Divergent Genotype of Hepatitis A Virus in Alpacas, Bolivia, 2019.

Hepatitis A virus (HAV) is a common human pathogen found exclusively in primates. In a molecular and serologic study of 64 alpacas in Bolivia, we detected RNA of distinct HAV in ≈9% of animals and HAV antibodies in ≈64%. Complete-genome analysis suggests a long association of HAV with alpacas.

Leveraging the Full Continuum of Care to Prevent Opioid Use Disorder.

Substance use disorder prevention programs are most effective when matched appropriately to the baseline risk of the population. Individuals who misuse opioids often have unique risk profiles different from those who use other substances such as alcohol or cannabis. However, most substance use prevention programs are geared toward universal audiences, neglecting key inflection points along the continuum of care. The HEAL Prevention Cooperative (HPC) is a unique cohort of research projects that represents a continuum of care, from community-level universal prevention to indicated prevention among older adolescents and young adults who are currently misusing opioids or other substances. This paper describes the theoretical basis for addressing opioid misuse and opioid use disorder across the prevention continuum, using examples from research projects in the HPC.

Modulation of the immune response by infection with Cryptosporidium spp. in children with allergic diseases.

It has been demonstrated that the allergic response can be ameliorated by the administration of pathogen derivatives that activate Toll-like receptors and induce a Th1-type immune response (IR). Cryptosporidium is a parasite that promotes an IR via Toll-like receptors and elicits the production of Th1-type cytokines, which limit cryptosporidiosis. The aim of this study was to investigate allergy-related immune markers in children naturally infected with Cryptosporidium. In a cross-sectional study, 49 children with or without clinical diagnosis of allergies, oocysts of Cryptosporidium spp. in the faeces were screened microscopically. We microscopically screened for leucocytes, examined T and B cells for allergy-related activation markers using flow cytometry and evaluated serum for total IgE using chemiluminescence. Children with allergies and Cryptosporidium in the faeces had significantly lower levels of total IgE, B cells, CD19(+) CD23(+) and CD19(+) CD124(+) cells as well as a greater percentage of interferon-gamma (IFN-γ(+) ) and IL-4(+) CD4(+) cells than children with allergies without Cryptosporidium. This is the first description of the modulation of the IR in children with allergic diseases in the setting of natural Cryptosporidium infection. Our findings suggest the involvement of CD4(+) cells producing IL-4 and IFN-γ in the IR to Cryptosporidium in naturally infected children.

Biodiversity and technological-functional potential of lactic acid bacteria isolated from spontaneously fermented quinoa sourdoughs.

AIMS: To analyse lactic acid bacteria (LAB) diversity and technological-functional and safety properties of strains present during spontaneous fermented quinoa sourdoughs. METHODS AND RESULTS: Fermentation was performed by daily backslopping at 30°C for 10 days. Autochthonous LAB microbiota was monitored by a biphasic approach combining random amplified polymorphic DNA (RAPD)-PCR and rRNA gene sequencing with PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Identification and intraspecies differentiation allowed to group isolates within nine LAB species belonging to four genera. A succession of LAB species occurred during 10-days backslopping; Lactobacillus plantarum and Lactobacillus brevis were detected as dominant species in the consortium. The characterization of 15 representative LAB strains was performed based on the acidifying capacity, starch and protein hydrolysis, γ-aminobutyric acid and exopolysaccharides production, antimicrobial activity and antibiotic resistance. CONCLUSION: Strains characterization led to the selection of Lact. plantarum CRL1905 and Leuconostoc mesenteroides CRL1907 as candidates to be assayed as functional starter culture for the gluten-free (GF) quinoa fermented products. SIGNIFICANCE AND IMPACT OF THE STUDY: Results on native LAB microbiota present during quinoa sourdough fermentation will allow the selection of strains with appropriate technological properties to be used as a novel functional starter culture for GF-fermented products.

Biodiversity and technological potential of lactic acid bacteria isolated from spontaneously fermented amaranth sourdough.

