Enhancement of Annexin V in response to combination of epigallocatechin gallate and quercetin as a potent arrest the cell cycle of colorectal cancer

Abstract Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.

Resumo O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.

Enhancement of Annexin V in response to combination of epigallocatechin gallate and quercetin as a potent arrest the cell cycle of colorectal cancer / Aumento da anexina V em resposta à combinação de galato de epigalocatequina e quercetina como uma potente parada do ciclo celular do câncer colorretal

Abstract Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.

Resumo O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.

In vitro cytotoxicity of curcuminoids against head and neck cancer HNO97 cell line

Abstract Oral squamous cell carcinoma (OSCC) is a malignant tumour of Head and Neck Cancer (HNC). The recent therapeutic approaches used to treat cancer have adverse side effects. The natural agents exhibiting anticancer activities are generally considered to have a robust therapeutic potential. Curcuminoids, one of the major active compounds of the turmeric herb, are used as a therapeutic agent for several diseases including cancer. In this study, the cytotoxicity of curcuminoids was investigated against OSCC cell line HNO97. Our data showed that curcuminoids significantly inhibits the proliferation of HNO97 in a time and dose-dependent manner (IC50=35 M). Cell cycle analysis demonstrated that curcuminoids increased the percentage of G2/M phase cell populations in the treated groups. Treating HNO97 cells with curcuminoids led to cell shrinking and increased detached cells, which are the typical appearance of apoptotic cells. Moreover, flow cytometry analysis revealed that curcuminoids significantly induced apoptosis in a time-dependent manner. Furthermore, as a response to curcuminoids treatment, comet tails were formed in cell nuclei due to the induction of DNA damage. Curcuminoids treatment reduced the colony formation capacity of HNO97 cells and induced morphological changes. Overall, these findings demonstrate that curcuminoids can in vitro inhibit HNC proliferation and metastasis and induce apoptosis.

Resumo O carcinoma de células escamosas oral (OSCC) é um tumor maligno do câncer de cabeça e pescoço (HNC). As recentes abordagens terapêuticas usadas para tratar o câncer têm efeitos colaterais adversos. Os agentes naturais que exibem atividades anticâncer são geralmente considerados como tendo um potencial terapêutico robusto. Curcuminoides, um dos principais compostos ativos da erva cúrcuma, são usados como agente terapêutico para várias doenças, incluindo câncer. Neste estudo, a citotoxicidade dos curcuminoides foi investigada contra a linha de células OSCC HNO97. Nossos dados mostraram que os curcuminoides inibem significativamente a proliferação de HNO97 de forma dependente do tempo e da dose (IC50 = 35 M). A análise do ciclo celular demonstrou que os curcuminoides aumentaram a porcentagem de populações de células da fase G2 / M nos grupos tratados. O tratamento das células HNO97 com curcuminoides levou ao encolhimento celular e ao aumento das células destacadas, que são a aparência típica das células apoptóticas. Além disso, a análise de citometria de fluxo revelou que os curcuminoides induziram significativamente a apoptose de uma maneira dependente do tempo. Além disso, em resposta ao tratamento com curcuminoides, caudas de cometa foram formadas nos núcleos das células devido à indução de danos ao DNA. O tratamento com curcuminoides reduziu a capacidade de formação de colônias das células HNO97 e induziu alterações morfológicas. No geral, esses achados demonstram que os curcuminoides podem inibir in vitro a proliferação e metástase de HNC e induzir apoptose.

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P > 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P > 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.(AU)

Photosynthetic modification of plants through recent technologies: a valuable way to ensure crop fortification / Modificação fotossintética de plantas por meio de tecnologias recentes: uma forma valiosa de garantir a fortificação das culturas

The 2030 Sustainable Development Goals of the United Nations include a strong emphasis on ending hunger worldwide. According to the 2019 Global Food Security Index, while 88% of countries claim there is sufficient food supply in their country, the sad reality is that 1 in 3 countries is facing insufficient availability of food supply, which means that in those countries, more than 10% of the population is malnourished. Since nutrition is crucial to leading a healthy life and satisfying food security needs, several governments have turned to national nutrition surveys to gauge the extent of malnutrition in their populations. Plants are able to grow, develop, and store nutrients by photosynthesis, which convert light into chemical energy through cell redox regulatory networks. A photosynthesis system's electron flow may be adjusted to accommodate varying light and environmental circumstances. Many techniques exist for controlling the flow of electrons emitted during light processes in order to save or waste energy. The two protein molecules TROL and flavoenzyme ferredoxin (oxidoreductase+NADP) (FNR) interact dynamically to form an excellent molecular switch capable of splitting electrons from the photosystem. The TROL-FNR bifurcation may be limited by either generating NADPH or preventing reactive oxygen species from propagating. TROL-based genome editing is an experimental method for enhancing plant stress and defensive responses, efficiency, and ultimately agricultural production.

