In Vivo Genotoxicity and Cytotoxicity Kinetics of Trimethoprim Sulfamethoxazole in Well-nourished and Undernourished Young Rats.

BACKGROUND/AIM: Undernutrition is a serious health problem prevalent in poor countries, affecting millions of people worldwide, especially young children, pregnant women, and sick elderly individuals. This condition increases vulnerability to infections, leading to widespread use of antibiotic treatments in undernourished populations. The objective of the present study was to determine the in vivo genotoxic and cytotoxic effects of trimethoprim-sulfamethoxazole (TMP-SMX) treatment according to nutritional conditions. MATERIALS AND METHODS: The effects of TMP-SMX treatment were measured by analyzing the kinetics of micronucleated reticulocytes (MN-RET) induced in the peripheral blood of young, well-nourished (WN) and undernourished (UN) rats. RESULTS: In the WN group, two distinct peaks of MN-RET were observed, while the UN group had a significantly higher basal frequency of MN-RET compared to the WN group and only a later peak. Reticulocyte (RET) frequency slightly decreased in WN, indicating a poor cytotoxic effect. In contrast, in the UN, the treatment caused a significant increase in RET frequency. The results indicate that SMX's aromaticity index decreases when formed with TMP, suggesting potentially fewer toxic effects. CONCLUSION: In vivo TMP-SMX produces two MN-RET induction peaks in WN animals, indicating two DNA damage induction mechanisms and consequent micronucleus production. The UN rats did not display the two peaks, indicating that the first MN induction mechanism did not occur in UN, possibly due to pharmacokinetic effects, decreased metabolism or effects on cell proliferation. TMP-SMX has a slight cytotoxic effect on WN. In contrast, in the UN, the antibiotic treatment seems to favor early erythropoiesis.

Hyperglycemia affects neuronal differentiation and Nestin, FOXO1, and LMO3 mRNA expression of human Wharton's jelly mesenchymal stem cells of children from diabetic mothers.

Pregestational Diabetes Mellitus (PDM) during pregnancy constitutes an unfavorable embryonic and fetal development environment, with a high incidence of congenital malformations (CM). Neural tube defects are the second most common type of CM in children of diabetic mothers (CDM), who also have an elevated risk of developing neurodevelopmental disorders. The mechanisms that lead to these neuronal disorders in CDM are not yet fully understood. The present study aimed to know the effect of hyperglycemia on proliferation, neuronal differentiation percentage, and expression of neuronal differentiation mRNA markers in human umbilical cord Wharton's jelly mesenchymal stem cells (hUCWJMSC) of children from normoglycemic pregnancies (NGP) and PDM. We isolated and characterized hUCWJMSC by flow cytometry, immunofluorescence, RT-PCR and were induced to differentiate into adipocytes, osteocytes, and neurons. Proliferation assays were performed to determine the doubling time, and Nestin, TUBB3, FOXO1, KCNK2, LMO3, and MAP2 mRNA gene expression was assessed by semiquantitative RT-PCR. Hyperglycemia significantly decreased proliferation and neuronal differentiation percentage in NGP and PDM cells treated with 40 mM d-glucose. Nestin mRNA expression decreased under control glycemic conditions, while FOXO1, KCNK2, LMO3, and MAP2 mRNA expression increased during neuronal differentiation in both NGP and PDM cells. On the other hand, under hyperglycemic conditions, Nestin was significantly decreased in cells from NGP but not in cells from PDM, while mRNA expression of FOXO1 and LMO3 was significantly increased in cells from NGP, but not in cells from PDM. We found evidence that maternal PDM, with hyperglycemia in culture, affects the biological properties of fetal cells. All these results could be part of fetal programming.

Class I Myosins, molecular motors involved in cell migration and cancer.

