Influences of Glabridin Treatment on Motor Function Recovery after Spinal Cord Injury / Influencia del Tratamiento con Glabridina en la Recuperación de la Función Motora después de una Lesión de la Médula Espinal

SUMMARY: Spinal cord injury (SCI) usually arises from compression due to traffic accidents and falls, resulting in varying degrees of movement, sensory loss, and possible paralysis. Glabridin (Gla) is a natural compound derived from licorice. It significantly affects drug development and medicine because of its anti-inflammatory, anti-oxidative, anti-tumoral, antibacterial, bone protective, cardiovascular protective, neuroprotective, liver protective, anti-obesity, and anti-diabetic properties. Various methods were employed to administer Gla to SCI mice in order to investigate its impact on the recovery of motor function. The mice were allocated into four cohorts using a randomization procedure. In the sham cohort, solely the lamina of vertebral arch was surgically exposed without causing any harm to the spinal cord tissue. Conversely, the injury cohort was subjected to spinal cord tissue damage and received no treatment thereafter. The mice in the remaining two cohorts received a dosage of 40 mg/kg Gla every two days via either intraperitoneal or intrathecal injection for a duration of 42 d following spinal cord injury. We conducted behavioral tests utilizing the Basso Mouse Scale score and gait analysis techniques. Magnetic resonance imaging and hematoxylin and eosin were employed to evaluate scar tissue formation. Systemic inflammation in mice was evaluated by employing an enzyme-linked immunosorbent assay. Gla promoted motor function recovery in mice following SCI and improved the pathological environment in the damaged area. These alterations were more evident in mice subjected to the intrathecal injection method. Intraperitoneal injections appear to be more beneficial for controlling systemic inflammatory responses. Although more intensive studies are required, Gla exhibits promising clinical potential as a cost-effective dietary phytochemical.

La lesión de la médula espinal (LME) generalmente surge de la compresión producto de caídas y accidentes de tránsito, lo que resulta en alteraciones del movimiento, pérdida sensorial y posible parálisis. La Glabridina (Gla) es un compuesto natural derivado del regaliz, constituyéndose en un aporte significativo para el desarrollo de fármacos y la medicina debido a sus propiedades antiinflamatorias, antioxidantes, antitumorales, antibacterianas, osteoprotectoras, cardioprotectoras, neuroprotectoras, hepatoprotectoras, antidiabéticas y contra la obesidad. En el presente trabajo se emplearon varios métodos para administrar Gla a ratones con lesión medular con el fin de investigar su impacto en la recuperación de la función motora. Los ratones fueron distribuidos en cuatro grupos mediante un procedimiento de aleatorización. En el grupo simulado, únicamente se expuso quirúrgicamente la lámina del arco vertebral sin causar ningún daño al tejido de la médula espinal. Por el contrario, el grupo lesionado fue sometido a daño del tejido de la médula espinal, sin recibir tratamiento posterior. Los ratones de los dos grupos restantes recibieron una dosis de 40 mg/kg de Gla cada dos días mediante inyección intraperitoneal o intratecal durante 42 días después de la lesión de la médula espinal. Fueron realizadas pruebas de comportamiento utilizando la puntuación de la escala Basso Mouse y técnicas de análisis de la marcha. Se emplearon imágenes por resonancia magnética y se aplicaron tinciones histológicas (Hematoxilina & Eosina) en muestras para evaluar la formación de tejido cicatricial. La inflamación sistémica en ratones se evaluó mediante el empleo de un ensayo inmunoabsorbente ligado a enzimas. Gla promovió la recuperación de la función motora en ratones después de una lesión medular y mejoró el entorno patológico en el área dañada. Estas alteraciones fueron más evidentes en ratones sometidos al método de inyección intratecal. Las inyecciones intraperitoneales parecen ser más beneficiosas para controlar las respuestas inflamatorias sistémicas. Aunque se requieren estudios más intensivos, Gla exhibe un potencial clínico prometedor como fitoquímico dietético rentable.

Novel approach to soft tissue regeneration: in vitro study of compound hyaluronic acid and horizontal platelet-rich fibrin combination.

OBJECTIVE: This study aims to develop a compound biomaterial to achieve effective soft tissue regeneration. METHODOLOGY: Compound hyaluronic acid (CHA) and liquid horizontal-platelet-rich fibrin (H-PRF) were mixed at a ratio of 1:1 to form a CHA-PRF gel. Human gingival fibroblasts (HGFs) were used in this study. The effect of CHA, H-PRF, and the CHA-PRF gel on cell viability was evaluated by CCK-8 assays. Then, the effect of CHA, H-PRF, and the CHA-PRF gel on collagen formation and deposition was evaluated by qRT‒PCR and immunofluorescence analysis. Finally, qRT‒PCR, immunofluorescence analysis, Transwell assays, and scratch wound-healing assays were performed to determine how CHA, H-PRF, and the CHA-PRF gel affect the migration of HGFs. RESULTS: The combination of CHA and H-PRF shortened the coagulation time of liquid H-PRF. Compared to the pure CHA and H-PRF group, the CHA-PRF group exhibited the highest cell proliferation at all time points, as shown by the CCK-8 assay. Col1a and FAK were expressed at the highest levels in the CHA-PRF group, as shown by qRT‒PCR. CHA and PRF could stimulate collagen formation and HGF migration, as observed by fluorescence microscopy analysis of COL1 and F-actin and Transwell and scratch healing assays. CONCLUSION: The CHA-PRF group exhibited greater potential to promote soft tissue regeneration by inducing cell proliferation, collagen synthesis, and migration in HGFs than the pure CHA or H-PRF group. CHA-PRF can serve as a great candidate for use alone or in combination with autografts in periodontal or peri-implant soft tissue regeneration.