Os Objetivos de Desenvolvimento Sustentável de 2030 da Organização das Nações Unidas incluem uma forte ênfase em acabar com a fome em todo o mundo. De acordo com o Índice Global de Segurança Alimentar de 2019, enquanto 88% dos países afirmam que há abastecimento alimentar suficiente em seu país, a triste realidade é que 1 em cada 3 países enfrenta disponibilidade insuficiente de alimentos, o que significa que, nesses países, mais de 10% da população está desnutrida. Uma vez que a nutrição é crucial para levar uma vida saudável e satisfazer as necessidades de segurança alimentar, vários governos recorreram a pesquisas nacionais de nutrição para avaliar a extensão da desnutrição em suas populações. As plantas são capazes de crescer, desenvolver e armazenar nutrientes pela fotossíntese, que converte luz em energia química por meio de redes reguladoras redox celulares. O fluxo de elétrons de um sistema de fotossíntese pode ser ajustado para acomodar luz variável e circunstâncias ambientais. Existem muitas técnicas para controlar o fluxo de elétrons emitidos durante os processos de luz, a fim de economizar ou desperdiçar energia. As duas moléculas de proteína TROL e a flavoenzima ferredoxina (oxidorredutase+NADP) (FNR) interagem dinamicamente para formar um excelente interruptor molecular capaz de separar elétrons do fotossistema. A bifurcação TROL-FNR pode ser limitada, gerando NADPH ou impedindo a propagação de espécies reativas de oxigênio. A edição do genoma baseada em TROL é um método experimental para aumentar o estresse da planta, as respostas defensivas, a eficiência e, finalmente, a produção agrícola.

Thecoma in an elderly crioulo mare

Background: Ovarian enlargement in mares can either be a physiological or pathological condition associated with neoplastic or non-neoplastic causes. Neoplasia is more prominent in elderly than young horses. Granulosa cell tumors are the most common neoplastic cause of ovarian enlargement, and thecoma is the rarest. Thecoma is a benign ovarian stromal neoplasia originating from the sexual cords of the embryonic gonad and can affect the production of steroid hormones, leading to changes in behavior and the estrous cycle. If only one ovary is affected, after unilateral ovariectomy, usually the mare can still be used for reproductive purposes. Herein, we report a case of ovarian thecoma in an elderly Crioulo mare. Case: A 15-year-old Crioulo mare was evaluated due to progressive weight loss. The mare presented a low body score, and on physical examination, all parameters were within reference limits. Hematological examination revealed anemia, hyperfibrinogenemia, and neutrophilia. Serum biochemistry examination were within the physiological limits. Rectal palpation revealed an increase in volume in the left ovary (4.3 × 2.9 × 3 cm), smooth consistency and no associated pain in either of the ovaries. Rectal ultrasonography revealed a heterogeneous pattern, suggesting a neoplasm. As a treatment modality and for definitive diagnosis, the mare underwent a unilateral flank ovariectomy laparoscopically in the quadrupedal position. On histopathological evaluation of the left ovary, the sample showed cysts associated with proliferating spindle cells arranged in a solid arrangement, occasionally forming eddies, in addition to hemosiderosis, with clear cytoplasm and lipid vacuoles. Macroscopically, the ovary presented with a regular surface, smooth to firm consistency, a homogeneous brownish appearance with blackened foci, and brownish fluid-filled cysts. These findings confirmed thecoma of the left ovary in the mare. Discussion: Ovarian enlargement in mares can be associated to the presence of neoplasia. Clinical signs of ovarian neoplasms can be nonspecific; therefore, to achieve a definitive diagnosis, it is imperative to evaluate and compare the patient's history, physical examination, and results of complementary examinations. Excessive production of hormones lead to behavioral changes in mares with ovarian tumors, such as masculinized or aggressive behavior, and anestrus persistence. Thecomas may or may not interfere with steroid production and levels. On ultrasound examination, ovarian neoplasms may present similar echogenicity patterns. Histopathological evaluation is the best diagnostic approach after an ovariectomy. The prognosis of ovariectomy is considerably successful with the mare regaining reproductive health, although some mares may become infertile, with a small, inactive contralateral ovary. Thecoma is a rare neoplasm in horses; however, owing to its potential to cause altered reproductive functions, it should be considered as a differential diagnosis in mares with increased ovarian volume.