UNLABELLED: Spontaneous fermented sourdoughs prepared from amaranth flour were investigated for the presence of autochthonous lactic acid bacteria (LAB) predominating microbiota. The doughs were fermented with daily backslopping on a laboratory scale at 30°C for 10 days. LAB counts ranged from 2·60 to 8·54 log CFU g(-1) with a pH declined from 6·2 to 3·8 throughout fermentation. The combined use of randomly amplified polymorphic DNA (RAPD)-PCR analysis and sequence analysis of 16S rRNA was applied for LAB intraspecies differentiation and taxonomic identification, respectively. Enterococcus, Pediococcus and Lactobacillus species were present in amaranth sourdoughs (AS). After the first refreshment step, Lactobacillus plantarum dominated AS until the end of fermentation. In coincidence, when DGGE analysis was performed, the occurrence of a progressive change in bacterial communities allowed the selection of Lact. plantarum as a dominant species. Moreover, technological, functional and safety characteristics of representative RAPD-biotypes were investigated. Lact. plantarum CRL1898 was selected as a potential candidate for gluten-free amaranth sourdough starter. SIGNIFICANCE AND IMPACT OF THE STUDY: Nowadays, there is an increasing interest in ancient noncereal gluten-free (GF) crops such as amaranth, due to their reported nutritional and health benefits. However, the use of these grains is still limited to traditional foods and bread making processes that are not yet well standardized. Results on the dynamics of autochthonous lactic acid bacteria (LAB) microbiota during laboratory spontaneous amaranth sourdoughs (AS) fermentation will contribute to overcome challenges for GF-fermented products development. In addition, knowledge about LAB diversity involving Enterococcus, Pediococcus and Lactobacillus species, with Lactobacillus plantarum predominating during AS fermentation, and their technological and functional properties provides the basis for the selection of autochthonous strains as starters cultures for novel gluten-free bakery products with enhanced nutritional, sensory and/or safety quality.

Aerobic and anaerobic enzymatic activity of orange roughy (Hoplostethus atlanticus) and alfonsino (Beryx splendens) from the Juan Fernandez seamounts area.

The aerobic and anaerobic enzymatic activity of two important commercial bathypelagic species living in the Juan Fernández seamounts was analyzed: alfonsino (Beryx splendens) and orange roughy (Hoplostethus atlanticus). These seamounts are influenced by the presence of an oxygen minimum zone (OMZ) located between 160 and 250 m depth. Both species have vertical segregation; alfonsino is able to stay in the OMZ, while orange roughy remains at greater depths. In this study, we compare the aerobic and anaerobic capacity of these species, measuring the activity of key metabolic enzymes in different body tissues (muscle, heart, brain and liver). Alfonsino has higher anaerobic potential in its white muscle due to greater lactate dehydrogenase (LDH) activity (190.2 µmol NADH min(-1) g ww(-1)), which is related to its smaller body size, but it is also a feature shared with species that migrate through OMZs. This potential and the higher muscle citrate synthase and electron transport system activities indicate that alfonsino has greater swimming activity level than orange roughy. This species has also a high MDH/LDH ratio in its heart, brain and liver, revealing a potential capacity to conduct aerobic metabolism in these organs under prolonged periods of environmental low oxygen conditions, preventing lactic acid accumulation. With these metabolic characteristics, alfonsino may have increased swimming activity to migrate and also could stay for a period of time in the OMZ. The observed differences between alfonsino and orange roughy with respect to their aerobic and anaerobic enzymatic activity are consistent with their characteristic vertical distributions and feeding behaviors.

Amblyomma parvitarsum (Acari: Ixodidae): localities, hosts and host-parasite ecology.

Only a few aspects of the biology of Amblyomma parvitarsum Neumann are known. Adults of this hard tick species are parasites of South American camelids in the Andean plateau of Argentina, Bolivia, Peru and Chile and also in the Argentine Patagonia, but they have been also rarely found on other artyodactils and two species of birds. The larva has been collected from reptiles in northern Chile, whereas the hosts for the nymph remain unknown. On nine localities included within Altitude Tropical and Perarid Mediterranean ecoregions in northern Chile, we analyzed 237 reptiles, 285 birds, 624 rodents and 52 camelids for infestation with A. parvitarsum to calculate seasonal prevalence of this tick. We also reviewed the literature of this tick and three entomological collections for obtaining and summarizing all the information to date about this tick. None of the analyzed birds and rodents were parasitized with A. parvitarsum; however, seven over a total of ten reptile species that we caught were infested with the larva. In the camelid species Lama glama and Vicugna pacos we collected adult specimens of this tick. Larval prevalence was higher during fall (75 %) in Liolaemus pleopholis in the Altitude Tropical ecoregion. We also collected adult specimens of A. parvitarsum from camelid manure heaps during summer in Salar de Surire and Llullaillaco localities. Additionally, we also reviewed the literature of this tick and examined specimens in three entomological collections for obtaining and summarizing all the information to date about this tick. By this study, nine localities and seven new hosts are added for A. parvitarsum and we confirm reptiles as specific hosts of this tick larva.