Comparison of 4 laboratory tests for the detection of bovine rabies viral infection in Paraguay: fluorescent antibody test, rapid detection test, histologic lesions, and RT-PCR.

Rabies virus (RABV; Lyssavirus rabies) is a neurotropic virus that can be transmitted to mammals by the hematophagous bat Desmodus rotundus. An accurate, accessible method for the detection of RABV in cattle is necessary in Paraguay; thus, we evaluated the detection of RABV using 4 techniques: fluorescent antibody test (FAT), immunochromatography rapid detection test (RDT; Anigen Rapid Rabies Ag test kit; Bionote), a reverse-transcription PCR (RT-PCR) assay, and histologic lesions in different portions of the CNS of 49 Paraguayan cattle to determine the most sensitive and specific technique. By FAT and RDT, 15 of 49 (31%) samples were positive. By RT-PCR amplification of N and G genes, 13 of 49 (27%) and 12 of 49 (25%) were positive, respectively. RDT had high agreement with FAT (kappa = 1); sensitivity was 100% (95% CI: 97-100%) and specificity was 100% (95% CI: 99-100%). The amplification of the N and G genes resulted in substantial agreement (kappa of 0.9 and 0.8, respectively) compared with FAT, and the sensitivity and specificity of the N gene were 87% (95% CI: 66-100%) and 100% (95% CI: 98-100%), respectively, and those of the G gene were 80% (95% CI: 56-100%) and 100% (95% CI: 98-100%), respectively. Histologic lesions observed were lymphoplasmacytic meningoencephalitis, gliosis, and neuronophagia. The agreement observed between the FAT and RDT tests suggests that RDT is an accurate tool for the detection of RABV. Histopathology can be used to confirm lesions caused by RABV and to rule out other conditions; the RT-PCR assay is useful for molecular epidemiology studies.

Acteoside Attenuates Recognition Memory Impairment in CUMS Rats via Regulating Synaptic Plasticity and mTOR Signaling Pathway / El Acteoside Atenúa el Deterioro de la Memoria de Reconocimiento en Ratas ELCI Mediante la Regulación de la Plasticidad Sináptica y la Vía de Señalización mTOR

SUMMARY: We evaluated the role and mechanism of acteoside in the regulation of memory impairment induced by chronic unpredictable mild stress (CUMS). CUMS was used to induce depression in rats and the successful establishment of CUMS model were verified by forced swimming test and sucrose preference test. The Y-maze test and novel object recognition test assessed memory functions. The structural changes in the cortex and hippocampus were observed by hematoxylin and eosin (HE) staining. Immunofluorescence staining and western blotting determined the protein levels. Y-maze test and novel object recognition test showed that there was memory performance impairment in rats of CUMS group, which was improved by the acteoside treatment. HE staining showed that CUMS exposure damaged the structure in the cortex and hippocampus, while the acteoside treatment alleviated the structural changes. Compared with the control group, the levels of BNDF and CREB in the cortex and hippocampus of the CUMS group were significantly decreased. Acteoside significantly reversed the expressions of these proteins in CUMS rats. Meanwhile, compared with the control group, the levels of p-mTOR and p- P70S6K in the cortex and hippocampus of the CUMS group were significantly increased, and these changes were significantly reversed by acteoside. Nevertheless, the effect of acteoside on mTOR signaling was markedly blocked by rapamycin, a specific inhibitor of mTOR signaling. Acteoside can attenuate memory impairment and ameliorate neuronal damage and synaptic plasticity in depression rats probably via inhibiting the mTOR signaling pathway. Acteoside may serve as a novel reagent for the prevention of depression.

Evaluamos el papel y el mecanismo del acteoside en la regulación del deterioro de la memoria inducido por estrés leve crónico impredecible (ELCI). Se utilizó ELCI para inducir depresión en ratas y el establecimiento exitoso del modelo ELCI se verificó mediante una prueba de natación forzada y una prueba de preferencia de sacarosa. La prueba del laberinto en Y y la prueba de reconocimiento de objetos novedosos evaluaron las funciones de la memoria. Los cambios estructurales en la corteza y el hipocampo se observaron mediante tinción con hematoxilina y eosina (HE). La tinción por inmunofluorescencia y la transferencia Western determinaron los niveles de proteína. La prueba del laberinto en Y y la prueba de reconocimiento de objetos novedosos mostraron que había un deterioro del rendimiento de la memoria en ratas del grupo ELCI, que mejoró con el tratamiento con acteósidos. La tinción con HE mostró que la exposición a ELCI dañó la estructura de la corteza y el hipocampo, mientras que el tratamiento con actósidos alivió los cambios estructurales. En comparación con el grupo de control, los niveles de BNDF y CREB en la corteza y el hipocampo del grupo ELCI disminuyeron significativamente. Acteoside revirtió significativamente las expresiones de estas proteínas en ratas ELCI. Mientras tanto, en comparación con el grupo control, los niveles de p-mTOR y p-P70S6K en la corteza y el hipocampo del grupo ELCI aumentaron significativamente, y estos cambios fueron revertidos significativamente ELCI por el acteoside. Sin embargo, el efecto del acteoside sobre la señalización de mTOR fue notablemente bloqueado por la rapamicina, un inhibidor específico de la señalización de mTOR. El acteoside puede atenuar el deterioro de la memoria y mejorar el daño neuronal y la plasticidad sináptica en ratas con depresión, probablemente mediante la inhibición de la vía de señalización mTOR. Acteoside puede servir como un reactivo novedoso para la prevención de la depresión.

Obtaining Tissues of Human Amniotic Membrane and Identification of Pluripotent Markers.

The immunofluorescence technique has been used to identify pluripotent markers in the human amniotic epithelial cells (hAEC). hAEC belonging to human fetal membranes, specificamently to amnion layer, and are arising by epiblast, this sugest that the hAEC have characteristics of epiblast cells, in other words, characteristcs of pluripotent stem cells. Here we describe obtaining human amnion tissue and identifying pluripotent markers by immunofluorescence.