In vitro cytotoxicity of curcuminoids against head and neck cancer HNO97 cell line / Citotoxicidade in vitro de curcuminoides contra a linhagem de células HNO97 de câncer de cabeça e pescoço

Oral squamous cell carcinoma (OSCC) is a malignant tumour of Head and Neck Cancer (HNC). The recent therapeutic approaches used to treat cancer have adverse side effects. The natural agents exhibiting anticancer activities are generally considered to have a robust therapeutic potential. Curcuminoids, one of the major active compounds of the turmeric herb, are used as a therapeutic agent for several diseases including cancer. In this study, the cytotoxicity of curcuminoids was investigated against OSCC cell line HNO97. Our data showed that curcuminoids significantly inhibits the proliferation of HNO97 in a time and dose-dependent manner (IC50=35 μM). Cell cycle analysis demonstrated that curcuminoids increased the percentage of G2/M phase cell populations in the treated groups. Treating HNO97 cells with curcuminoids led to cell shrinking and increased detached cells, which are the typical appearance of apoptotic cells. Moreover, flow cytometry analysis revealed that curcuminoids significantly induced apoptosis in a time-dependent manner. Furthermore, as a response to curcuminoids treatment, comet tails were formed in cell nuclei due to the induction of DNA damage. Curcuminoids treatment reduced the colony formation capacity of HNO97 cells and induced morphological changes. Overall, these findings demonstrate that curcuminoids can in vitro inhibit HNC proliferation and metastasis and induce apoptosis.

O carcinoma de células escamosas oral (OSCC) é um tumor maligno do câncer de cabeça e pescoço (HNC). As recentes abordagens terapêuticas usadas para tratar o câncer têm efeitos colaterais adversos. Os agentes naturais que exibem atividades anticâncer são geralmente considerados como tendo um potencial terapêutico robusto. Curcuminoides, um dos principais compostos ativos da erva cúrcuma, são usados como agente terapêutico para várias doenças, incluindo câncer. Neste estudo, a citotoxicidade dos curcuminoides foi investigada contra a linha de células OSCC HNO97. Nossos dados mostraram que os curcuminoides inibem significativamente a proliferação de HNO97 de forma dependente do tempo e da dose (IC50 = 35 μM). A análise do ciclo celular demonstrou que os curcuminoides aumentaram a porcentagem de populações de células da fase G2 / M nos grupos tratados. O tratamento das células HNO97 com curcuminoides levou ao encolhimento celular e ao aumento das células destacadas, que são a aparência típica das células apoptóticas. Além disso, a análise de citometria de fluxo revelou que os curcuminoides induziram significativamente a apoptose de uma maneira dependente do tempo. Além disso, em resposta ao tratamento com curcuminoides, caudas de cometa foram formadas nos núcleos das células devido à indução de danos ao DNA. O tratamento com curcuminoides reduziu a capacidade de formação de colônias das células HNO97 e induziu alterações morfológicas. No geral, esses achados demonstram que os curcuminoides podem inibir in vitro a proliferação e metástase de HNC e induzir apoptose.

In vitro cytotoxicity of curcuminoids against head and neck cancer HNO97 cell line / Citotoxicidade in vitro de curcuminoides contra a linhagem de células HNO97 de câncer de cabeça e pescoço

Oral squamous cell carcinoma (OSCC) is a malignant tumour of Head and Neck Cancer (HNC). The recent therapeutic approaches used to treat cancer have adverse side effects. The natural agents exhibiting anticancer activities are generally considered to have a robust therapeutic potential. Curcuminoids, one of the major active compounds of the turmeric herb, are used as a therapeutic agent for several diseases including cancer. In this study, the cytotoxicity of curcuminoids was investigated against OSCC cell line HNO97. Our data showed that curcuminoids significantly inhibits the proliferation of HNO97 in a time and dose-dependent manner (IC50=35 μM). Cell cycle analysis demonstrated that curcuminoids increased the percentage of G2/M phase cell populations in the treated groups. Treating HNO97 cells with curcuminoids led to cell shrinking and increased detached cells, which are the typical appearance of apoptotic cells. Moreover, flow cytometry analysis revealed that curcuminoids significantly induced apoptosis in a time-dependent manner. Furthermore, as a response to curcuminoids treatment, comet tails were formed in cell nuclei due to the induction of DNA damage. Curcuminoids treatment reduced the colony formation capacity of HNO97 cells and induced morphological changes. Overall, these findings demonstrate that curcuminoids can in vitro inhibit HNC proliferation and metastasis and induce apoptosis.(AU)

