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BACKGROUND AND OBJECTIVE: Despite curative-intent radical cystectomy (RC), patients with muscle-invasive bladder cancer (MIBC) are at high risk of recurrence. Biomarkers are urgently needed to refine prognostication and selection of appropriate perioperative systemic therapies. Our aim was to evaluate the prognostic and predictive value of tumor-informed circulating tumor DNA (ctDNA) results in a multicenter cohort of patients with bladder cancer who underwent RC. METHODS: We performed a retrospective analysis of real-world data for a commercial ctDNA test (Signatera; Natera, Austin, TX, USA) performed in 167 patients (852 plasma samples) before RC and during molecular residual disease (MRD; adjuvant decision) and surveillance windows. We assessed the correlation between recurrence and ctDNA status before and after RC using Cox regression analysis. RESULTS AND LIMITATIONS: During study-defined postoperative MRD and surveillance windows, detectable ctDNA was associated with shorter disease-free survival (DFS) when compared to undetectable ctDNA (MRD: hazard ratio 6.93; p < 0.001; surveillance: hazard ratio 23.02; p < 0.001). Of note, patients with undetectable ctDNA did not appear to benefit from adjuvant therapy (p = 0.34). Detectable ctDNA in the pre-RC (p = 0.045), MRD (p = 0.002), and surveillance (p < 0.001) windows was the only risk factor independently associated with shorter DFS. Limitations include the retrospective and nonrandomized nature of the study. CONCLUSIONS: ctDNA testing in patients with bladder cancer undergoing RC was prognostic and potentially predictive. Identification of patients at high risk of recurrence may aid in patient counseling and decision-making. PATIENT SUMMARY: We found that outcomes for patients with muscle-invasive bladder cancer are strongly linked to detection of tumor DNA in blood samples. The results show the value of tumor-informed testing for tumor DNA in blood for decisions on the best treatment for each individual patient.
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INTRODUCTION: Personalized and tumor-informed circulating tumor DNA (ctDNA) testing is feasible and allows for molecular residual disease (MRD) identification in patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: In this retrospective analysis of commercial cases from multiple US institutions, personalized, tumor-informed, whole-exome sequenced, and germline-controlled ctDNA levels were quantified and analyzed in patients with PDAC. Plasma samples (nâ =â 1329) from 299 clinically validated patients were collected at diagnosis, perioperatively (MRD-window; within 2-12 weeks after surgery, before therapy), and during surveillance (>12 weeks post-surgery if no ACT or starting 4 weeks post-ACT) from November 2019 to March 2023. RESULTS: Of the initially diagnosed patients with stages I-III PDAC who went for resection, the median follow-up time from surgery was 13 months (range 0.1-214). Positive ctDNA detection rates were 29% (29/100) and 29.6% (45/152) during the MRD and surveillance windows, respectively. Positive ctDNA detection was significantly associated with shorter DFS within the MRD window (median DFS of 6.37 months for ctDNA-positive vs 33.31 months for ctDNA-negative patients; HR: 5.45, Pâ <â .0001) as well as during the surveillance period (median DFS: 11.40 months for ctDNA-positive vs NR for ctDNA-negative; HR: 12.38, Pâ <â .0001). Additionally, DFS was significantly better with KRAS wildtype status followed by KRASG12R (HR: 0.99, Pâ =â .97), KRASG12D (HR: 1.42, Pâ =â .194), and worse with KRASG12V (HR: 2.19, Pâ =â .002) status. In multivariate analysis, ctDNA detection at surveillance was found to be the most significant prognostic factor for recurrence (HR: 24.28, Pâ <â .001). CONCLUSIONS: Perioperative tumor-informed ctDNA detection in PDAC is feasible across all stages and is associated with patient survival outcomes.