Telocytes in Human Embryonic Development: A Preliminary Microscopic Anatomy Study / Telocitos en el Desarrollo Embrionario Humano: Un Estudio Preliminar de Anatomía Microscópica

SUMMARY: Telocytes are a cell population described in 2011 with a multitude of functions such as tissue support, regulation of stem cell niches or intercellular signal transmission. However, there are no studies about their embryonic origin, their function in development, or their moment of appearance. The objective of this work is to try to answer these questions through histological and immunofluorescence studies with samples from the embryological collection of the Department of Anatomy of the University of Granada. In the results obtained, as demonstrated by immunofluorescence for CD34, the presence of these cells can be seen in the fourth week of embryonic development in the perinotochordal region. Its presence is evident from the sixth week of development in a multitude of organs such as the heart, skeletal muscle tissue and supporting tissue of various organs such as the kidney, brain or pericardium. Its function seems to be when the embryonic histological images are analyzed in an evolutionary way, to act as a scaffold or scaffold for the subsequent population by mature tissue elements. In conclusion, telocytes appear at a very early stage of embryonic development and would have a fundamental role in it as scaffolding and directors of organ and tissue growth.

Los telocitos son una población celular descrita en 2011 con multitud de funciones como el sostén tisular, la regulación de los nichos de células madre o la transmisión de señales intercelulares. Sin embargo, no existen estudios acerca del origen embrionario de los mismos, su función en el desarrollo ni su momento de aparición. El objetivo de este trabajo es tratar de responder a estos interrogantes mediante estudios histológicos y por inmunofluorescencia con muestras de la colección embriológica del Departamento de Anatomía de la Universidad de Granada. En los resultados se puede observar como se demuestra mediante inmunofluorescencia para CD34, la presencia de estas células en la cuarta semana del desarrollo embrionario en la región perinotocordal. Su presencia se evidencia a partir de la sexta semana del desarrollo en multitud de órganos como corazón, tejidos músculo esqueléticos y tejidos de sostén de diversos órganos como riñón, encéfalo o pericardio. Su función parece ser al ser analizadas las imágenes histológicas embrionarias de forma evolutiva, la de actuar como un andamiaje o scafold para el posterior poblamiento por elementos tisulares maduros. Como conclusión, los telocitos aparecen en un momento muy precoz del desarrollo embrionario y presentarían una función fundamental en el mismo como andamiajes y directores del crecimiento de los órganos y tejidos.

Microcirculatory changes as a hallmark of aging in the heart and kidney / Cambios microcirculatorios como sello distintivo del envejecimiento en el corazón y riñón

SUMMARY: Changes in the microcirculation of multiple tissues and organs have been implicated as a possible mechanism in physiological aging. In particular, vascular endothelial growth factor is a secretory protein responsible for regulating angiogenesis via altering endothelial proliferation, survival, migration, extracellular matrix degradation and cell permeability. The aim of the present study was to evaluate the role of vascular endothelial growth factor in the progression of morphological alterations caused by physiological aging in the heart and kidney and to examine its relation to changes in capillary density. We used two age groups of healthy Wistar rats - 6- and 12-month- old. The expression of vascular endothelial growth factor was examined through immunohistochemistry and immunofluorescence and assessed semi-quantitatively. Changes in capillary density were evaluated statistically and correlated with the expression of vascular endothelial growth factor. We reported stronger immunoreactivity for vascular endothelial growth factor in the left compared to the right ventricle and also observed an increase in its expression in both ventricles in older animals. Contrasting results were reported for the renal cortex and medulla. Capillary density decreased statistically in all examined structures as aging progressed. The studied correlations were statistically significant in the two ventricles in 12-month-old animals and in the renal cortex of both age groups. Our results shed light on some changes in the microcirculation that take place as aging advances and likely contribute to impairment in the function of the examined organs.

Los cambios en la microcirculación de múltiples tejidos y órganos se han implicado como un posible mecanismo en el envejecimiento fisiológico. En particular, el factor de crecimiento endotelial vascular es una proteína secretora responsable de regular la angiogénesis mediante la alteración de la proliferación endotelial, la supervivencia, la migración, la degradación de la matriz extracelular y la permeabilidad celular. El objetivo del presente estudio fue evaluar el papel del factor de crecimiento del endotelio vascular en la progresión de las alteraciones morfológicas causadas por el envejecimiento fisiológico en el corazón y riñón y examinar su relación con los cambios en la densidad capilar. Utilizamos dos grupos de ratas Wistar sanas: 6 y 12 meses de edad. La expresión del factor de crecimiento del endotelio vascular se examinó mediante inmunohistoquímica e inmunofluorescencia y se evaluó semicuantitativamente. Los cambios en la densidad capilar se evaluaron estadísticamente y se correlacionaron con la expresión del factor de crecimiento del endotelio vascular. Informamos una inmunorreactividad más fuerte para el factor de crecimiento endotelial vascular en el ventrículo izquierdo en comparación con el derecho y también observamos un aumento en su expresión en ambos ventrículos en animales mayores. Se informaron resultados contrastantes para la corteza renal y la médula. La densidad capilar disminuyó estadísticamente en todas las estructuras examinadas a medida que avanzaba el envejecimiento. Las correlaciones estudiadas fueron estadísticamente significativas en los dos ventrículos en animales de 12 meses y en la corteza renal de ambos grupos de edad. Nuestros resultados arrojan luz sobre algunos cambios en la microcirculación que tienen lugar a medida que avanza el envejecimiento y probablemente contribuyan a un deterioro en la función de los órganos examinados.