O carcinoma de células escamosas oral (OSCC) é um tumor maligno do câncer de cabeça e pescoço (HNC). As recentes abordagens terapêuticas usadas para tratar o câncer têm efeitos colaterais adversos. Os agentes naturais que exibem atividades anticâncer são geralmente considerados como tendo um potencial terapêutico robusto. Curcuminoides, um dos principais compostos ativos da erva cúrcuma, são usados como agente terapêutico para várias doenças, incluindo câncer. Neste estudo, a citotoxicidade dos curcuminoides foi investigada contra a linha de células OSCC HNO97. Nossos dados mostraram que os curcuminoides inibem significativamente a proliferação de HNO97 de forma dependente do tempo e da dose (IC50 = 35 μM). A análise do ciclo celular demonstrou que os curcuminoides aumentaram a porcentagem de populações de células da fase G2 / M nos grupos tratados. O tratamento das células HNO97 com curcuminoides levou ao encolhimento celular e ao aumento das células destacadas, que são a aparência típica das células apoptóticas. Além disso, a análise de citometria de fluxo revelou que os curcuminoides induziram significativamente a apoptose de uma maneira dependente do tempo. Além disso, em resposta ao tratamento com curcuminoides, caudas de cometa foram formadas nos núcleos das células devido à indução de danos ao DNA. O tratamento com curcuminoides reduziu a capacidade de formação de colônias das células HNO97 e induziu alterações morfológicas. No geral, esses achados demonstram que os curcuminoides podem inibir in vitro a proliferação e metástase de HNC e induzir apoptose.(AU)

Isoliquiritigenin regulates the circ_0002860/miR-431-5p/RAB9A axis to function as a tumor inhibitor in melanoma

Background: Isoliquiritigenin (ISL) presents antitumor effects against melanoma cells. It is known that various circular RNAs (circRNAs) are involved in the development of melanoma. Therefore, the present study aims to investigate the molecular mechanisms of ISL and circ_0002860. Methods: Circ_0002860, microRNA-431-5p (miR-431-5p) and member RAS oncogene family (RAB9A) were detected through reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay. Cell viability was examined via cell counting kit-8 assay. The proliferation ability was assessed using colony formation assay. Cell apoptosis and cell cycle were determined by flow cytometry. Transwell assay was used for detection of migration and invasion. Western blot was conducted for protein analysis. Target binding was confirmed via dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. In vivo research was performed through xenograft tumor assay. Results: Circ_0002860 was downregulated by ISL in melanoma cells. ISL-induced inhibitory effects on cell proliferation, cell cycle progression, migration and invasion were alleviated by circ_0002860 overexpression. MiR-431-5p was a target of circ_0002860. Circ_0002860 eliminated the ISL-induced tumor inhibition via sponging miR-431-5p in melanoma cells. Circ_0002860 elevated the RAB9A level by targeting miR-431-5p. The function of ISL was related to miR-431-5p/RAB9A axis in melanoma progression. Tumor growth was reduced by ISL in vivo through downregulating circ_0002860 to regulate miR-431-5p and RAB9A levels. Conclusion: The current data indicates that ISL suppressed cell malignant progression of melanoma via targeting the circ_0002860/miR-431-5p/RAB9A pathway.(AU)

Pregnancy success in bitches - evaluation of interactions between artificial insemination method, serum progesterone concentration and vaginal cytology parameters

