Matrix Stiffness Modulates Metabolic Interaction between Human Stromal and Breast Cancer Cells to Stimulate Epithelial Motility.

Breast tumors belong to the type of desmoplastic lesion in which a stiffer tissue structure is a determinant of breast cancer progression and constitutes a risk factor for breast cancer development. It has been proposed that cancer-associated stromal cells (responsible for this fibrotic phenomenon) are able to metabolize glucose via lactate production, which supports the catabolic metabolism of cancer cells. The aim of this work was to investigate the possible functional link between these two processes. To measure the effect of matrix rigidity on metabolic determinations, we used compliant elastic polyacrylamide gels as a substrate material, to which matrix molecules were covalently linked. We evaluated metabolite transport in stromal cells using two different FRET (Fluorescence Resonance Energy Transfer) nanosensors specific for glucose and lactate. Cell migration/invasion was evaluated using Transwell devices. We show that increased stiffness stimulates lactate production and glucose uptake by mammary fibroblasts. This response was correlated with the expression of stromal glucose transporter Glut1 and monocarboxylate transporters MCT4. Moreover, mammary stromal cells cultured on stiff matrices generated soluble factors that stimulated epithelial breast migration in a stiffness-dependent manner. Using a normal breast stromal cell line, we found that a stiffer extracellular matrix favors the acquisition mechanistical properties that promote metabolic reprograming and also constitute a stimulus for epithelial motility. This new knowledge will help us to better understand the complex relationship between fibrosis, metabolic reprogramming, and cancer malignancy.

The overexpression of TcAP1 endonuclease confers resistance to infective Trypanosoma cruzi trypomastigotes against oxidative DNA damage.

Trypanosoma cruzi, the causative agent of Chagas' disease survives to DNA damage generated by ROS/RNS inside to their different hosts. In recent eukaryotes, oxidative DNA damage is repaired mainly by the Base Excision Repair (BER) pathway, being essential the apurinic/apyrimidinic endonuclease activity. Using a pTREX-gfp vector, the nucleotide sequence that encodes T. cruzi AP endonuclease TcAP1 (orthologue of human APE1) and a putative TcAP1 dominant negative (TcAP1DN), were transfectedand expressed in T. cruzi epimastigotes. TcAP1-GFP and TcAP1DN-GFP were expressed in those modified epimastigotes and found in the parasite nucleus. The endonucleases were purified under native conditions and the AP endonuclease activity was evaluated. While TcAP1 presents the expected AP endonuclease activity TcAP1DN does not. Moreover, TcAP1DN partially inhibits in vitro TcAP1 enzymatic activity. Transfected epimastigotes expressing TcAP1-GFP and TcAP1DN-GFP were differentiated to infective trypomastigotes. The infective parasites maintained both proteins (TcAP1-GFP and TcAP1DN-GFP) in the nucleus. The overexpression of TcAP1-GFP in epimastigotes and trypomastigotes increases the viability of both parasite forms when exposed to oxidative stress while the expression of TcAP1DN-GFP did not show any in vivo inhibitory effect, suggesting that endogenous TcAP1 constitutive expression overcomes the TcAP1DN inhibitory activity. Our results show that TcAP1 is important for trypomastigote survival under oxidative conditions similar to those found in infected mammalian cells, then increasing its permanence in the infected cells and the possibility of development of Chagas disease.

Importance of adhesins in the recurrence of pharyngeal infections caused by Streptococcus pyogenes.

PURPOSE: Pharyngo-amygdalitis is the most common infection caused by Streptococcus pyogenes (S. pyogenes). Reinfection with strains of different M types commonly occurs. However, a second infection with a strain of the same M type can still occur and is referred to as recurrence. We aimed to assess whether recurrence of S. pyogenes could be associated to erythromycin resistance, biofilm formation or surface adhesins like fibronectin-binding proteins and pilus proteins, both located in the fibronectin-binding, collagen-binding, T-antigen (FCT) region. METHODOLOGY: We analyed clinical isolates of S. pyogenes obtained from children with multiple positive cultures of throat swabs. We analysed potential associations between M types, clonal patterns, biofilm production and FCT types with their capacity of producing a recurrent infection. We genetically defined recurrence as an infection with the same M type (same strain) and reinfection as an infection with a different M type. RESULTS: No differences were observed between recurrent and reinfection isolates in relation to erythromycin resistance, presence and number of domains of prtF1 gene, and biofilm formation capacity; the only significant difference was the higher frequency of FCT-4 type among recurrent isolates. However, when all the factors that could contribute to recurrence (erythromycin resistance, biofilm production, presence of prtF1 gene and FCT-4 type) were analysed together, we observed that recurrent isolates have a higher number of factors than reinfection isolates. CONCLUSIONS: Recurrence seems not to be associated with biofilm formation. However, pili and fibronectin-binding proteins could be associated with recurrence because FCT-4 isolates which harbour two fibronectin-binding proteins are more frequent among recurrent isolates.