Over-expression of gastrin inhibits apoptosis of gastric cancer cells via reactive oxygen species and annexin A2-mediated mitochondrial dysfunction / La sobreexpresión de gastrina inhibe la apoptosis de las células de cáncer gástrico a través de especies reactivas de oxígeno y disfunción mitocondrial mediada por anexina A2

SUMMARY: Gastrin plays a vital role in the development and progression of gastric cancer (GC). Its expression is up-regulated in GC tissues and several GC cell lines. Yet, the underlying mechanism remains to be investigated. Here, we aim to investigate the role and mechanism of gastrin in GC proliferation. Gastrin-overexpressing GC cell model was constructed using SGC7901 cells. Then the differentially expressed proteins were identified by iTRAQ analysis. Next, we use flow cytometry and immunofluorescence to study the effect of gastrin on the mitochondrial potential and mitochondria-derived ROS production. Finally, we studied the underlying mechanism of gastrin regulating mitochondrial function using Co-IP, mass spectrometry and immunofluorescence. Overexpression of gastrin promoted GC cell proliferation in vitro and in vivo. A total of 173 proteins were expressed differently between the controls and gastrin- overexpression cells and most of these proteins were involved in tumorigenesis and cell proliferation. Among them, Cox17, Cox5B and ATP5J that were all localized to the mitochondrial respiratory chain were down-regulated in gastrin-overexpression cells. Furthermore, gastrin overexpression led to mitochondrial potential decrease and mitochondria-derived ROS increase. Additionally, gastrin-induced ROS generation resulted in the inhibition of cell apoptosis via activating NF-kB, inhibiting Bax expression and promoting Bcl-2 expression. Finally, we found gastrin interacted with mitochondrial membrane protein Annexin A2 using Co-IP and mass spectrometry. Overexpr ession of gastrin inhibits GC cell apoptosis by inducing mitochondrial dysfunction through interacting with mitochondrial protein Annexin A2, then up-regulating ROS production to activate NF-kB and further leading to Bax/Bcl-2 ratio decrease.

La gastrina juega un papel vital en el desarrollo y progresión del cáncer gástrico (CG). Su expresión está regulada al alza en tejidos de CG y en varias líneas celulares de CG. Sin embargo, el mecanismo subyacente aun no se ha investigado. El objetivo de este estudio fue investigar el papel y el mecanismo de la gastrina en la proliferación de CG. El modelo de células CG que sobre expresan gastrina se construyó usando células SGC7901. Luego, las proteínas expresadas diferencialmente se identificaron mediante análisis iTRAQ. A continuación, utilizamos la citometría de flujo y la inmunofluorescencia para estudiar el efecto de la gastrina en el potencial mitocondrial y la producción de ROS derivada de las mitocondrias. Finalmente, estudiamos el mecanismo subyacente de la gastrina que regula la función mitocondrial utilizando Co-IP, espectrometría de masas e inmunofluorescencia. La sobreexpresión de gastrina promovió la proliferación de células CG in vitro e in vivo. Un total de 173 proteínas se expresaron de manera diferente entre los controles y las células con sobreexpresión de gastrina y la mayoría de estas proteínas estaban implicadas en la tumorigenesis y la proliferación celular. Entre estas, Cox17, Cox5B y ATP5J, todas localizadas en la cadena respiratoria mitocondrial, estaban reguladas a la baja en las células con sobreexpresión de gastrina. Además, la sobreexpresión de gastrina provocó una disminución del potencial mitocondrial y un aumento de las ROS derivadas de las mitocondrias. Por otra parte, la generación de ROS inducida por gastrina resultó en la inhibición de la apoptosis celular mediante la activación de NF-kB, inhibiendo la expresión de Bax y promoviendo la expresión de Bcl-2. Finalmente, encontramos que la gastrina interactuaba con la proteína de membrana mitocondrial Anexina A2 usando Co-IP y espectrometría de masas. La sobreexpresión de gastrina inhibe la apoptosis de las células CG al inducir la disfunción mitocondrial a través de la interacción con la proteína mitocondrial Anexina A2, luego regula el aumento de la producción de ROS para activar NF-kB y conduce aún más a la disminución de la relación Bax/Bcl-2.

Herd-level based seroprevalence and associated factors for Toxoplasma gondii in cows in the state of Paraíba, Northeastern Brazil / Soroprevalência em nível de rebanho e fatores associados para Toxoplasma gondii em vacas no estado da Paraíba, Nordeste do Brasil

We aimed to determine the herd and animal levels seroprevalence and associated factors for Toxoplasma gondii infections in cattle from the state of Paraíba, Northeastern Brazil. Herds (n = 434) and cows aged ≥ 24 months (n = 1,895) were randomly selected, and serum samples were tested with the immunofluorescence antibody test (IFAT) using as cutoff of 64. Of the 434 farms investigated, 197 had at least one seropositive cow (prevalence of 49.0%; 95% CI = 44.3%-53.8%), and the prevalence at animal level was 18.0% (95% CI = 5.3%-21.1%). The antibody titers ranged from 64 to 1024, with the most frequent titers being 64 (10.8%) and 128 (3.7%). The risk factors identified were property located in Sertão region (odds ratio [OR] = 3.07), property located in Agreste/Zona da Mata regions (OR = 2.00), animal purchasing (OR = 2.68), herd size of 34-111 animals (OR = 2.91) and herd size > 111 animals (OR = 6.97). The results suggest the wide distribution of T. gondii infections in cattle throughout the state of Paraíba, and the risk factors identified are not possible to correct.(AU)