Background: The artificial insemination has become a well-established method in the breeding of bitches, and evaluation of the factors that may potentially affect pregnancy success is essential. For this reason, it is essential to evaluate the factors that may affect fertility of the bitch when artificial insemination is performed. Serum progesterone concentrations and vaginal cytology have been used to determine the time of ovulation and stage of the estrus cycle. This study aimed to evaluate the artificial insemination method, the serum progesterone concentration, the breed size, age, the whelping number, vaginal cytology parameters, and their interactions on pregnancy success in bitches. Materials, Methods & Results: A total of 607 bitches that had undergone reproductive consultation with the Mexican Canine Federation from January to December 2016 were enrolled in the present study and assigned to one of 2 artificial insemination methods (intravaginal and transcervical) using fresh semen. Determination of the estrus cycle phase and the time of Artificial insemination was based on vaginal cytology and serum progesterone concentrations. Bitches inseminated by the transcervical technique had a higher pregnancy rate with respect to females inseminated by the intravaginal technique (P < 0.05). Moreover, females with a serum progesterone concentration of 5-10 ng/mL had a greater probability (> 4 times) of getting pregnant than animals with lower or higher progesterone concentrations (P < 0.05). Bitches inseminated by the intravaginal technique and with serum progesterone concentrations >10 ng/mL had a considerable reduction in pregnancy (P < 0.05) compared with females with < 10 ng/mL serum progesterone or with bitches inseminated by the transcervical technique. Discusion: Serum progesterone concentration, the artificial insemination method, and superficial cells without a nucleus modified the pregnancy rate in bitches. Females inseminated by transcervical semen deposition had a higher pregnancy rate than females inseminated by the intravaginal technique. Using fresh or frozen-thawed semen produced a higher pregnancy rate in bitches inseminated by transcervical semen deposition than females inseminated by the intravaginal technique. Differences in the pregnancy rate between transcervical and intravaginal insemination could be associated with the correct semen disposition, the distance that the sperm must travel to reach the oocyte, as well as the number of sperm that reach the oviduct ampulla. Exist evidences that after ovulation, as progesterone rises, the cervix is closed, which may compromise the passage of the sperm deposited into the vagina. Therefore, it is likely that in females with a serum progesterone concentration > 10 ng/mL, the cervix was closed, compromising the ability of the sperm to access the oviduct. Thus, the use of intravaginal insemination should be done in bitches with a serum progesterone concentrations < 11 ng/mL to reduce the possibility of cervical closure and to increase the odds of pregnancy. It is well documented that the serum progesterone concentration and vaginal cytology parameters have a great influence on pregnancy success, and the results confirm these findings. In the present study, 96% of the bitches inseminated with a serum progesterone concentration of 5-10 ng/mL got pregnant and had higher odds of pregnancy than bitches with lower or higher serum progesterone concentrations.

Enhancement of Annexin V in response to combination of epigallocatechin gallate and quercetin as a potent arrest the cell cycle of colorectal cancer / Aumento da anexina V em resposta à combinação de galato de epigalocatequina e quercetina como uma potente parada do ciclo celular do câncer colorretal

Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.

O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.

Enhancement of Annexin V in response to combination of epigallocatechin gallate and quercetin as a potent arrest the cell cycle of colorectal cancer / Aumento da anexina V em resposta à combinação de galato de epigalocatequina e quercetina como uma potente parada do ciclo celular do câncer colorretal

Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.(AU)

O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.(AU)

Molecular characteristics and transcriptional regulatory of spermatogenesis-related gene RFX2 in adult Banna mini-pig inbred line (BMI)

RFX2 plays critical roles in mammalian spermatogenesis and cilium maturation. Here, the testes of 12-month-old adult boars of Banna mini-pig inbred line (BMI) were subjected to whole-transcriptome sequencing. The results indicated that the average expression (raw count) of RFX2 gene in BMI testes was 16138.25, and the average expression value of the corresponding transcript ENSSSCT00000043271.2 was 123.1898. The CDS of RFX2 obtained from BMI testes was 2,817 bp (GenBank accession number: OL362242). Gene structure analysis showed that RFX2 was located on chromosome 2 of the pig genome with 19 exons. Protein structure analysis indicated that RFX2 contains 728 amino acids with two conserved domains. Phylogenetic analysis revealed that RFX2 was highly conserved with evolutionary homologies among mammalian species. Other analyses, including PPI networks, KEGG, and GO, indicated that BMI RFX2 had interactions with 43 proteins involving various functions, such as in cell cycle, spermatid development, spermatid differentiation, cilium assembly, and cilium organization, etc. Correlation analysis between these proteins and the transcriptome data implied that RFX2 was significantly associated with FOXJ1, DNAH9, TMEM138, E2F7, and ATR, and particularly showed the highest correlation with ATR, demonstrating the importance of RFX2 and ART in spermatogenesis. Functional annotation implied that RFX2 was involved in 17 GO terms, including three cellular components (CC), six molecular functions (MF), and eight biological processes (BP). The analysis of miRNA-gene targeting indicated that BMI RFX2 was mainly regulated by two miRNAs, among which four lncRNAs and five lncRNAs competitively bound ssc-miR-365-5p and ssc-miR-744 with RFX2, respectively. Further, the dual-luciferase report assay indicated that the ssc-miR-365-5p and ssc-miR-744 significantly reduced luciferase activity of RFX2 3'UTR in the 293T cells, suggesting that these two miRNAs regulated the expression of RFX2. Our results revealed the important role of RFX2 in BMI spermatogenesis, making it an intriguing candidate for follow-up studies.(AU)

