction that catenates E-cadherin to the actin-microfilament network. CONCLUSIONS: Results provide evidence for the location of TRPV4 in human bladder urothelium. TRPV4 is molecularly connected to adherence junctions on the urothelial cell membrane. TRPV4 coupling to a rigid intracellular and intercellular structural network would agree with the hypothesis that TRPV4 can be activated by bladder stretch.

Biomarkers for the diagnosis of prostatic inflammation in benign prostatic hyperplasia.

BACKGROUND: Chronic prostatic inflammation could be a central mechanism in benign prostatic hyperplasia (BPH) progression. Currently, the histological examination of prostate biopsies remains the only way to diagnose prostatic inflammation. Our objective was to find new noninvasive biomarkers for the diagnosis of prostatic inflammation. METHODS: Ninety BPH samples were investigated in two steps. First, a hypothesis was generated using a profiling procedure with a panel of 96 genes on an initial set of 30 samples. Then, the candidate biomarkers were validated on a large number of samples (n = 90). Gene expression was compared with the histological prostatic inflammation score based on the density and the confluence of lymphoid nodules. Finally, protein transcripts of the candidate biomarkers were investigated in urine samples and compared with clinical data. RESULTS: Of the 96 genes, nine were significantly correlated with the inflammation score on the initial set of patients. Four of them were validated on the complete set of patients: CCR7, CD40LG, CTLA4, and ICOS. ICOS and CTLA4 protein levels were readily measured in urine samples using a conventional ELISA procedure. High-ICOS expression in urine was associated with a higher post-void residual and a lower maximum urinary flow rate. CONCLUSIONS: Four genes were significantly upregulated at the mRNA level in the prostate tissue of patients with severe inflammation score. Two proteins were measured in urine samples, and were associated with maximum uroflowmetry and post-void residual. A prospective clinical study is needed to confirm their clinical relevance.

Pesticide use in banana and plantain production and risk perception among local actors in Talamanca, Costa Rica.

The Talamanca County in Costa Rica has large-scale banana and small-scale plantain production, probably causing pesticide exposure in indigenous children. We explored to what extent different community actors are aware of children's pesticide hazards and how their awareness related to socio-economical and cultural conditions. Methods comprised eight focus groups with fathers and mothers separately, 27 semi-structured interviews to key actors, and field observations. As a whole, the indigenous plantain farmers and banana plantation workers had some general knowledge of pesticides concerning crop protection, but little on acute health effects, and hardly any on exposure routes and pathways, and chronic effects. People expressed vague ideas about pesticide risks. Inter-community differences were related to pesticide technologies used in banana and plantain production, employment status on a multinational plantation versus smallholder status, and gender. Compared to formalized practices on transnational company plantations, where workers reported to feel protected, pesticide handling by plantain smallholders was not perceived as hazardous and therefore no safety precautions were applied. Large-scale monoculture was perceived as one of the most important problems leading to pesticide risks in Talamanca on banana plantations, and also on neighboring small plantain farms extending into large areas. Plantain farmers have adopted use of highly toxic pesticides following banana production, but in conditions of extreme poverty. Aerial spraying in banana plantations was considered by most social actors a major determinant of exposure for the population living nearby these plantations, including vulnerable children. We observed violations of legally established aerial spraying distances. Economic considerations were most mentioned as the underlying reason for the pesticide use: economic needs to obtain the production quantity and quality, and pressure to use pesticides by other economic agents such as middlemen. Risk perceptions were modulated by factors such as people's tasks and positions in the production process, gender, and people's possibilities to define their own social conditions (more fatalistic perceptions among banana workers). The challenge for the future is to combine these insights into improved health risk assessment and management that is culturally adequate for each particular community and agricultural context.

Biofuel, dairy production and beef in Brazil: competing claims on land use in São Paulo state.

This paper examines the competing claims on land use resulting from the expansion of biofuel production. Sugarcane for biofuel drives agrarian change in So Paulo state, which has become the major ethanol-producing region in Brazil. We analyse how the expansion of sugarcane-based ethanol in So Paulo state has impacted dairy and beef production. Historical changes in land use, production technologies, and product and land prices are described, as well as how these are linked to changing policies in Brazil. We argue that sugarcane/biofuel expansion should be understood in the context of the dynamics of other agricultural sectors and the long-term national political economy rather than as solely due to recent global demand for biofuel. This argument is based on a meticulous analysis of changes in three important sectors - sugarcane, dairy farming, and beef production - and the mutual interactions between these sectors.

Non-commercial surrogacy: an account of patient management in the first Dutch Centre for IVF Surrogacy, from 1997 to 2004.

BACKGROUND: Surrogacy was prohibited in the Netherlands until 1994, at which time the Dutch law was changed from the general prohibition of surrogacy to the prohibition of commercial surrogacy. This paper describes the results from the first and only Dutch Centre for Non-commercial IVF Surrogacy between 1997 and 2004. METHODS: A prospective study was conducted of all intended parents, and surrogate mothers and their partners (if present), in which medical, psychological and legal aspects of patient selection were assessed by questionnaires and interviews developed for this study. RESULTS: More than 500 couples enquired about surrogacy by telephone or e-mail. More than 200 couples applied for surrogacy in the Centre, of which, after extensive screening, 35 couples actually entered the IVF programme and 24 completed the treatment, resulting in 16 children being born to 13 women. Recommendations for non-commercial surrogacy are given, including abandoning the 1-year waiting period before adoption, currently dictated by law, avoiding a period of unnecessary psychological distress. CONCLUSIONS: Our study has shown that non-commercial IVF surrogacy is feasible, with good results in terms of pregnancy outcome and psychological outcome for all parents, and with no legal problems relating to the adoption procedures arising. The extensive screening of medical, psychological and legal aspects was a key element in helping to ensure the safety and success of the procedure.