Divergent Gut Microbiota: Archaeal and Bacterial Signatures Unveil Unique Patterns in Colombian Cyclists Compared to Weightlifters and Non-Athletes.

Engagement in physical activity, across various sports, promotes a diverse microbiota in active individuals. This study examines the gut microbiota of Colombian athletes, specifically weightlifters (n = 16) and road cyclists (n = 13), compared to non-athletes (n = 15). Using Kruskal-Wallis tests, the physical activity level of a group of non-athletic individuals and the sports experience of a group of professional athletes is analyzed. The median age of participants is 24 years, comprising 25 men and 19 women. The microbiota is collected using fecal samples. Participants provided these samples during their pre-competitive stage, specifically during the concentration phase occurring two weeks prior to national competitions. This timing is chosen to capture the microbial composition during a period of heightened physical preparation. Questionnaire responses and microbial composition assessments identify disparities among groups. Microbial composition analysis explores core microbiome, abundance, and taxonomy using Pavian, MicrobiomeAnalyst 2.0, and GraPhlAn. ANCOM-BC2 reveals differentially abundant species. Road cyclists exhibit decreased Bacteria and increased Archaea abundance. Phylum-level variations included Planctomycetes, Acidobacteria, and Proteobacteria, while Bacteroidetes prevailed. Key families influencing gut microbiota are Bacteroidaceae, Muribaculaceae, and Selenomonadaceae. Weightlifters exhibit unique viral and archaeal community connections, while cyclists showed specialized microbial interplay influenced by endurance exercise. Correlation network analysis emphasizes distinctive microbial interactions within athlete groups, shedding light on the impact of physical activities on gut microbiota and athlete health.

Self-report and urine drug screen concordance among women with co-occurring PTSD and substance use disorders participating in a clinical trial: Impact of drug type and participant characteristics.

BACKGROUND: Self-report measures are important in substance use assessment, yet they are susceptible to reporting errors. Urine drug screens (UDS) are often considered a more valid alternative. However, collecting in-person UDS may not always be feasible, contributing to the need to understand factors that influence the validity of self-reported substance use. METHODS: In this secondary analysis of data from 295 women with co-occurring PTSD and substance use disorders (SUD) who participated in a clinical trial testing behavioral interventions, we examined concordance and discordance between self-reported drug use and associated UDS results. Generalized linear mixed models were used to examine the impact of treatment type and participant characteristics on the associations between self-reported drug use and UDS results. RESULTS: Findings revealed higher disagreement between self-report and UDS for opioids and sedatives (ranging from.77 to.90) and lower disagreement rates for cannabis and cocaine (ranging from.26 to.33). Treatment type was not a significant moderator of the associations between self-report and UDS across all drugs. Among those with a positive opioid UDS, those who reported employment in the past three years were more likely to self-report no opioid use compared to their counterparts without employment in the past three years. CONCLUSIONS: Findings add to the literature that supports the validity of self-reported cannabis and cocaine use. The greater discrepancies between self-report and UDS test results of opioids and sedatives suggest adjunctive UDS may be required, although a variety of factors other than inaccurate self-report may be associated with this discrepancy.

Dosimetric evaluation of radiopharmaceuticals in kidneys and uterine wall of a woman with early pregnancy using Stabin/Segars representations.

MIRD method with the Stabin/Segars anthropomorphic representations were used to calculate the absorbed doses in kidneys and uterine wall of an early-stage pregnant woman when 99mTc (DTPA), 99mTc (DMSA) and 99mTc (MAG3) are used for renal studies. Stabin and Segars anatomical representations are phantoms that are used in Monte Carlo calculations to determine the SAF, that with the pharmaceutical residence time are used to calculate the absorbed dose, from source organs, on target organs. Concerns about the impact on the absorbed dose due to the use of the three 99mTc-based compounds as well as the use of different phantoms were here treated for the case of a female at early pregnant state. The lowest absorbed dose in the kidneys was obtained with 99mTc (MAG3), and the relative difference of using Stabin and Segars anthropomorphic representations was 2.5%. For bladder and rest of organs the relative difference 14.63%. The lowest absorbed dose by uterine wall was obtained with 99mTc(DMSA), however the relative difference of using Stabin and Segars anthropomorphic representations was 12%.