Objetivou-se determinar a soroprevalência do rebanhos e de animais e os fatores associados à infecção por Toxoplasma gondii em bovinos do estado da Paraíba, Nordeste do Brasil. Rebanhos (n = 434) e vacas com idade ≥ 24 meses (n = 1.895) foram selecionados aleatoriamente, e as amostras de soro foram testadas com o teste de imunofluorescência (RIFI), usando-se como ponto de corte 64. Das 434 fazendas investigadas, 197 tiveram pelo menos uma vaca soropositiva (prevalência de 49%; IC 95% = 44,3%-53,8%), e a prevalência em nível de animais foi de 18% (IC 95% = 5,3%-21,1%). Os títulos de anticorpos variaram de 64 a 1024, sendo os títulos mais frequentes 64 (10,8%) e 128 (3,7%). Os fatores de risco identificados foram na propriedade localizada na região do Sertão ("odds ratio" [OR] = 3,07), na propriedade localizada na região do Agreste/Zona da Mata (OR = 2,00), na compra de animais (OR = 2,68), no tamanho do rebanho de 34-111 animais (OR = 2,91) e no tamanho do rebanho > 111 animais (OR = 6,97). Os resultados sugerem a ampla distribuição de infecções por T. gondii em bovinos em todo o estado da Paraíba, e os fatores de risco identificados não são passíveis de correção.(AU)

Immunofluorescent distribution of SIRT1, SIRT2, SIRT6 protein in the skin of SKH1 mouse after ultraviolet B irradiation for 10 Weeks / Distribución inmunofluorescente de la proteína SIRT1, SIRT2, SIRT6 en la piel del ratón SKH1 después de la irradiación ultravioleta B durante 10 semanas

SUMMARY: The skin, located on the outermost part of the body, is always exposed to external stimuli such as sunlight. The exposure of skin to ultraviolet B (UVB) radiation from sunlight is known to be a major environmental factor in inducing photoaging. After exposure to UVB, an increase in reactive oxygen species can affect the expression and activity of many critical proteins depending on the duration and dose of the UVB radiation. Mammalian sirtuins (SIRTs), which are nicotinamide dinucleotide-dependent protein deacetylases, are well known for playing a role in cellular longevity. However, little is known about SIRT protein alterations in keratinocytes upon UVB irradiation according to SIRT subtypes. Therefore, in this study, the distribution of non-mitochondrial SIRT1, SIRT2, and SIRT6 proteins was investigated by immunofluorescence (IF) staining of the skin of SKH-1 mice (n=12) after UVB irradiation for 10 weeks. After UVB irradiation for 10 weeks, the IF of both SIRT1 and SIRT6 was significantly increased in the UVB-irradiated mice group (UG), but the difference in SIRT2 IF was not statistically significant between the control group (CG) and the UG. The translocation of both SIRT1 and SIRT6 IF from the nucleus to the cytoplasm of keratinocytes was observed in the upper epidermis of the UG, whereas SIRT2 IF was localized in the cytoplasm of keratinocytes in the epidermis in both the CG and the UG. The translocation of SIRT1 and SIRT6 IF from the nucleus to the cytoplasm of keratinocytes may account for the physiologically protective action of keratinocytes against UVB irradiation. However, the exact role of SIRT1 and SIRT6 translocation in keratinocytes, where SIRT1 and SIRT6 shuttle from the nucleus to the cytoplasm, is not well known. Therefore, further studies are needed to understand the molecular mechanisms of SIRT1 and SIRT6 translocation in keratinocytes upon UVB irradiation.

La piel, situada en la parte más externa del cuerpo, está siempre expuesta a estímulos externos como la luz solar. Se sabe que la exposición de la piel a la radiación ultravioleta B (UVB) de la luz solar es un factor ambiental importante en la inducción del fotoenvejecimiento. Después de la exposición a los rayos UVB, un aumento en las especies reactivas de oxígeno puede afectar la expresión y la actividad de muchas proteínas críticas según la duración y la dosis de la radiación UVB. Las sirtuinas de mamíferos (SIRT), que son proteínas desacetilasas dependientes de dinucleótidos de nicotinamida, son bien conocidas por desempeñar un papel en la longevidad celular. Sin embargo, se sabe poco sobre las alteraciones de la proteína SIRT en los queratinocitos tras la irradiación UVB según los subtipos de SIRT. Por lo tanto, en este estudio, se investigó la distribución de las proteínas SIRT1, SIRT2 y SIRT6 no mitocondriales mediante tinción de inmunofluorescencia (IF) de la piel de ratones SKH-1 (n = 12), después de la irradiación con UVB durante 10 semanas. Posterior a la irradiación, el IF de SIRT1 y SIRT6 aumentaron significativamente en el grupo de ratones irradiados con UVB (UG), pero la diferencia en SIRT2 IF no fue estadísticamente significativa entre el grupo control (CG) y el UG. La translocación de SIRT1 y SIRT6 IF desde el núcleo al citoplasma de los queratinocitos se observó en la epidermis superior de la UG, mientras que SIRT2 IF se localizó en el citoplasma de los queratinocitos en la epidermis, tanto en el GC, como en la UG. La translocación de SIRT1 y SIRT6 IF del núcleo al citoplasma de los queratinocitos puede explicar la acción protectora fisiológica de estos contra la radiación UVB. Sin embargo, el papel exacto de la translocación de SIRT1 y SIRT6 en los queratinocitos, donde SIRT1 y SIRT6 se trasladan desde el núcleo al citoplasma, no se conoce bien. Por lo tanto, se necesitan más estudios para comprender los mecanismos moleculares de la translocación SIRT1 y SIRT6 en los queratinocitos tras la irradiación UVB.

