Large area fractional laser treatment of mouse skin increases energy expenditure.

Fractional laser (FL) treatment is a common dermatologic procedure that generates arrays of microscopic treatment zones separated by intact tissue, promoting fast wound healing. Using a mouse model, we introduced a large area fractional laser treatment (LAFLT) method to study metabolic effects. Using two laser modalities, ablative FL (AFL) and non-ablative FL (NAFL), and exposing different percentages of mice's total body surface area (TBSA), we followed changes in metabolic parameters in real time using metabolic cages. Additionally, body composition, markers of inflammation, neurohormonal signaling, and browning of adipocytes were investigated. LAFLT, especially in high TBSA groups, had specific metabolic effects such as significantly increased average daily energy expenditure, increased fat mass loss, systemic browning of adipocytes, and inflammatory states, without compromising other organs. The ability of LAFLT to stimulate metabolism in a controlled way could develop into a promising therapeutic treatment to induce positive metabolic changes that replace or augment systemic drugs.

Mouse model of selective cryolipolysis.

BACKGROUND: Cryolipolysis is a noninvasive method of destroying adipocytes using controlled cooling, thereby enabling localized and targeted fat reduction. Due to their greater vulnerability to cold injury, adipocytes are selectively targeted, while other cell types are spared. OBJECTIVES: This study aims to develop a mouse model of cryolipolysis to offer a reliable and convenient alternative to human models, providing a methodology to validate clinical hypotheses in-depth with relative ease, low cost, and efficiency. This further facilitates comprehensive studies of the molecular mechanisms involved in cryolipolysis. MATERIALS AND METHODS: Mice (C57BL/6J) were placed under general anesthesia and were treated using our custom, miniaturized cryolipolysis system. A thermoelectric cooling probe was applied to the inguinal (ING) area for either a cold exposure of -10°C, or for a room temperature exposure for 10 minutes. The thickness of the subcutaneous fat of the mice was quantified using an optical coherence tomography (OCT) imaging system before and after the treatment. Histological analyses were performed before and after cryolipolysis at multiple time points. RESULTS: OCT analysis showed that mice that underwent cold cryolipolysis treatment induced a significantly greater reduction of subcutaneous fat thickness 1 month after treatment than the control mice. The mice that received cold treatment had no skin injuries. The selective damage of adipocytes stimulated cold panniculitis that was characterized histologically by infiltration of immune cells 2 and 3 days after treatment. CONCLUSION: This study shows that cryolipolysis performed in mice yields reproducible and measurable subcutaneous fat reduction, consistent with previous studies conducted in humans and pigs. Future studies can utilize the model of selective cryolipolysis developed by our group to further elucidate the cellular and molecular mechanisms of fat cell loss and improve clinical outcomes in humans.

Segmentation of mouse skin layers in optical coherence tomography image data using deep convolutional neural networks.

Optical coherence tomography (OCT) enables the non-invasive acquisition of high-resolution three-dimensional cross-sectional images at micrometer scale and is mainly used in the field of ophthalmology for diagnosis as well as monitoring of eye diseases. Also in other areas, such as dermatology, OCT is already well established. Due to its non-invasive nature, OCT is also employed for research studies involving animal models. Manual evaluation of OCT images of animal models is a challenging task due to the lack of imaging standards and the varying anatomy among models. In this paper, we present a deep learning algorithm for the automatic segmentation of several layers of mouse skin in OCT image data using a deep convolutional neural network (CNN). The architecture of our CNN is based on the U-net and is modified by densely connected convolutions. We compared our adapted CNN with our previous algorithm, a combination of a random forest classification and a graph-based refinement, and a baseline U-net. The results showed that, on average, our proposed CNN outperformed our previous algorithm and the baseline U-net. In addition, a reduction of outliers could be observed through the use of densely connected convolutions.

Enhanced quantification of metabolic activity for individual adipocytes by label-free FLIM.

Fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) allows for label-free quantification of metabolic activity of individual cells over time and in response to various stimuli, which is not feasible using traditional methods due to their destructive nature and lack of spatial information. This study uses FLIM to measure pharmacologically induced metabolic changes that occur during the browning of white fat. Adipocyte browning increases energy expenditure, making it a desirable prospect for treating obesity and related disorders. Expanding from the traditional two-lifetime model of NADH to a four-lifetime model using exponential fitting and phasor analysis of the fluorescence decay results in superior metabolic assessment compared to traditional FLIM analysis. The four lifetime components can also be mapped to specific cellular compartments to create a novel optical ratio that quantitatively reflects changes in mitochondrial and cytosolic NADH concentrations and binding states. This widely applicable approach constitutes a powerful tool for studies where monitoring cellular metabolism is of key interest.