Class I Myosins are a subfamily of motor proteins with ATPase activity and a characteristic structure conserved in all myosins: A N-Terminal Motor Domain, a central Neck and a C terminal Tail domain. Humans have eight genes for these myosins. Class I Myosins have different functions: regulate membrane tension, participate in endocytosis, exocytosis, intracellular trafficking and cell migration. Cell migration is influenced by many cellular components including motor proteins, like myosins. Recently has been reported that changes in myosin expression have an impact on the migration of cancer cells, the formation of infiltrates and metastasis. We propose that class I myosins might be potential markers for future diagnostic, prognostic or even as therapeutic targets in leukemia and other cancers.Abbreviations: Myo1g: Myosin 1g; ALL: Acute Lymphoblastic Leukemia, TH1: Tail Homology 1; TH2: Tail Homology 2; TH3: Tail Homology 3.

High expression of Myosin 1g in pediatric acute lymphoblastic leukemia.

Acute Lymphoblastic Leukemia (ALL) is the most frequent cancer in pediatric population. Although the treatment has improved and almost 85% of the children are cured about 20% suffer relapse, therefore finding molecules that participate in the pathogenesis of the disease for the identification of relapse and patients at risk is an urgent unmet need. Class I myosins are molecular motors involved in membrane tension, endocytosis, phagocytosis and cell migration and recently they have been shown important for development and aggressiveness of diverse cancer types, however Myo1g an hematopoietic specific myosin has not been studied in cancer so far. We evaluated the expression of Myo1g by qRT-PCR, Immunocytochemistry and Immunofluorescence in a cohort of 133 ALL patients and correlated the expression at diagnosis and after treatment with clinical features and treatment outcomes. We found high expression levels of Myo1g in Peripheral Blood Mononuclear Cells (PBMCs) from patients with ALL at diagnosis and those levels decreased after complete remission; furthermore, we found an increase in Myo1g expression on patients with 9:22 translocation and those who relapse. This study show that Myo1g is over expressed in ALL and that may participate in the pathogenesis of the disease specially in high-risk patients.

Assessment of micronucleus and oxidative stress in peripheral blood from malnourished children.

INTRODUCTION: malnutrition is one of the most common health problems among children in underdeveloped countries, including Mexico. Previous studies have indicated increased genetic damage in malnourished humans and animal models, but the essential mechanisms remain unclear. In the present study, we assessed the effects of malnutrition on the frequency of micronucleus (MN) in reticulocytes (RET) from the peripheral blood of well-nourished uninfected (WN), well-nourished infected (WNI), moderately malnourished infected (UNM) and severely malnourished infected (UNS) children. Moreover, lipid peroxidation and the antioxidant status were evaluated to investigate the role of oxidative processes in malnutrition-associated genotoxicity. METHODS: the antioxidant status of the study population was determined by measuring superoxide dismutase (SOD) in the red blood cells and glutathione peroxidase (GPX) in whole blood. RESULTS: the UNS and UNM groups have increased percentages of MN-RET compared to the WNI group. Moreover, the data showed a significant increase in lipid peroxidation and a decrease in erythrocyte SOD activity and GPX activity in the malnourished group compared to the well-nourished infected children. CONCLUSION: the data suggest that the antioxidant system was impaired in the cells of malnourished children and that oxidative stress causes a significant increase in DNA damage, as evaluated by the MN-RET frequency.

Assessment of micronucleus and oxidative stress in peripheral blood from malnourished children / Evaluación de micronúcleos y estrés oxidante en sangre periférica de niños desnutridos

Introduction: malnutrition is one of the most common health problems among children in underdeveloped countries, including Mexico. Previous studies have indicated increased genetic damage in malnourished humans and animal models, but the essential mechanisms remain unclear. In the present study, we assessed the effects of malnutrition on the frequency of micronucleus (MN) in reticulocytes (RET) from the peripheral blood of well-nourished uninfected (WN), well-nourished infected (WNI), moderately malnourished infected (UNM) and severely malnourished infected (UNS) children. Moreover, lipid peroxidation and the antioxidant status were evaluated to investigate the role of oxidative processes in malnutrition-associated genotoxicity. Methods: the antioxidant status of the study population was determined by measuring superoxide dismutase (SOD) in the red blood cells and glutathione peroxidase (GPX) in whole blood. Results: the UNS and UNM groups have increased percentages of MN-RET compared to the WNI group. Moreover, the data showed a significant increase in lipid peroxidation and a decrease in erythrocyte SOD activity and GPX activity in the malnourished group compared to the well-nourished infected children. Conclusion: the data suggest that the antioxidant system was impaired in the cells of malnourished children and that oxidative stress causes a significant increase in DNA damage, as evaluated by the MN-RET frequency