A Flap Endonuclease (TcFEN1) Is Involved in Trypanosoma cruzi Cell Proliferation, DNA Repair, and Parasite Survival.

FLAP endonucleases (FEN) are involved both in DNA replication and repair by processing DNA intermediaries presenting a nucleotide flap using its phosphodiesterase activity. In spite of these important functions in DNA metabolism, this enzyme was not yet studied in Trypanosomatids. Trypanosoma cruzi, the ethiological agent of Chagas disease, presents two dividing cellular forms (epimastigote and amastigote) and one non-proliferative, infective form (trypomastigote). The parasite survives DNA damage produced by reactive species generated in its hosts. The activity of a T. cruzi FLAP endonuclease (TcFEN1) was determined in the three cellular forms of the parasite using a DNA substrate generated by annealing three different oligonucleotides to form a double-stranded DNA with a 5' flap in the middle. This activity showed optimal pH and temperature similar to other known FENs. The substrate cut by the flap endonuclease activity could be ligated by the parasite generating a repaired DNA product. A DNA flap endonuclease coding sequence found in the T. cruzi genome (TcFEN1) was cloned, inserted in parasite expression vectors and transfected to epimastigotes. The purified native recombinant protein showed DNA flap endonuclease activity. This endonuclease was found located in the parasite nucleus of transfected epimastigotes and its over-expression increased both parasite proliferation and survival to H2 O2 . The presence of a flap endonuclease activity in T. cruzi and its nuclear location are indicative of the participation of this enzyme in DNA processing of flap fragments during DNA replication and repair in this parasite of ancient evolutive origin. J. Cell. Biochem. 118: 1722-1732, 2017. © 2016 Wiley Periodicals, Inc.

Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase.

Trypanosoma cruzi, the etiological agent of Chagas' disease, presents three cellular forms (trypomastigotes, epimastigotes and amastigotes), all of which are submitted to oxidative species in its hosts. However, T. cruzi is able to resist oxidative stress suggesting a high efficiency of its DNA repair machinery.The Base Excision Repair (BER) pathway is one of the main DNA repair mechanisms in other eukaryotes and in T. cruzi as well. DNA glycosylases are enzymes involved in the recognition of oxidative DNA damage and in the removal of oxidized bases, constituting the first step of the BER pathway. Here, we describe the presence and activity of TcNTH1, a nuclear T. cruzi DNA glycosylase. Surprisingly, purified recombinant TcNTH1 does not remove the thymine glycol base, but catalyzes the cleavage of a probe showing an AP site. The same activity was found in epimastigote and trypomastigote homogenates suggesting that the BER pathway is not involved in thymine glycol DNA repair. TcNTH1 DNA-binding properties assayed in silico are in agreement with the absence of a thymine glycol removing function of that parasite enzyme. Over expression of TcNTH1 decrease parasite viability when transfected epimastigotes are submitted to a sustained production of H2O2.Therefore, TcNTH1 is the only known NTH1 orthologous unable to eliminate thymine glycol derivatives but that recognizes and cuts an AP site, most probably by a beta-elimination mechanism. We cannot discard that TcNTH1 presents DNA glycosylase activity on other DNA base lesions. Accordingly, a different DNA repair mechanism should be expected leading to eliminate thymine glycol from oxidized parasite DNA. Furthermore, TcNTH1 may play a role in the AP site recognition and processing.