Tfe3 and Tfeb Transcriptionally Regulate Peroxisome Proliferator-Activated Receptor γ2 Expression in Adipocytes and Mediate Adiponectin and Glucose Levels in Mice.

Members of the MiT transcription factor family are pivotal regulators of several lineage-selective differentiation programs. We show that two of these, Tfeb and Tfe3, control the regulator of adipogenesis, peroxisome proliferator-activated receptor γ2 (Pparγ2). Knockdown of Tfeb or Tfe3 expression during in vitro adipogenesis causes dramatic downregulation of Pparγ2 expression as well as adipogenesis. Additionally, we found that these factors regulate Pparγ2 in mature adipocytes. Next, we demonstrated that Tfeb and Tfe3 act directly by binding to consensus E-boxes within the Pparγ transcriptional regulatory region. This transcriptional control also exists in vivo, as we discovered that wild-type mice in the fed state increased their expression of Tfe3, Tf3b, and Pparγ in white adipose tissue. Furthermore, Tfe3 knockout (Tfe3KO) mice in the fed state failed to upregulate Pparγ and the adiponectin gene, a Pparγ-dependent gene, confirming the in vivo role for Tfe3. Lastly, we found that blood glucose is elevated and serum adiponectin levels are suppressed in the Tfe3KO mice, indicating that the Tfe3/Tfeb/Pparγ2 axis may contribute to whole-body energy balance. Thus, we offer new insights into the upstream regulation of Pparγ by Tfe3/Tf3b and propose that targeting these transcription factors may offer opportunities to complement existing approaches for the treatment of diseases that have dysregulated energy metabolism.

Transcription Factor Tfe3 Directly Regulates Pgc-1alpha in Muscle.

The microphthalmia (MiT) family of transcription factors is an important mediator of metabolism. Family members Mitf and Tfeb directly regulate the expression of the master regulator of metabolism, peroxisome-proliferator activated receptor gamma coactivator-1 alpha (Pgc-1alpha), in melanomas and in the liver, respectively. Pgc-1alpha is enriched in tissues with high oxidative capacity and plays an important role in the regulation of mitochondrial biogenesis and cellular metabolism. In skeletal muscle, Pgc-1alpha affects many aspects of muscle functionally such as endurance, fiber-type switching, and insulin sensitivity. Tfe3 also regulates muscle metabolic genes that enhance insulin sensitivity in skeletal muscle. Tfe3 has not yet been shown to regulate Pgc-1alpha expression. Our results reported here show that Tfe3 directly regulates Pgc-1alpha expression in myotubes. Tfe3 ectopic expression induces Pgc-1alpha, and Tfe3 silencing suppresses Pgc-1alpha expression. This regulation is direct, as shown by Tfe3's binding to E-boxes on the Pgc-1alpha proximal promoter. We conclude that Tfe3 is a critical transcription factor that regulates Pgc-1alpha gene expression in myotubes. Since Pgc-1alpha coactivates numerous biological programs in diverse tissues, the regulation of its expression by upstream transcription factors such Tfe3 implies potential opportunities for the treatment of diseases where modulation of Pgc-1alpha expression may have important clinical outcomes.

Chromatin accessibility and transcription factor binding at the PPARγ2 promoter during adipogenesis is protein kinase A-dependent.

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor that specifies formation of the adipocyte lineage. PPARγ also serves as a primary target for the treatment of type 2 diabetes, illustrating both its medical relevance as well as the need to understand fundamental aspects of PPARγ expression and function. Here, we characterize molecular changes that occur at the PPARγ2 promoter within the first several hours of adipocyte differentiation in culture. Our results demonstrate that changes in chromatin accessibility at the PPARγ2 promoter and occupancy of the promoter by the c-Fos transcription factor occur within an hour of the onset of differentiation, followed closely by the binding of the CCAAT/enhancer binding protein beta (C/EBPß) transcription factor. All three events show a remarkable dependency on protein kinase A (PKA) activity. These results reflect novel requirements for the PKA signaling pathway and reinforce the importance of PKA function during the onset of adipocyte differentiation.

Phenotypic transcription factors epigenetically mediate cell growth control.