Analysis of Nerve Endings in the Superior Labrum-Biceps Complex by Fluorescence Immunohistochemistry and Confocal Laser Microscopy / Análise de terminações nervosas no complexo labrum-bíceps superior usando imunohistoquímica de fluorescência e microscopia a laser confocal

Abstract Objectives The capsuloligamentous structures of the shoulder work as static stabilizers, together with the biceps and rotator cuff muscles, increasing the contact surface of the glenoid cavity. Free nerve endings and mechanoreceptors have been identified in the shoulder; however, there are a few studies that describe the presence of these nerves in the biceps' insertion. The present study aimed to describe the morphology and distribution of nerve endings using immunofluorescence with protein gene product 9.5 (PGP 9.5) and confocal microscopy. Methods Six labrum-biceps complexes from six fresh-frozen cadavers were studied. The specimens were coronally cut and prepared using the immunofluorescence technique. In both hematoxylin and eosin (H&E) and immunofluorescence, the organization of the connective tissue with parallel collagen fibers was described. Results In the H&E study, vascular structures and some nerve structures were visualized, which were identified by the elongated presence of the nerve cell. All specimens analyzed with immunofluorescence and confocal microscopy demonstrated poor occurrence of morphotypes of sensory corpuscles and free nerve endings. We identified free nerve endings located in the labrum and in the bicipital insertion, and sparse nerve endings along the tendon. Corpuscular endings with fusiform, cuneiform, and oval aspect were identified in the tendon. Conclusion These findings support the hypothesis that the generation of pain in the superior labral tear from Anterior to posterior (SLAP) lesions derives from the more proximal part of the long biceps cord and even more from the upper labrum. Future quantitative studies with a larger number of specimens may provide more information on these sensory systems.

Resumo Objetivos As estruturas capsulo-ligamentares do ombro funcionam como estabilizadores estáticos, juntamente com os músculos do bíceps e do manguito rotador, aumentando a superfície de contato da cavidade glenoide. Terminações nervosas livres e mecanorreceptores foram identificados no ombro; no entanto, existem alguns estudos que descrevem a presença desses nervos na inserção do bíceps. Este estudo teve como objetivo descrever a morfologia e distribuição de terminações nervosas utilizando imunofluorescência com protein gene product 9.5 (PGP 9.5) e microscopia confocal. Métodos Foram estudados seis complexos labrum-bíceps de seis cadáveres congelados frescos. Os espécimes foram cortados coronalmente e preparados pelo método de imunofluorescência. Tanto em hematoxilina e eosina (H&E) quanto em imunofluorescência, foi descrita a organização do tecido conjuntivo com fibras paralelas de colágeno. Resultados No estudo de H&E, foram visualizadas estruturas vasculares e algumas estruturas nervosas, que foram identificadas pela presença alongada da célula nervosa. Todas as amostras analisadas com imunofluorescência e microscopia confocal demonstraram baixa ocorrência de morfotipos de corpúsculos sensoriais e terminações nervosas livres. Identificamos terminações nervosas livres localizadas no labrum, inserção bicipital e terminações nervosas esparsas ao longo do tendão. Terminais corpusculares com aspecto fusiforme, cuneiforme e oval foram identificados no tendão. Conclusão Esses achados corroboram a hipótese de que a geração de dor nas lesões labrais superiores de anterior a posterior (SLAP, na sigla em inglês) deriva da parte mais proximal do cabo longo do bíceps e ainda mais do labrum superior. Estudos quantitativos futuros com um número maior de espécimes podem fornecer mais informações sobre esses sistemas sensoriais.

Low-dose cisplatin exposure and SNAIL, Vimentin, E-cadherin expression in HEPG2 cell line / Exposición a dosis bajas de cisplatino y expresión de SNAIL, Vimentina, E-cadherina en línea celular HEPG2

Cisplatin, the first platinum compound approved for cancer treatment, is widely used in the treatment of various cancers including hepatocellular carcinoma (HCC). HCC incidence rates rise globally. Epithelial mesenchymal transition (EMT) is implicated in cancer invasion and metastasis, which are associated with increased mortality. Cisplatin dose might influence cancer invasion and metastatic behavior of the cells. The aim of the study was to investigate the effect of low-dose cisplatin treatment on EMT- related changes in HepG2 cells. Following treatment with 4 µM cisplatin, HepG2 cells were evaluated morphologically. Gene expression of E-cadherin, Vimentin, Snail1 was assessed by quantitative PCR. Immunofluorescence analyses of NA-K ATPase were performed. Although the low-dose cisplatin treated cells exhibited a more stretched morphology, no statistical difference was detected in gene expression of E-cadherin, Vimentin, Snail1 and immunofluorescence of NA-K ATPase. Findings on low-dose cisplatin effects in HepG2 might contribute to the knowledge of antineoplastic inefficacy by further understanding the molecular mechanisms of drug action.

El cisplatino, el primer compuesto de platino aprobado para el tratamiento del cáncer, es ampliamente utilizado en el tratamiento de varios tipos de cáncer, incluido el carcinoma hepatocelular (CHC). Las tasas de incidencia de CHC aumentan a nivel mundial. La transición mesenquimal epitelial (EMT) está implicada en la invasión del cáncer y la metástasis, que se asocian con un aumento de la mortalidad. La dosis de cisplatino podría influir en la invasión del cáncer y el comportamiento metastásico de las células. El objetivo del estudio fue investigar el efecto del tratamiento con dosis bajas de cisplatino en los cambios relacionados con la EMT en las células HepG2. Tras el tratamiento con cisplatino de 4 µM, se evaluaron morfológicamente las células HepG2. La expresión génica de E-cadherina, vimentina, caracol1 se evaluó mediante PCR cuantitativa. Se realizaron análisis de inmunofluorescencia de NA-K ATPasa . Aunque las células tratadas con cisplatino en dosis bajas exhibieron una morfología más estirada, no se detectaron diferencias estadísticas en la expresión génica de E-cadherina, vimentina, Snail1 e inmunofluorescencia de NA-K ATPasa. Los hallazgos sobre los efectos del cisplatino en dosis bajas en HepG2 podrían contribuir al conocimiento de la ineficacia antineoplásica al comprender mejor los mecanismos moleculares de la acción del fármaco.