Environmental refuges increase performance of juvenile mussels Mytilus chilensis: Implications for mussel seedling and farming strategies.

Estuarine ecosystems are characterized by a wide physical-chemical variation that in the context of global change scenarios may be exacerbated in the future. The fitness of resident organisms is expected to be influenced by such variation and, hence, its study is a priority. Some of that variation relates to water vertical stratification, which may create "environmental refuges" or distinct layers of water with conditions favoring the fitness of some individuals and species. This study explored the performance of juvenile mussels (M. chilensis) settled in two distinctive water depths (1 m and 4 m) of the Reloncaví fjord (southern Chile) by conducting a reciprocal transplants experiment. Salinity, saturation state and the contents of CO3 in seawater were among the factors that best explained the differences between the two layers. In such environmental conditions, the mussel traits that responded to such variation were growth and calcification rates, with significantly higher values at 4 m deep, whereas the opposite, increased metabolic stress, was higher in mussels raised and transplanted to the surface waters (1 m). Such differences support the notion of an environmental refuge, where species like mussels can find better growth conditions and achieve higher performance levels. These results are relevant considering the importance of M. chilensis as a shellfish resource for aquaculture and a habitat forming species. In addition, these results shed light on the variable responses exhibited by estuarine organisms to small-scale changes in the characteristics of the water column, which in turn will help to better understand the responses of the organisms to the projected scenarios of climate global change.

Combined effect of synthetic enterocin CRL35 with cell wall, membrane-acting antibiotics and muranolytic enzymes against Listeria cells.

AIMS: To evaluate the inhibition effectiveness of enterocin CRL35 in combination with cell wall, membrane-acting antibiotics and muranolytic enzymes against the foodborne pathogen Listeria. METHODS AND RESULTS: Synthetic enterocin CRL35 alone and in combination with monensin, bacitracin, gramicidin, mutanolysin and lysozyme were used in this study. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) index assays were performed using Listeria innocua 7 and Listeria monocytogenes FBUNT as sensitive strains. Antibiotics showed positive interactions with the bacteriocin in both strains tested. On the other hand, when mutanolysin and enterocin CRL35 were added to resting cells in a buffer system, the lytic effect of mutanolysin was enhanced. However, the addition of mutanolysin showed no effect on the growth of L. innocua 7 cells in a culture medium. Moreover, mutanolysin allowed the overgrowth of L. innocua 7 cells to an OD similar to control cells in the presence of inhibitory concentration of enterocin CRL35. In contrast, the combination of lysozyme and enterocin CRL35 resulted in a 50% inhibition of the L. innocua 7 growth. CONCLUSIONS: Based on our results, we conclude that the combination of synthetic enterocin CRL35 with some antibiotics is effective against L. innocua 7 and L. monocytogenes FBUNT cells, and more importantly the amount of these agents to be used was considerably reduced. The effectiveness of the combination of synthetic enterocin CRL35 with muramidases seems to depend on complex environments, and more detailed studies need to be performed to elucidate this issue. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterocin CRL35 represents a promising agent that not only can ensure the quality and safety of food but it can also be combined with several antimicrobial agents important in the medical field.

Metagenomics-based approach for studying and selecting bioprotective strains from the bacterial community of artisanal cheeses.