Ribosomal RNA (rRNA) genes are down-regulated during osteogenesis, myogenesis, and adipogenesis, necessitating a mechanistic understanding of interrelationships between growth control and phenotype commitment. Here, we show that cell fate-determining factors [MyoD, myogenin (Mgn), Runx2, C/EBPbeta] occupy rDNA loci and suppress rRNA expression during lineage progression, concomitant with decreased rRNA expression and reciprocal loss of occupancy by c-Myc, a proliferation-specific activator of rRNA transcription. We find interaction of phenotypic factors with the polymerase I activator upstream binding factor UBF-1 at interphase nucleoli, and this interaction is epigenetically retained on mitotic chromosomes at nucleolar organizing regions. Ectopic expression and RNA interference establish that MyoD, Mgn, Runx2, and C/EBPbeta each functionally suppress rRNA genes and global protein synthesis. We conclude that epigenetic control of ribosomal biogenesis by lineage-specific differentiation factors is a general developmental mechanism for coordinate control of cell growth and phenotype.

Temporal recruitment of transcription factors and SWI/SNF chromatin-remodeling enzymes during adipogenic induction of the peroxisome proliferator-activated receptor gamma nuclear hormone receptor.

The peroxisome proliferator-activated receptor gamma (PPARgamma) regulates adipogenesis, lipid metabolism, and glucose homeostasis, and roles have emerged for this receptor in the pathogenesis and treatment of diabetes, atherosclerosis, and cancer. We report here that induction of the PPARgamma activator and adipogenesis forced by overexpression of adipogenic regulatory proteins is blocked upon expression of dominant-negative BRG1 or hBRM, the ATPase subunits of distinct SWI/SNF chromatin-remodeling enzymes. We demonstrate that histone hyperacetylation and the binding of C/EBP activators, polymerase II (Pol II), and general transcription factors (GTFs) initially occurred at the inducible PPARgamma2 promoter in the absence of SWI/SNF function. However, the polymerase and GTFs were subsequently lost from the promoter in cells expressing dominant-negative SWI/SNF, explaining the inhibition of PPARgamma2 expression. To corroborate these data, we analyzed interactions at the PPARgamma2 promoter in differentiating preadipocytes. Changes in promoter structure, histone hyperacetylation, and binding of C/EBP activators, Pol II, and most GTFs preceded the interaction of SWI/SNF enzymes with the PPARgamma2 promoter. However, transcription of the PPARgamma2 gene occurred only upon subsequent association of SWI/SNF and TFIIH with the promoter. Thus, induction of the PPARgamma nuclear hormone receptor during adipogenesis requires SWI/SNF enzymes to facilitate preinitiation complex function.

Presencia de IgG-Anti-helicobacter pylori en mujeres embarazadas y muestras sanguineas de cordon umbilical / The prevalence of IgG anti.H.pylori in pregnant women and serum samples from umbilical cord blood

En 50 sueros obtenidos de sangre venosa de mujeres embarazadas, sin afecciones gastroduodenales, y 50 muestras de sangre cordón umbilical, se analizó la presencia de IgG-anti H.pylori. En el 54 por ciento de las madres, se observaron títulos de IgG anti H.pylori. Todas las muestras de cordón umbilical obtenidas durante el parto del grupo de mujeres seropositivas para H.pylori. El 46 por ciento de las madres fueron negativas para IgG antio H.pylori al igual que las muestras apareadas obtenidas de cordón umbilical. Estos resultados sugieren que existe una transmisión de anticuerpos IgG anti.pylori a través de la barrera placentaria lo cual contribuye a la protección de los recién nacidos en contraer la infección con H.pylori. En la población pediátrica estudiada en edades comprendidas entre 10 meses y 10 años, se determinó la presencia de IgG anti H.pylori en un 62.5 por ciento de los niños, el 37.5 por ciento de esta población infantil fue seronegativa para H.pylori (P<0.0001)(

Detección de virus papiloma humano (VPH) mediante biología molecular y su asociación con neoplasia cervical uterina / Human papiloma virus detection(HPV) through molecular biology and its association with cervical neoplasms