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Importance: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignant tumor, and durable disease control is rare with the current standard of care, even for patients who undergo surgical resection. Objective: To assess whether neoadjuvant modified 5-fluorouracil, leucovorin, oxaliplatin, and irinotecan (mFOLFIRINOX) leads to early control of micrometastasis and improves survival. Design, Setting, and Participants: This open-label, single-arm, phase 2 nonrandomized controlled trial for resectable PDAC was conducted at the Yale Smilow Cancer Hospital from April 3, 2014, to August 16, 2021. Pancreatic protocol computed tomography was performed at diagnosis to assess surgical candidacy. Data were analyzed from January to July 2023. Interventions: Patients received 6 cycles of neoadjuvant mFOLFIRINOX before surgery and 6 cycles of adjuvant mFOLFIRINOX. Whole blood was collected and processed to stored plasma for analysis of circulating tumor DNA (ctDNA) levels. Tumors were evaluated for treatment response and keratin 17 (K17) expression. Main Outcomes and Measures: The primary end point was 12-month progression-free survival (PFS) rate. Additional end points included overall survival (OS), ctDNA level, tumor molecular features, and K17 tumor levels. Survival curves were summarized using Kaplan-Meier estimator. Results: Of 46 patients who received mFOLFIRINOX, 31 (67%) were male, and the median (range) age was 65 (46-80) years. A total of 37 (80%) completed 6 preoperative cycles and 33 (72%) underwent surgery. A total of 27 patients (59%) underwent resection per protocol (25 with R0 disease and 2 with R1 disease); metastatic or unresectable disease was identified in 6 patients during exploration. Ten patients underwent surgery off protocol. The 12-month PFS was 67% (90% CI, 56.9-100); the median PFS and OS were 16.6 months (95% CI, 13.3-40.6) and 37.2 months (95% CI, 17.5-not reached), respectively. Baseline ctDNA levels were detected in 16 of 22 patients (73%) and in 3 of 17 (18%) after 6 cycles of mFOLFIRINOX. Those with detectable ctDNA levels 4 weeks postresection had worse PFS (hazard ratio [HR], 34.0; 95% CI, 2.6-4758.6; P = .006) and OS (HR, 11.7; 95% CI, 1.5-129.9; P = .02) compared with those with undetectable levels. Patients with high K17 expression had nonsignificantly worse PFS (HR, 2.7; 95% CI, 0.7-10.9; P = .09) and OS (HR, 3.2; 95% CI, 0.8-13.6; P = .07). Conclusions and Relevance: This nonrandomized controlled trial met its primary end point, and perioperative mFOLFIRINOX warrants further evaluation in randomized clinical trials. Postoperative ctDNA positivity was strongly associated with recurrence. K17 and ctDNA are promising biomarkers that require additional validation in future prospective studies. Trial Registration: ClinicalTrials.gov Identifier: NCT02047474.
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Protocolos de Quimioterapia Combinada Antineoplásica , Fluoruracila , Irinotecano , Leucovorina , Oxaliplatina , Neoplasias Pancreáticas , Humanos , Masculino , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Fluoruracila/uso terapêutico , Fluoruracila/administração & dosagem , Leucovorina/uso terapêutico , Leucovorina/administração & dosagem , Feminino , Irinotecano/uso terapêutico , Irinotecano/administração & dosagem , Pessoa de Meia-Idade , Oxaliplatina/uso terapêutico , Oxaliplatina/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Idoso , Terapia Neoadjuvante , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/cirurgia , Carcinoma Ductal Pancreático/mortalidade , Idoso de 80 Anos ou mais , Intervalo Livre de ProgressãoRESUMO
Cellular senescence is a tightly regulated pathophysiologic process and is caused by replicative exhaustion or external stressors. Since naturally derived bioactive compounds with anti-ageing properties have recently captured scientific interest, we analysed the anti-ageing and antioxidant efficacy of Cryptomphalus aspersa egg extract (CAEE). Its effects on stemness, wound-healing properties, antioxidant defense mechanisms, and DNA damage repair ability of Human Wharton's jelly mesenchymal stem cells (WJ-MSCs) were analysed. Our results revealed that CAEE fortifies WJ-MSCs stemness, which possibly ameliorates their wound-healing ability. Additionally, we show that CAEE possesses a strong antioxidant capacity as demonstrated by the elevation of the levels of the basic antioxidant molecule, GSH, and the induction of the NRF2, a major antioxidant regulator. In addition, CAEE alleviated cells' oxidative stress and therefore prevented stress-induced premature senescence (SIPS). Furthermore, we demonstrated that the prevention of SIPS could be mediated via the extract's ability to induce autophagy, as indicated by the elevation of the protein levels of all basic autophagic molecules and the increase in formation of autophagolysosomes in CAEE-treated WJ-MSCs. Moreover, CAEE-treated cells exhibited decreased Caveolin-1 levels. We propose that Cryptomphalus aspersa egg extract comprises bioactive compounds that can demonstrate strong antioxidant/anti-ageing effects by regulating the Caveolin-1-autophagy-senescence molecular axis.