A metagenome-based approach was used to assess the taxonomic affiliation and functional potential for bacteriocin production of the bacterial community in cow's milk artisanal cheeses from Northwestern Argentina. Three different samples were analyzed by high-throughput sequencing of the V4 region of the 16S rRNA gene and shotgun metagenomics. Taxonomic analysis showed that cheese A and C were quite similar whereas cheese B displayed a rather different bacterial composition. Overall, two families, Streptococceae and Enterococceae, dominated the artisanal cheese microbiota, being the former family prevalent in cheese B and the later family the most important in samples A and C. Besides the usual species associated to cheeses, a number of bacterial taxa that have not been previously found in Argentinean artisanal cheeses were reported in the present work such as Macrococcus caseolyticus and Streptococcus macedonicus Functional metagenomics analysis using the bacteriocin mining software BAGEL3, identified 2 ORFs encoding antimicrobial peptides in cheese B and 42 different peptides in sample C. The bacteriocin genes found showed good correlation with taxonomy. Based on the microbial diversity and functional features found through shotgun metagenomic sequencing, a culture-dependent approach was applied aiming to isolate bacteriocin-producing bacteria able to inhibit the growth of the foodborne pathogen Listeria monocytogenes. From 151 bacterial colonies derived from the cheese samples, 10 were associated to high anti-Listeria activity. Based on partial 16S rRNA gene sequencing and RAPD-PCR analysis, all bacteriocinogenic isolates were identified as Enterococcus faecium. Finally, we carried out a pilot experiment with L. monocytogenes-contaminated cheese using one of the enterococcal isolates as a bioprotective adjunct culture. The use of E. faecium CRL1879 during artisanal cheese manufacturing did not alter the main organoleptic properties of the cheese and ensured an efficient control of the foodborne pathogen up to 30 days. This finding supports the use of E. faecium CRL1879 as an adjunct culture in the cheese-making process with a combination of both safety and minimal processing.

Oral administration of Lactobacillus fermentum CRL1446 improves biomarkers of metabolic syndrome in mice fed a high-fat diet supplemented with wheat bran.

This work aimed to evaluate the effect of oral administration of probiotic Lactobacillus (L.) fermentum CRL1446, with feruloyl esterase (FE) activity, on metabolic biomarkers and intestinal microbiota of mice with high fat diet-induced Metabolic Syndrome (MS) and supplemented with wheat bran as a source of esterified ferulic acid. Six-week-old male Swiss albino mice developed the components of MS when fed with high fat diet supplemented with wheat bran (HFD + WB) for 14 weeks. Positive impact of L. fermentum CRL1446 administration on these animals was reflected in a decrease in body weight gain and adiposity index compared to the animals that did not receive the probiotic strain. In addition, a decrease in plasma leptin levels, improvement of inflammatory profile, reduction of fatty infiltration in hepatocytes and modification of lipid profile (increased HDL-cholesterol and decreased LDL-cholesterol and triglyceride levels) were observed. On the other hand, L. fermentum CRL1446 reduced fasting glucose and insulin levels, improving the HOMA index in mice with MS. Postprandial glucose levels were also reduced in the oral glucose tolerance test. Consumption of L. fermentum CRL1446 with HFD + WB (HFD + WB-Lf mice group) had a great impact on host metabolism, modulating intestinal microbiota, with an increase in Bacteroidetes and a decrease in Firmicutes abundance being observed. Increased intestinal FE activity, improved oxidative status and increased abundance of 3-hydroxyphenylpropionic acid and butyric acid concentration in colonic content, were also demonstrated in HFD + WB-Lf mice. Results obtained suggest that supplementation with L. fermentum CRL1446 enhances beneficial effects of a bran diet, attenuating the risk factors associated with MS.

New immunobiotics from highly proteolytic Lactobacillus delbrueckii strains: their impact on intestinal antiviral innate immune response.

Proteolytic starter cultures with intrinsic immunomodulatory activities are desirably features for the development of functional foods, which would significantly reduce the cost of their production (one-strain starter) having an additional beneficial effect on the host. In this work, Lactobacillus delbrueckii strains were selected according to their ability to efficiently hydrolyse ß-casein and to modulate the immune system. Among 36 strains evaluated, the highest proteolytic activities were found for L. delbrueckii subsp. lactis CRL581 and L. delbrueckii subsp. bulgaricus CRL656. The immunomodulatory effect of both strains and their ß-casein hydrolysates (CRL581 and CRL656 hydrolysates, respectively) were studied in a murine model. Balb/c mice were fed lactobacilli or their hydrolysates for three days. One day after the last lactobacilli or hydrolysate treatments, mice were challenged with the Toll-like receptor 3 (TLR3) agonist poly(I:C) by intraperitoneal injection. Before and after poly(I:C) challenge the phagocytic and microbicidal activity of peritoneal macrophages, intestinal immunoglobulin A (IgA), cytokine profile, and histological analysis of the intestine were analysed. L. delbrueckii subsp. lactis CRL581 significantly increased the activation of peritoneal macrophages as well as the levels of intestinal IgA, interleukin (IL)-10 and interferon (IFN)-γ when compared to untreated controls. In addition, the CRL581 strain was able to significantly reduce the intestinal inflammatory damage triggered by TLR3 activation. L. delbrueckii CRL581 increased the levels of IL-10, IFN-γ and IFN-ß, and reduced tumour necrosis factor alpha and IL-6 concentrations in the intestine of poly(I:C)-challenged mice. No immunomodulatory effects were observed for the CRL656 strain or for the CRL581 or CRL656 hydrolysates. The results of this work show that the technologically relevant and high proteolytic strain L. delbrueckii CRL581 is able to beneficially modulate the intestinal innate antiviral immune response. Although further studies with the CRL581 strain are required to corroborate and deepen its immunological effects, this bacterium is an interesting alternative for the development of new functional foods with antiviral capabilities.