ß-micrustoxin (Mlx-9), a PLA2 from Micrurus lemniscatus snake venom: biochemical characterization and anti-proliferative effect mediated by p53

Background: Endogenous phospholipases A2 (PLA2 ) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of ß-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The ß-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: ß-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM médium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on ß-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. ß-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that ß-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.(AU)

Vaginal cytology in mares in the different stages of the estral cycle associated with the seasons of the year / Citologia vaginal em éguas nas diferentes fases do ciclo estral associadas as estações do ano

The reproductive biotechnologies directly assist in the development of different methods of reproductive evaluation. The objective of this study was to develop a new method to determine the estrous cycle phase in equine females through vaginal cytology throughout the seasons, as well as to establish an estimate of inflammatory cells present in the vagina. Six mares were evaluated at different ages without a defined breed and reproductive activity and were subjected to ultrasound evaluation of the reproductive system and cytological analysis of the vaginal region. In the summer, there was a predominance of keratinized epithelial cells in the estrus and intermediate in the diestrus. In the autumn, there was a predominance of keratinized cells in estrus and parabasal cells in estrus and diestrus. In anestrus (winter), a greater number of parabasal cells was identified in relation to the other cell types. In estrus (spring), there was a predominance of parabasal and intermediate cells. Conversely, in diestrus, parabasals were found in greater numbers. The evaluation of inflammatory cells of the vaginal epithelium of mares showed greater activity in the summer; however, there are no reference values for healthy animals in the literature, and it is necessary to conduct studies on the subject. It is concluded that there is an influence of cyclicity on the vaginal epithelium of the equine species, varying according to the season. Additionally, vaginal cytological evaluation is an important complementary tool in the diagnosis of vaginitis in mares, requiring further research on the subject.(AU)

As biotecnologias reprodutivas auxiliam diretamente no desenvolvimento de diferentes métodos de avaliação repro-dutiva. O objetivo deste estudo foi desenvolver um novo método para determinar a fase do ciclo estral em fêmeas equinas através da citologia vaginal ao longo das estações, bem como estabelecer uma estimativa das células inflamatórias presentes na vagina. Seis éguas foram avaliadas em diferentes idades, sem raça definida e em atividade reprodutiva, e foram submetidas à avaliação ultrassonográfica do aparelho reprodutor e análise citológica da região vaginal. No verão, houve predomínio de células epiteliais queratinizadas no estro e intermediárias no diestro. No outono, houve predomínio de células queratinizadas no estro e células parabasais no estro e diestro. No anestro (inverno), foi identificado maior número de células parabasais em relação aos demais tipos celulares. No estro (primavera), houve predomínio de células parabasais e intermediárias. Por outro lado, no diestro, os parabasais foram encontrados em maior número. A avaliação das células inflamatórias do epitélio vaginal de éguas mostrou maior atividade no verão; entretanto, não há valores de referência para animais saudáveis na literatura, sendo necessário a rea-lização de estudos sobre o assunto. Conclui-se que há influência da ciclicidade no epitélio vaginal da espécie equina, variando de acordo com a estação do ano. Além disso, a avaliação citológica vaginal é uma importante ferramenta complementar no diagnóstico da vaginite em éguas, necessitando de mais pesquisas sobre o assunto.(AU)

Maturation and quality of 'BRS Magna' grapes influenced by rootstocks in rainy season