Algunos aspectos clínicos-epidemiológicos del cafeinismo en una comunidad rural

Se realizó un estudio descriptivo transversal con el objetivo de determinar la prevalencia y algunos aspectos clínicos-epidemiológicos del cafeinismo en la comunidad rural correspondiente al consultorio 76 del policlínico Ramón Heredia Umpierre de Veguitas, municipio Yara, durante el período 1ro. de julio del 2000 hasta diciembre del 2001. El universo estuvo dado por 62 pacientes identificados como enfermos (cafeinómanos) a partir de la aplicación del mismo modelo diagnóstico utilizado para el alcoholismo; se utilizó como medida de resumen el porcentaje y parámetros como la media y desviación estándar. Existió una alta prevalencia de cafeinismo (12,6 por ciento) y sobre todo fueron más afectadas las mujeres con marcada tendencia a incrementarse en ambos sexos paralelo a la edad. La satisfacción fue el móvil mayormente referido por los pacientes para el consumo de café (41,9 por ciento); se destacan las manifestaciones físicas (principalmente la acidez y al epigastralgia), por encima de las psíquicas, dentro de las cuales la ansiedad figuró como la más importante. En el marco actual de una importante batalla de carácter coyuntural que alcanza su mayor connotación en Cuba, esta investigación puede convertirse en una referencia que da medida de la necesidad de intervenir frente a un hábito tóxico que para los efectos es parte de una vieja costumbre social, ignorando casi siempre su nocividad(AU)

Riesgo pediátrico, enfoque institucional / Pediatric risk. Institutional focus

Con el objetivo de evaluar el comportamiento de algunas variables epidemiológicas como la edad, el sexo, estado clínico al ingreso institucional, complicaciones nosocomiales y estadia hospitalaria de niños ingresados en el hospital pediátrico docente Hermanos Cordové de la ciudad de Manzanillo, se realizó un estudio comparativo durante el primer semestre del año 2001. El universo se constituyó por 260 niños con algún riesgo, que a la vez representaron los casos; los controles se seleccionaron en la misma proporción bajo criterio fundamental de estar libres de riesgos; se hizo comparación a través del estadígrafo chi cuadrado con a = 0,05. La condición de riesgo predominó en los varones y en el grupo de menores de un año, asimismo, esos niños con riesgos asociados tenían un estado clínico más desfavorable al ingreso que los niños sin riesgo (p<0,05), además sufrieron mayor número de complicaciones (p<0,05) y prolongaron más la estadia. Los resultados pudieran ser muy útiles al efecto de establecer pautas específicas para intervenir sobre el fenómeno riesgo pediátrico desde el ámbito comunitario(AU)

With the aim to evaluate the behavior of some epidemiological variables like the age, the sex, clinical state to the institutional entry, nosocomial complications and hospital stay of children at the Pediatric Teaching Hospital Hermanos Cordové in Manzanillo. It was performed a comparative study during the first semester of the year 2001. The universe was constituted by 260 children with some risk, that at the same time represented the cases; the controls were selected in the same proportion under the main criterion to be free of risks; the comparison was made through the estadigraph chi squared with a = 0,05. The condition of risk predominated in the men and in the group of minors of a year, likewise, these boys with risks associated had a clinical state more desfavorable to the entry that the boys without risk (p<0,05), besides suffered greater number of complications (p<0,05) and prolonged more the estadia. The results could be very useful to the effect to establish specific guidelines to take part on the phenomenon. Risk pediátrico?From the community field(EU)

El factor alcoholismo en la conducta suicida / Alcoholism factor in the suicidal behavior

Se realizó un estudio de casos y controles para conocer la influencia del alcoholismo en la conducta suicida de un grupo de adultos del área de salud de Veguita, policlínico Ramón Heredia Umpierre, durante el último semestre del año 2000 y el primero del 2001. Se seleccionó por cada caso (n=51) un control estableciéndose un apareamiento 1:1 y se procedió a calcular el estadígrafo McNemar y odds ratio (OR) del otro para analizar la fuerza de asociación entre el factor y el suceso consecuente; luego se determinó a través del coeficiente f la correlación entre el alcoholismo y otras condiciones de riesgo en el grupo de casos. El factor alcoholismo influyó como un riesgo real de la conducta suicida (OR=5,25); otros riesgos como falta de apoyo y atención familiar y expresión manifiesta del intento suicida mostraron una correlación moderada con el alcoholismo (f=0.50 en ambos casos)(AU)

A case-control study was conducted to determine the influence of alcoholism in the suicidal behavior of a group of adults in the health area of Veguita, Ramón Heredia Umpierre Polyclinic during the last semester of the year 2000 and the first semester of 2001. Fore each case it was selected (n = 51) a control establishing a mating 1: 1 and it was calculated the McNemar and Odds Ratio (OR) of the other to analyze the strength of association between the factor and the consequent event; then it was determined the correlation between alcoholism and other conditions of risk in the Group of cases through the f coefficient. The alcoholism factor influenced as a real risk of suicidal behavior (OR = 5, 25); other risks such as lack of support and family care and expression of the suicide attempt showed a moderate correlation with alcoholism (f = 0. 50 in both cases)(EU)