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Antioxidantes , Caveolina 1 , Humanos , Antioxidantes/farmacologia , Senescência Celular , Células-Tronco , EnvelhecimentoRESUMO
Affective states are long lasting mood states resulting from an accumulation of experiences. The knowledge of the affective state of animals can significantly help maintain and/or increase animal welfare. The aim of the study was to recognize the affective state of 13 adult rams reared under an intensive system and to further associate their affective state with hierarchy, sociability order and maintenance and social behaviour. The affective state was estimated by exposing them to an attention bias test and two novel object tests. Individuals with negative affective states performed reduced head hanging (p = 0.014), reduced agonistic behaviour (p = 0.033), increased social proximity (p = 0.009) and received less affiliative behaviours (p = 0.006). The study estimated the affective state of the rams and indicated easily recognizable maintenance and social behaviours correlated with negative affective state. Such behaviours could increase the awareness of the animals' welfare and thus improve management practices.
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Carneiro Doméstico , Comportamento Social , Humanos , Ovinos , Masculino , Animais , Emoções , Criação de Animais Domésticos/métodos , Bem-Estar do AnimalRESUMO
PURPOSE: To investigate whether circulating tumor DNA (ctDNA) assessment in patients with muscle-invasive bladder cancer predicts treatment response and provides early detection of metastatic disease. EXPERIMENTAL DESIGN: We present full follow-up results (median follow-up: 68 months) from a previously described cohort of 68 neoadjuvant chemotherapy (NAC)-treated patients who underwent longitudinal ctDNA testing (712 plasma samples). In addition, we performed ctDNA evaluation of 153 plasma samples collected before and after radical cystectomy (RC) in a separate cohort of 102 NAC-naïve patients (median follow-up: 72 months). Total RNA sequencing of tumors was performed to investigate biological characteristics of ctDNA shedding tumors. RESULTS: Assessment of ctDNA after RC identified metastatic relapse with a sensitivity of 94% and specificity of 98% using the expanded follow-up data for the NAC-treated patients. ctDNA dynamics during NAC was independently associated with patient outcomes when adjusted for pathologic downstaging (HR = 4.7; P = 0.029). For the NAC-naïve patients, ctDNA was a prognostic predictor before (HR = 3.4; P = 0.0005) and after RC (HR = 17.8; P = 0.0002). No statistically significant difference in recurrence-free survival for patients without detectable ctDNA at diagnosis was observed between the cohorts. Baseline ctDNA positivity was associated with the Basal/Squamous (Ba/Sq) subtype and enrichment of epithelial-to-mesenchymal transition and cell cycle-associated gene sets. CONCLUSIONS: ctDNA is prognostic in NAC-treated and NAC-naïve patients with more than 5 years follow-up and outperforms pathologic downstaging in predicting treatment efficacy. Patients without detectable ctDNA at diagnosis may benefit significantly less from NAC, but additional studies are needed.
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Carcinoma de Células de Transição , DNA Tumoral Circulante , Neoplasias da Bexiga Urinária , Humanos , Carcinoma de Células de Transição/tratamento farmacológico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , DNA Tumoral Circulante/genética , Seguimentos , Recidiva Local de Neoplasia/genética , Terapia Neoadjuvante/métodosRESUMO
Recognizing the identity of conspecifics is important for survival and social interactions. In sheep, vocal individuality enhances postnatal recognition and strengthens the mother-offspring bond. Although previous studies report vocal individuality in an early postnatal period (3-15 days of life), scarce information exists on whether individuality occurs at a later postnatal time point. The aim of the study was to identify whether individuality in acoustic cues is expressed in ewes' and their lambs' bleats at 40 days post-partum. Dairy ewes (N = 21) and their suckling offspring (n = 30) were isolated separately without hearing or seeing each other, and vocalizations were recorded. Different approaches for estimating individuality on 18 determined acoustic parameters were implemented. All parameters showed individuality, but higher individuality appeared in those related with source and spectral characteristics. A discriminant function analysis showed similar levels of individuality between ewes and lambs, but lower than that reported in an earlier postnatal time, suggesting that ewes and lambs do not need strong individualized cues at the examined time point. In both cases, jitter was the only common parameter, suggesting its importance. Distinctive cues were noted between siblings. Ewes displayed vocal individuality at their dry season (a later time point than suckling period), which was based on amplitude-related acoustic parameters.