Cinomose em canídeos silvestres no Brasil / Distemper in wild canids in Brazil

A cinomose é uma doença viral multissistêmica causada por um Morbillivirus. No Brasil, existem seis espécies de canídeos silvestres vulneráveis a essa enfermidade. Dessa forma, este trabalho tem como objetivo apresentar uma revisão sobre a situação da cinomose em canídeos silvestres no Brasil e os impactos causados na fauna brasileira. A transmissão do vírus ocorre através do contato com amostras contaminadas, a exemplo dos aerossóis, excretas e secreções de indivíduos infectados. Os sinais clínicos mais prevalentes são de ordem neurológica, entretanto, também podem ser identificadas manifestações respiratórias, cutâneas e gastrointestinais. Portanto, o diagnóstico consiste na avaliação da sintomatologia apresentada em conjunto com testes específicos, como isolamento viral, ELISA, imunofluorescência e RT-PCR. Atualmente, não existe tratamento específico. Desta forma, são realizados apenas cuidados paliativos. Os grandes carnívoros são os mamíferos mais ameaçados do mundo, sobretudo, em consequência dos impactos causados pela redução do habitat natural, associada à expansão territorial humana. As epidemias também justificam as altas taxas de mortalidades desses animais, o que pode estar relacionado com comportamentos sociais e de farejamento, assim como o crescente contato entre animais silvestres e domésticos devido à urbanização e à proximidade genética que os canídeos silvestres têm com os cães domésticos, tornando-os suscetíveis às infecções. Visto que a cinomose é uma patologia emergente em populações de carnívoros silvestres e que a presença de cães domésticos não vacinados em áreas de conservação representa um grande risco de contaminação, conclui-se que a não vacinação está diretamente associada à perpetuação do vírus no meio selvagem.

Distemper is a multisystem viral disease caused by a Morbillivirus. In Brazil, there are six species of wild canids, vulnerable to this disease. Therefore, the present work aims to develop a review on the situation of distemper in wild canids in Brazil and the impacts caused on the Brazilian fauna. The virus transmission occurs through contact with contaminated samples, such as aerosols, excreta, and secretions from infected individuals. The most prevalent clinical signs are neurological; however, respiratory, cutaneous and gastrointestinal manifestations can also be identified. Thus, the diagnosis consists of evaluating the symptoms presented together with specific tests, such as viral isolation, ELISA, immunofluorescence, and RT-PCR. Currently, there is no specific treatment. Therefore, only palliative care is performed. Large carnivores are the most threatened mammals in the world, mainly as a result of the impacts caused by the reduction of natural habitat, associated with human territorial expansion. Epidemics also justify the high mortality rates of these animals, which may be related to social and sniffing behaviors, as well as the increasing contact between wild and domestic animals due to urbanization and the genetic proximity that wild canids have with domestic dogs, making them susceptible to infections. Since distemper is an emerging pathology in populations of wild carnivores and the presence of unvaccinated domestic dogs in conservation areas represents a great risk of contamination, it is concluded that non-vaccination is directly associated with the perpetuation of the virus in the wild.

Comparison between nucleic acid amplification tests, antigen immunofluorescence assay, and in vitro infectivity in SARS-CoV-2 diagnosis.

The number of SARS-CoV-2 detection tests requested to the laboratories has dramatically increased together with an urgent need to release reliable responses in a very short time. The two options taken into consideration and analyzed in the current study were the point-of-care test (POCT) based on the nucleic acid amplification test (NAAT) and the Antigen (Ag) rapid test. The POCT-NAAT-based assay was compared with a rapid antigen test of nasopharyngeal swab samples. If the specimen tested positive, it was followed by viral load quantification and by the functional assessment of the residual infectivity. When the initial cycle threshold (Ct) was below 20 (100%), and in the range of 20-25 (92%) and of 25-30 (88%), a great concordance between the POCT-NAAT and the Ag test was observed. Moreover, the positivity of the antigen test was well correlated to a successful infection in vitro (78%), with greater concordance when the initial Ct below 20 or above 35 (100%) and in the range 20-25 (83%). Our findings showed that most of the swabs which tested positive using the antigen test were able to infect the cells in vitro, suggesting that probably only these samples hold residual infectivity and therefore an increased risk of virus transmission at the moment of being tested.

Reply to Sheval, E.V. Comment on "Piña et al. Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence. Int. J. Mol. Sci. 2022, 23, 1426".

We have carefully read the interesting explanatory comment by Eugene V. Sheval [...].

Investigation of Galectins in Frozen Tissue and Mammalian Cell Culture Using Confocal Miccroscopy.

Galectins are multifunctional glycan-binding proteins present in various tissues that participate in multiple physiological and pathological processes and are considered as not only biomarkers of human diseases but also molecular targets for treating cancer and inflammatory illnesses in many organs. In the glycobiology field, it is crucial to determine the pattern of galectin expression and location in cells and tissues. Confocal microscopy is a powerful imaging technology that represents a unique approach to investigate the expression and location of biomolecules in various tissues and cells. The confocal microscope acquires images of the specimen through the reflected or fluorescent light from the objective's focal plane, using laser light focused on a small spot inside the tissue or cell. This technique provides high-resolution and high-contrast images without artifacts generated by conventional microscopy and enables reconstruction of virtual tridimensional images by acquiring multiple sections from several focal planes, which makes it possible to obtain the precise spatial location of any cellular structure or molecule. Furthermore, confocal microscopy is a non-invasive tissue imaging strategy used in clinical practices. We describe herein the immunofluorescence confocal method for examining galectins in frozen tissue sections and mammalian cell culture.

Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence.

Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles. The application of fluorochromes during immunolabeling is referred to as immunofluorescence, a method coupled to widefield or confocal microscopy and extensively applied in basic research and clinical diagnosis. Notwithstanding, there are still disadvantages associated with the application of this technique due to technical challenges in the process, such as sample fixation, permeabilization, antibody incubation times, and fluid exchange, etc. These disadvantages call for continuous updates and improvements to the protocols extensively described in the literature. This review contributes to protocol optimization, outlining 10 current methods for improving sample processing in different stages of immunofluorescence, including a section with further recommendations. Additionally, we have extended our own antibody signal enhancer method, which was reported to significantly increase antibody signals and is useful for cervical cancer detection, to improve the signals of fluorochrome-conjugated staining reagents in fibrous tissues. In summary, this review is a valuable tool for experienced researchers and beginners when planning or troubleshooting the immunofluorescence assay.

Mitochondrial energy metabolism in baby hamster kidney (BHK-21/C13) cells treated with karnozin extra® / Metabolismo energético mitocondrial en células de riñón de hámster bebé (BHK-21 / C13) tratadas con karnozin extra®

SUMMARY: Carnosine is known as a natural dipeptide, which inhibits the proliferation of tumor cells throughout its action on mitochondrial respiration and cell glycolysis. However, not much is known about its effects on the metabolism of healthy cells. We explored the effects of Karnozin EXTRA® capsule with different concentrations of L-carnosine, on the cell viability and the expressions of intermediate filament vimentin (VIM) and superoxide dismutase (SOD2) in normal fibroblasts BHK-21/C13. Furthermore, we investigated its action on the energy production of these cells. Cell viability was quantified by the MTT assay. The Clark oxygen electrode (Oxygraph, Hansatech Instruments, England) was used to measure the "intact cell respiration rate", state 3 of ADP-stimulated oxidation, maximum oxidation capacity and the activities of complexes I, II and IV. Results showed that Karnozin EXTRA® capsule in concentrations of 2 and 5 mM of L-carnosine did not induce toxic effects and morphological changes in treated cells. Our data revealed a dose-dependent immunofluorescent signal amplification of VIM and SOD2 in the BHK-21/C13 cell line. This supplement substantially increased the recorded mitochondrial respiration rates in the examined cell line. Due to the stimulation of mitochondrial energy production in normal fibroblasts, our results suggested that Karnozin EXTRA® is a potentially protective dietary supplement in the prevention of diseases with altered mitochondrial function.

RESUMEN: La carnosina se conoce como dipéptido natural, que inhibe la proliferación de células tumorales a través de su acción sobre la respiración mitocondrial y la glucólisis celular. Sin embargo, no se sabe mucho de sus efectos sobre el metabolismo de las células sanas. Exploramos los efectos de la cápsula Karnozin EXTRA® con diferentes concentraciones de L-carnosina, sobre la viabilidad celular y las expresiones de vimentina de filamento intermedio (VIM) y superóxido dismutasa (SOD2) en fibroblastos normales BHK-21 / C13. Además, estudiamos su acción sobre la producción de energía de estas células. La viabilidad celular se cuantificó mediante el ensayo MTT. Se utilizó el electrodo de oxígeno Clark (Oxygraph, Hansatech Instruments, Inglaterra) para medir la "tasa de respiración de células intactas", el estado 3 de oxidación estimulada por ADP, la capacidad máxima de oxidación y las actividades de los complejos I, II y IV. Los resultados mostraron que la cápsula de Karnozin EXTRA® en concentraciones de 2 y 5 mM de L- carnosina no indujo efectos tóxicos ni cambios morfológicos en las células tratadas. Nuestros datos revelaron una amplificación de señal inmunofluorescente dependiente de la dosis de VIM y SOD2 en la línea celular BHK-21 / C13. Este suplemento aumentó sustancialmente las tasas de respiración mitocondrial registradas en la línea celular examinada. Debido a la estimulación de la producción de energía mitocondrial en fibroblastos normales, nuestros resultados sugirieron que Karnozin EXTRA® es un suplemento dietético potencialmente protector en la prevención de enfermedades con función mitocondrial alterada.

Albumin Expands Albumin Reabsorption Capacity in Proximal Tubule Epithelial Cells through a Positive Feedback Loop between AKT and Megalin.

Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1+ endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a phenomenon that we refer to as "albumin-induced albumin endocytosis". Mice treated with L-lysine present decreased albumin endocytosis leading to proteinuria and albuminuria associated with inhibition of AKT activity. Renal cortex explants obtained from control mice treated with MK-2206 decreased albumin uptake and promoted megalin internalization. Our data highlight the mechanism behind the capacity of PTECs to adapt albumin reabsorption to physiologic fluctuations in its filtration, avoiding urinary excretion.

Costain 1 (ARM19800.1) - The first identified protein of the costa of the pathogenic protozoan Tritrichomonas foetus.

Protists members of the Trichomonadidae and Tritrichomonadidae families include agents of trichomoniasis that constitute important parasitic diseases in humans and in animals of veterinary interest. One of the characteristic features of these eukaryotic microorganisms is that they contain a fibrous structure known as the costa as an important cytoskeleton structure, that differs in several aspects from other cytoskeleton structures found in eukaryotic cells. Previous proteomic analysis of an enriched costa fraction revealed the presence of several hypothetical proteins. Here we describe the localization of one of the most prevalent protein found in this previously made proteomic assay to confirm its presence in the costa of Tritrichomonas foetus. A peptide sequence of the hypothetical protein ARM19800.1 was selected for the production of specific polyclonal antibodies and its specificity was confirmed by Western Blotting using an enriched costa fraction. Next, the specific localization of the selected protein was evaluated by immunofluorescence and electron microscopy immunocytochemistry. Our observations clearly showed that the ARM 19800.1 protein is indeed localized in the costa and displays an almost periodic labeling pattern. Since this is the first protein identified in the costa, it was designated as costain 1. A better understanding of a structure as peculiar as the costa is of great biological and evolutionary importance due to the fact that it contains unique proteins, it may represent a possible chemotherapy target and it may correspond to antigens of interest in immunodiagnosis and/or vaccine development.