Estudios epidemiológicos indican que uno de los factores de mayor riesgo en el desarrollo de lesiones preinvasoras o invasoras de cervix, es la lesión de VPH independientemente de otros factores de riesgo conocidos tales como: edad del inicio de actividad sexual, número de compañeros sexuales, estado socio económico y hábitos tabáquicos. La mayor incidencia de cáncer cervical a nivel mundial ocurre en mujeres en edades comprendidas entre 40-55 años y en el 90 por ciento de los casos se encuentra asociada a VPH como agente etiológico. En Venezuela el cáncer de cuello uterino representa la primera causa de muerte oncológica en mujeres en el grupo etario mencionado anteriormente. El objetivo del presente estudio fue determinar la presencia de VPH de bajo, intermedio y alto riesgo oncogénico en especímenes cervicales mediante técnicas de biología molecular y su asociación con neoplasia cervical. Selección de Pacientes y Metodología: Se estudiaron 120 pacientes las cuales presentaron alteraciones clínicas, cambios citológicos, ambos o lesión en su pareja. El promedio de edad de la población estudiada fue de 24 años. Las muestras se tomaron mediante hisopado del cuello uterino(endo y exocervix) con hisopo estéril de dacrón, colocado en un medio de transporte (Viratype, Digene). El ADN total fue obtenido mediante lisis celular, desproteinazación con cloroformo y presipitación con etanol 95 por ciento. Para la reacción en cadena de la polimerasa (PCR) se utilizaron oligonucleóticos cebadores MY09 y MY011., que reconocen un segmento conservador de la región viral L1. La tipificación viral se realizó utilizando el sistema "Sharp Signal" (Digene) que reconoce virus de bajo riesgo oncogénico: 6,11,42,43,44: e intermedio/alto riesgo oncogénico: 16,18,31,33,35,45,51,52,53,56 y 58. En el 66,6 por ciento (80/120) de las muestras de ADN de pacientes se observó la presencia de genoma viral y el 33,3 por ciento (40/120) de los pacientes fueron negativos para VPH (p<0,0001). En el subgrupo de pacientes sin cambios morfológicos sugestivos de infección por VPH se encontró ADN viral en el 40 por ciento de las muestras, de las cuales 23,9 por ciento presentaron VPH del tipo AR oncogénico, 3,3 por ciento fueron del tipo BR y 13,3 por ciento fueron no tipificables. En el subgrupo de pacientes con LIEbg se determinó la presencia de VPH en el 70 por ciento cuya tipificación permitió determinar el que 56,25 por ciento fue de...

Ensayo inmunoenzimático para el serodiagnóstico de la infección de helicobacter pylori / Inmunoenzymatic essay for serodiagnosis of helicobacter pylori infection

Actualmente se encuentran disponibles un gran número de ensayos inmunoenzimáticos para la determminación de la respuesta humoral generada durante la infección por Helicobacter Pylori. Sin embargo, no existe un concenso sobre el método ideal a utilizar para evaluar la infección debida a este microorganismo. En el presente estudio se evaluó la sensibilidad de un ensayo inmunoenzimático desarrollado en el laboratorio, empleando 38 sueros de pacientes con úlcera duodenal y cultivo positivo para H. pylori y 41 sueros de niños sanos menores de 10 años. En el ensayo se utilizaron extractos proteícos obtenidos de la sonicación de cinco cepas aisladas de los pacientes con úlcera duodenal. Los niveles de la IgG del grupo de pacientes fueron significativamente mayores (p < 0.001) que en el grupo control. El límite de positividad de 0.231 fue determinado empleando la media + 3ds, de los títulos obtenidos en el grupo control. Los resultados del ensayo inmunoenzimático mostraron que una dilución de 1:300 de los sueros y una concentración de antígeno de 5 ug/ml sensibilidad del 92 por ciento en la determinación de la infección por H. pylori

Caracterización de antígenos Paracoccidioides brasiliensis: inmunodiagnóstico serológico mediante western blotting / Characterization of Paracoccidioides brasiliensis antigens. Serological immunodiagnosis using western blotting

The electrophoretic characterization on SDS-PAGE gels of Paracoccidioides brasiliensis antigens, strains 2511 and 6688; and the additional use of immunoblotting has permitted in this study to identify the immunogenic, sensitive and specific antigen fractions for the diagnosis of Paracoccidioidomycosis. The antigenic preparations showed differences depending on the morphologic form of the fungus and the method utilized. The procedure revealed heterogeneity in the humoral immune response of the patients studied and permitted us to establish indices of activity of Paracoccidioidomycosis by the sequential analysis of sera obtained during different stages of the disease. The high sensitivity of this method makes it useful as an additional technique for the serologic immunodiagnosis of Paracoccidioidomycosis