Molecular and Serologic Survey of Pathogens in an Endangered Andean Cat ( Leopardus jacobita) of the High Andes of Bolivia.

The Andean cat ( Leopardus jacobita) is one of the most threatened and least known wild felids in the world. Using molecular and serologic tests, we screened a free-ranging Andean cat for 17 pathogens of conservation concern. Results suggested no evidence of infection or exposure. Whether pathogens are a threat for Andean cat populations remains currently unknown.

Compatibility and safety of five lectin-binding putative probiotic strains for the development of a multi-strain protective culture for poultry.

The ban on the use of antibiotics as feed additives for animal growth promotion in the European Union and United States and the expectation of this trend to further expand to other countries in the short term have prompted a surge in probiotic research. Multi-species probiotics including safe and compatible strains with the ability to bind different nutritional lectins with detrimental effects on poultry nutrition could replace antibiotics as feed additives. Lactobacillus salivarius LET201, Lactobacillus reuteri LET210, Enterococcus faecium LET301, Propionibacterium acidipropionici LET103 and Bifidobacterium infantis CRL1395 have proved to be compatible as evaluated through three different approaches: the production and excretion of antimicrobial compounds, growth inhibition by competition for essential nutrients and physical contact, and a combination of both. The safety of P. acidipropionici LET103 was confirmed, since no expression of virulence factors or antibiotic resistance was detected. The innocuity of E. faecium LET301 should be further evaluated, since the presence of genes coding for certain virulence factors (gelE, efaAfm and efaAfs) was observed, albeit no expression of gelE was previously detected for this strain and there are no reports of involvement of efaAfm in animal pathogenicity. Finally, a combination of the five strains effectively protected intestinal epithelial cells of broilers from the cytotoxicity of mixtures of soybean agglutinin, wheat germ agglutinin and concanavalin A. To our knowledge, this is the first time that a combination of strains is evaluated for their protection against lectins that might be simultaneously present in poultry feeds.

Chromatography-based on- and in-line pre-concentration methods in capillary electrophoresis.

Capillary electrophoresis (CE) poses unique challenges in many different analytical applications, mainly to biological and complex samples and when only small amounts of sample are available, due to its low sample consumption. As a consequence, poor limits of detection are usually observed with this technique, especially with UV photodetectors. Minimal or no sample treatment is desirable in any analytical method to avoid external sources of contamination or errors and to provide a high throughput. On- and in-capillary sample pre-concentration strategies, based on solid-phase extraction (SPE) technology can take advantage of both techniques (SPE and CE), while avoiding sample contamination and tedious manipulations when the sample amount is an issue. Moreover, the combination can provide two-dimensional separations. This review collects the most recent strategies that merge SPE technology built on- and in-capillary pre-concentration for increasing sensitivity and/or selectivity.

Development of a frit-free SPE-based in-column preconcentration system for capillary electrophoresis.

An in-capillary sample preconcentration strategy based on solid phase extraction (SPE) technology coupled with capillary electrophoresis (CE) has been developed taking advantage of both techniques (SPE and CE). An in-line frit-free preconcentration device for capillary electrophoresis containing MCX beads, obtained from the corresponding Waters OASIS cartridges, was prepared. The retention of the particles was based on the relative diameters of the particles, carefully selected, and the capillaries. An experimental preconcentration factor of 100 was found for the system. Conditions were optimised for 3-nitrotyrosine measurement in rat urine being 4.4 microM spiked in the urine the lowest value detectable.