Seasonal variations and production factors can influence the quality of grapes. Considering the Sub-middle region of the São Francisco Valley, Brazil, differentiated by production in rainy and warmer periods of the year, the climatic conditions in each season affect the duration of the phenological phases and quality of the grapes. The objective of this study was to characterize the changes in the quality components during maturation of 'BRS Magna' grapes on different rootstocks, in rainy seasons under tropical conditions, as a support to define the harvest time. The experiment was conducted in Petrolina, Pernambuco, Brazil, from Jan to Apr 2017 (cycle 1) and Dec 2017 to Apr 2018 (cycle 2) evaluating rootstocks and age of the fruits. Bunches were picked from the start of maturation to the harvest: 49, 56, 61, 64 and 68 days after fruit set (DAF) for cycle 1, and 35, 41, 48, 55, 61, and 66 DAF for cycle 2. In cycle 1 'IAC 572' and 'Paulsen 1103' rootstocks provided higher sugar contents in grapes, which was only repeated in cycle 2 for 'Paulsen 1103'. The accumulation of anthocyanins and yellow flavonoids was favored by 'IAC 313' and 'IAC 572' rootstocks in cycle 1, and by 'IAC 313' and 'IAC 766' in cycle 2. A higher content of polyphenols was conferred on 'IAC 572' in cycle 1 and on 'IAC 766' in cycle 2, which also had a greater antioxidant capacity. Although there were differences between rootstocks, harvesting at approximately 64 DAF maximized grape quality for all treatments.(AU)

Early puberty in short-haired Guinea pigs kept in laboratory animal facilities

Lab animals, such as Guinea pigs (Cavia porcellus), are crucial for scientific development, as they play an important role in the development and quality control chain of vaccines and drugs distributed by the Brazilian public health system. Investigating their biological and physiological parameters is fundamental to raise and keep these animals, so the handling of the facilities that hold them can be updated whenever new information comes up, with the well-being of the animals and alignment with the 3 Rs in mind. In the search for understanding reproductive aspects of Guinea pigs, the present study had the main goal of studying puberty by means of estrous cycle analysis in short-haired Guinea pigs. Guinea pigs have a vaginal occlusive membrane that covers the vaginal orifice. Its rupture takes place gradually and naturally, moments before labor and during estrus. The present study followed 42 females as for the presentation of the vaginal occlusive membrane. Once the membranes ruptured spontaneously, a swab was collected to study vaginal cytology. Membrane rupture was observed in 39 females; six females showed membrane rupture with less than 21 days of age (17 to 21 days). Twenty-three females were characterized as being in estrus due to cytology showing a prevalence of anucleated superficial cells. One of these females was younger than 21 days old. The opening of the vaginal occlusive membrane took place most frequently in intervals between 17 and 18 days, and the membrane remained open between one and three consecutive days. It was possible to follow three cycles of membrane opening on six females. The present study showed the need to adapt handling guidelines for C. porcellus kept in research animal facilities. The early age of puberty imposes the need of separate the female daughters from their fathers at 16 days old.(AU)

Effects of ß-glucan from S. cerevisiae on the expression of Casp9, Ccna2 and Sod1 genes in MCF-7 cells

ß-Glucans (ßG) are polysaccharides widely distributed in nature with chemopreventive properties. The aim of this study was to investigate the effects of ßG and the combined treatment with doxorubicin (Dox) on cell viability and mRNA levels of genes involved in cell cycle, apoptosis and antioxidant response. ßG was not cytotoxic. The mRNA levels of CCNA2of cells exposed to ß-glucan was upregulated and the exposure to Dox decreased the expression, while the combination led to an upregulation. Modulation of mRNA levels of CASP9suggest that ßG could inhibit promotion and progression steps of carcinogenesis, eliminatingneoplastic cells. The upregulation of CCNA2gene in combined treatment could be occurred due to ability of ßG in restoring the cell cycle distribution pattern after treatment with Dox. The upregulation of SOD1suggests that ßG can enhance the intracellular antioxidant defense, reducing the levels of superoxide dismutase induced by Dox. This response could reduce oxidative damage and attenuate tissue damage during chemotherapeutic treatment. Our data suggest that the drug combination may be less effective in killing tumor cells than the treatment with Dox alone. Thus, future studies should carefully consider this effect on indication of ßG during chemotherapy.Keywords:caspase-9; cyclin A2; superoxide dismutase 1; cell cycle; antioxidant.Received on July 2, 2020.Accepted on February 7, 2022.IntroductionGlucans are polysaccharides widely distributed in nature and oftenstudied due to chemopreventive properties. They are constituent of the cell wall of plants (oats and barley), algae, bacteria and fungi. ß-glucans (ßG)have a common structure comprising a main chain of ß-(1,3) and/or ß-(1,4) D-glucopyranosyl unit and they differ in length and branching structures. ßG of Saccharomyces cerevisiaehave 1→6 side branches while those of bacteria have 1→4 side branches (Chan, Chan, & Sze, 2009). ßGcan prevent DNA damage induced by chemical and physical agents (Ghavami,Goliaei, Taghizadeh, & Nikoofar, 2014). Some authors showed its significant efficacy in preventing mutagenic effects caused by doxorubicin, cyclophosphamide and cisplatin (Tohamy, El-Ghor, El-Nahas, & Noshy, 2003), methyl methanesulfonate (Oliveira et al., 2007)and hydrogen peroxide (Slamenová, 2003). Moreover, some studies have related the antioxidant ability of ßGagainst reactive free radicals formed by endogenous metabolic processes or exogenous chemicals (Tsiapali et al., 2001; Slamenová,2003; Sener, Eksioglu-Demiralp, Cetiner, Ercan, & Yegen, 2006; Guerra Dore et al., 2007; Kofuji et al., 2012; Lei et al., 2015). Yeast-derived ßGhave modulating action of humoral and cellular immune responses (Vetvicka et al., 2007).This activity provides protection to the organism against infections and cancer development (Samuelsen, Schrezenmeir, & Knutsen, 2014; Roudbary, Daneshmand, Hajimorad, Roudbarmohammadip, & Hassan, 2015). Despite postulated modes of action by which ß-glucan works are lacking information about the molecular mechanisms involved in the chemopreventive activity of this polysaccharide. In addition, compounds with chemopreventive properties can contribute to reduce side effects and toxicity during the chemotherapeutic treatment. Therefore, the aim of this study was to investigate the effects of ßG and the combined treatment with doxorubicin (Dox) on the expression of genes related with apoptosis (CASP9), cell cycle control (CCNA2)and antioxidant defense (SOD1)in human breast cancer MCF-7 cells. Doxorubicin (Dox) was chosen because it is one of the most used chemotherapeutic agent for cancer treatment. The limitation on the use of Dox in cancer treatment is the lack of selectivity against cancer cells and, consequently, its toxicity to patients.(AU)