Introducción: la desnutrición es uno de los principales problemas de salud entre los niños de los países en desarrollo, incluido México. Estudios previos han mostrado que existe un incremento en el daño genético en humanos y modelos animales desnutridos, pero los mecanismos por los que se desencadena aún son poco claros. En el presente estudio, evaluamos los efectos de la desnutrición en la frecuencia de micronúcleos (MN) en reticulocitos (RET) de sangre periférica de niños bien nutridos no infectados (WN), bien nutridos infectados (WNI), desnutridos moderados infectados (UNM) y con desnutrición severa e infecciones (UNS). Asimismo, se evaluaron la lipoperoxidación y la capacidad antioxidante para investigar el papel del proceso oxidativo en la genotoxicidad asociada a la desnutrición. Métodos: la capacidad antioxidante de la población de estudio fue determinada midiendo la superóxido-dismutasa (SOD) en los eritrocitos y glutatión peroxidasa (GPX) en sangre completa. Resultados: los niños UNS y UNM tienen alto porcentaje de MN-RET comparados con el grupo WNI. Además, los datos mostraron un incremento significativo en la lipoperoxidación y disminución en la actividad de SOD y GPX en el grupo de niños desnutridos comparados con el grupo de niños bien nutridos infectados. Conclusión: los datos sugieren que el sistema antioxidante está deteriorado en las células de los niños desnutridos y que el estrés oxidante causa un incremento significativo en el daño al ADN, el cual se refleja en el incremento en la frecuencia de RET-MN

Multiple DNA Markers for Identification of Xanthomonas arboricola pv. juglandis Isolates and its Direct Detection in Plant Samples.

Xanthomonas arboricola pv. juglandis (Xaj) is the etiological agent of walnut (Juglans regia L.) bacterial blight (WBB), and has been associated to other walnut emerging diseases, namely brown apical necrosis (BAN) and vertical oozing canker (VOC), altogether severely affecting the walnut production worldwide. Despite the research efforts carried out to disclose Xaj genetic diversity, reliable molecular methods for rapid identification of Xaj isolates and culture-independent detection of Xaj in infected plant samples are still missing. In this work, we propose nine novel specific DNA markers (XAJ1 to XAJ9) selected by dedicated in silico approaches to identify Xaj isolates and detect these bacteria in infected plant material. To confirm the efficacy and specificity of these markers, dot blot hybridization was carried out across a large set of xanthomonads. This analysis, which confirmed the pathovar specificity of these markers, allowed to identify four broad-range markers (XAJ1, XAJ4, XAJ6, and XAJ8) and five narrow-range markers (XAJ2, XAJ3, XAJ5, XAJ7, and XAJ9), originating 12 hybridization patterns (HP1 to HP12). No evident relatedness was observed between these hybridization patterns and the geographic origin from which the isolates were obtained. Interestingly, four isolates that clustered together according the gyrB phylogenetic analysis (CPBF 1507, 1508, 1514, and 1522) presented the same hybridization pattern (HP11), suggesting that these nine markers might be informative to rapidly discriminate and identify different Xaj lineages. Taking into account that a culture-independent detection of Xaj in plant material has never been described, a multiplex PCR was optimized using markers XAJ1, XAJ6, and XAJ8. This triplex PCR, besides confirming the dot blot data for each of the 52 Xaj, was able to detect Xaj in field infected walnut leaves and fruits. Altogether, these nine Xaj-specific markers allow conciliating the specificity of DNA-detection assays with typing resolution, contributing to rapid detection and identification of potential emergent and acutely virulent Xaj genotypes, infer their distribution, disclose the presence of this phytopathogen on potential alternative host species and improve phytosanitary control.