Tumor ovariano de células de Sertoli em fêmea canina / Ovarian tumor of Sertoli cells in a female dog

O tumor de células de Sertoli, classificado como tumor do cordão sexual puro, é caracterizado por provocar manifestações clínicas endócrinas. É encontrado mais comumente em machos e é raro em fêmeas. O presente estudo objetivou relatar um caso de tumor ovariano de células de Sertoli em uma cadela. A paciente era uma fêmea canina, de cinco anos de idade da raça Shi Tzu, que apresentava queixa de "posição de prece" com frequência e anestro há dois anos. O exame físico determinou alopecia difusa e simétrica, hiperpigmentação de pele, pelos frágeis e comedões, com aumento de volume no flanco esquerdo e sensibilidade à palpação abdominal. O exame ultrassonográfico evidenciou estrutura arredondada, de bordas definidas em topografia de ovário esquerdo, medindo aproximadamente 5,27cm em seu maior eixo; além de piometra incipiente. Após ovariossalpingohisterectomia, realizou-se avaliação histopatológica do ovário, com diagnóstico de tumor do cordão sexual/estroma, com padrão de células de Sertoli infiltrativo. Conclui-se que, apesar de incomum, é importante incluir o tumor de células de Sertoli como diagnóstico diferencial, em casos de sinais de desregulação hormonal e alterações no ciclo estral e do pelame do animal. Ressalta-se a importância dos exames de imagem e histopatológicos para o diagnóstico, prognóstico e tratamento adequado.

Sertoli cells tumor, classified as a pure sexual cord tumor, is characterized by causing endocrine clinical manifestations. It is commonly found in males and is rare in females. The present study aimed to report a case of a Sertoli cell ovarian tumor in a female dog. The patient was a five-year-old female Shi Tzu dog, who complained of prayer position frequently and anestrus for two years. The physical examination determined diffuse and symmetrical alopecia, skin hyperpigmentation, fragile hair, and comedones, with increased volume in the left flank and sensitivity to abdominal palpation. The ultrasound examination evidenced the presence of a rounded structure, with defined edges in the topography of the left ovary, measuring approximately 5.27cm in its longest axis, in addition to incipient pyometra. After ovariosalpingohysterectomy, a histopathological evaluation of the ovary was performed with a diagnosis of sexual cord/stroma with an infiltrative Sertoli cell pattern. It is concluded that, although uncommon, it is important to include the Sertoli cell tumor as a differential diagnosis in cases of signs of hormonal dysregulation and changes in the estrous cycle, and animal's coat. The importance of imaging and histopathological exams for diagnosis, prognosis, and appropriate treatment is highlighted.

New multienzymatic complex formed between human cathepsin D and snake venom phospholipase A2

Background Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.