Moderate malnutrition in rats induces somatic gene mutations.

The relationship between malnutrition and genetic damage has been widely studied in human and animal models, leading to the observation that interactions between genotoxic exposure and micronutrient status appear to affect genomic stability. A new assay has been developed that uses the phosphatidylinositol glycan class A gene (Pig-a) as a reporter for measuring in vivo gene mutation. The Pig-a assay can be employed to evaluate mutant frequencies (MFs) in peripheral blood reticulocytes (RETs) and erythrocytes (RBCs) using flow cytometry. In the present study, we assessed the effects of malnutrition on mutagenic susceptibility by exposing undernourished (UN) and well-nourished (WN) rats to N-ethyl-N-nitrosourea (ENU) and measuring Pig-a MFs. Two week-old UN and WN male Han-Wistar rats were treated daily with 0, 20, or 40mg/kg ENU for 3 consecutive days. Blood was collected from the tail vein one day before ENU treatment (Day-1) and after ENU administration on Days 7, 14, 21, 28, 35, 42, 49, 56 and 63. Pig-a MFs were measured in RETs and RBCs as the RET(CD59-) and RBC(CD59-) frequencies. In the vehicle control groups, the frequencies of mutant RETs and RBCs were significantly higher in UN rats compared with WN rats at all sampling times. The ENU treatments increased RET and RBC MFs starting at Day 7. Although ENU-induced Pig-a MFs were consistently lower in UN rats than in WN rats, these differences were not significant. To understand these responses, further studies should use other mutagens and nucleated surrogate cells and examine the types of mutations induced in UN and WN rats.

Cytokine patterns in paediatric patients presenting serious gastrointestinal and respiratory bacterial infections.

In the adaptive immune response, the types of cytokines produced define whether there is a cellular (T1) or a humoral (T2) response. Specifically, in the T1 response, interleukin 2 (IL-2), interferon γ (IFN-γ) and tumor necrosis factor ß (TNF-ß) are produced, whereas in the T2 response, IL-4, IL-5, IL- 6, IL-10 and IL-13 are primarily produced. Cytokines are primarily involved in the regulation of immune system cells. The aim of the present study was to evaluate the cytokine patterns (Type 1/Type 2) and TNF-α expression levels in children with severe gastrointestinal and respiratory bacterial infections. The enzyme-linked immunosorbent assay (ELISA) technique was used to identify the cytokines and the infectious agents. The results obtained demonstrated that, in general, children with bacterial infections experienced an increase in IL-2, IFN-γ and IL-4 concentrations and a decrease in TNF-α, IL-5 and IL-6 concentrations when compared to healthy children. Specifically, type 1 cytokines and an increased TNF-α concentration were found in children with gastrointestinal infections. However, patients with respiratory infections showed increased concentrations of both T2 (IL-4, IL-6 and IL-10) and T1 (IL-2 and IFN-γ) components. Thus, it was concluded that children with gastrointestinal infections exclusively developed a T1 response, whereas children with respiratory infections developed a T1/T2 response to fight the infection.

Evolutionary and experimental assessment of novel markers for detection of Xanthomonas euvesicatoria in plant samples.

BACKGROUND: Bacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens. METHODOLOGY: In this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants. SIGNIFICANCE: This study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics.

Malnutrition and infection influence the peripheral blood reticulocyte micronuclei frequency in children.

Malnutrition is a serious public health problem that affects approximately one third of all children. Developing countries have the highest incidence of malnourished children, and approximately 60% of deaths that occur in children under five are directly related to malnutrition and associated diseases. The relationship between malnutrition and genetic damage has been widely studied in humans and animal models. The micronucleus (MN) assay is useful in detecting chromosome damage induced by several factors. The aim of this study was to evaluate the effects of infection and malnutrition on the frequency of MN in erythrocytes from the peripheral blood of well-nourished, uninfected (WN) and well-nourished, infected (WNI) children, and moderately malnourished (UNM) and severely malnourished (UNS) children, both with infection, using a flow cytometric analysis technique. The percentage of reticulocytes (RETs) was significantly higher (1.5-fold) in WNI children than well-nourished controls. In addition, the UNS group had a 2.2-fold increase in the percentage of RETs compared to the WNI group. The frequency of micronucleated reticulocytes (MN-RETs) was 2.5 times greater, in WNI group compared to the WN group. These frequencies were significantly higher (1.7- and 2.1-fold) in UNM and UNS, respectively, compared to the WNI group. The results suggest that infection and malnutrition induce DNA damage in children.

Identification of Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans with novel markers and using a dot blot platform coupled with automatic data analysis.

Phytosanitary regulations and the provision of plant health certificates still rely mainly on long and laborious culture-based methods of diagnosis, which are frequently inconclusive. DNA-based methods of detection can circumvent many of the limitations of currently used screening methods, allowing a fast and accurate monitoring of samples. The genus Xanthomonas includes 13 phytopathogenic quarantine organisms for which improved methods of diagnosis are needed. In this work, we propose 21 new Xanthomonas-specific molecular markers, within loci coding for Xanthomonas-specific protein domains, useful for DNA-based methods of identification of xanthomonads. The specificity of these markers was assessed by a dot blot hybridization array using 23 non-Xanthomonas species, mostly soil dwelling and/or phytopathogens for the same host plants. In addition, the validation of these markers on 15 Xanthomonas spp. suggested species-specific hybridization patterns, which allowed discrimination among the different Xanthomonas species. Having in mind that DNA-based methods of diagnosis are particularly hampered for unsequenced species, namely, Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans, for which comparative genomics tools to search for DNA signatures are not yet applicable, emphasis was given to the selection of informative markers able to identify X. fragariae, X. axonopodis pv. phaseoli, and X. fuscans subsp. fuscans strains. In order to avoid inconsistencies due to operator-dependent interpretation of dot blot data, an image-processing algorithm was developed to analyze automatically the dot blot patterns. Ultimately, the proposed markers and the dot blot platform, coupled with automatic data analyses, have the potential to foster a thorough monitoring of phytopathogenic xanthomonads.

Cashew nut extract (De BCC) in the treatment of basal cell carcinoma

PURPOSE OF THE STUDY: Basal cell carcinoma (BCC) remains as the most common cutaneous neoplasm in the Philippines consisting of more than 60% of all skin cancers. Anacardium occidentale (Linn.) cashew extract, which had been used successfully in the removal of warts and moles in previous studies, is presented as a therapeutic option in BCC. METHODS: An open-label prospective study was conducted on 36 patients (mean age of 65 years) with documented BCC lesions on middle third of the face. Cashew nut extract (DeBCC) application was offered to these patients as an alternative option to very unacceptable extirpative surgery offered by general and plastic surgeons as treatment for their lesions. Lesion size ranged from 7.5- 64 mm. (26.26 mm). Topical treatment was applied every 1-2 weeks, as needed. Follow-up examinations with photographic documentation were made every week to evaluate success of the treatment. RESULTS: After a mean of 7 treatment applications (range of 1-20) all the lesions were undetectable on clinical examinations. Mild tingling sensation, which was reported by all patients during the treatment applications, was tolerable on all occasions. With follow-up ranging from 5-60 months (38.72 months), 16 patients completed the planned 5 - year post-treatment follow-up period. There were no recurrences detected. CONCLUSIONS: Anacardium occidentale (Linn.) cashew extract (DeBCC) presents a viable and acceptable treatment option in primary BCC. The importance of this treatment option could be stressed in patients with lesions not amenable to the prescribed wide margin of resection needed in surgery.