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Acústica , Sinais (Psicologia) , Animais , Ovinos , Feminino , Humanos , Análise Discriminante , Audição , Período Pós-PartoRESUMO
Regardless of the advances in our ability to detect early and treat breast cancer, it is still one of the common types of malignancy worldwide, with the majority of patients decease upon metastatic disease. Nevertheless, due to these advances, we have extensively characterized the drivers and molecular profiling of breast cancer and further dividing it into subtypes. These subgroups are based on immunohistological markers (Estrogen Receptor-ER; Progesterone Receptor-PR and Human Epidermal Growth Factor Receptor 2-HER-2) and transcriptomic signatures with distinct therapeutic approaches and regiments. These therapeutic approaches include targeted therapy (HER-2+), endocrine therapy (HR+) or chemotherapy (TNBC) with optional combination radiotherapy, depending on clinical stage. Technological and scientific advances in the identification of molecular pathways that contribute to therapy-resistance and establishment of metastatic disease, have provided the rationale for revolutionary targeted approaches against Cyclin-Dependent Kinases 4/6 (CDK4/6), PI3 Kinase (PI3K), Poly ADP Ribose Polymerase (PARP) and Programmed Death-Ligand 1 (PD-L1), among others. In this review, we focus on the comprehensive overview of epidemiology and current standard of care treatment of metastatic breast cancer, along with ongoing clinical trials. Towards this goal, we utilized available literature from PubMed and ongoing clinical trial information from clinicaltrials.gov to reflect the up to date and future treatment options for metastatic breast cancer.
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This e research focused on the detection and identification of genetic polymorphisms in exon 7 of the ß-casein CSN2 gene in blood samples from Greek Holstein cows and from local breeds of cattle, such as Vrachykeratiki, Katerinis, and Sykias. For this purpose, DNA was isolated from 780 blood samples obtained from Greek Holstein cows, 86 from three local breeds of cattle, namely Brachyceros, Katerinis, and Sykias, and 14 from Greek buffalo. The desired region of exon 7 was amplified by PCR, resulting in 121 and 251 bp products in bovine and buffalo samples. The PCR product was digested with restriction fragment length polymorphism (RFLP) on agarose gels. The restriction enzymes DdeI and TaqI were used. All of the blood samples had the amplified size. The results showed that 74.4% of the Greek Holstein cows had the A2A2 ß-casein genotype, the three native breads Vrachykeratiki had 57.7%, and the other two had 100% of the A2A2 ß-casein. From the 14 Greek buffalo, 100% had the A2A2 ß-casein.
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INTRODUCTION: The aim of this study was to determine changes of reactive oxygen species (ROS), serum antioxidant capacity (SAC), oxidative stress index (OSi), and α-tocopherol (α-T) during the periparturient period in healthy and mastitic cows and to further investigate whether these parameters can be used as a tool for identifying cows at higher risk of developing mastitis. MATERIAL AND METHODS: Blood samples from 110 dairy cows from two commercial farms were obtained at dry-off, calving, and 30 days post-partum. Healthy cows formed group A (n = 90) and mastitic cows B (n = 20). Blood serum was obtained by centrifugation, and the aforementioned parameters were determined. A general linear model was used for analysing the associations among the determined blood parameters, the health of the animals' udder, and the sampling time. RESULTS: ROS and OSi values were higher (P < 0.001) by a respective 14% and 26%, and SAC values lower (P < 0.001) by 10% in group B than in group A at calving. ROC curve analysis revealed that all determined parameters at calving and α-T at dry-off and 30 days post-partum had excellent or acceptable predicting ability for mastitis incidence. CONCLUSION: This information provides a tool for early identification of cows at high risk of developing mastitis, allowing the implementation of intervention strategies.
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BACKGROUND: The indigenous cattle populations from Greece and Cyprus have decreased to small numbers and are currently at risk of extinction due to socio-economic reasons, geographic isolation and crossbreeding with commercial breeds. This study represents the first comprehensive genome-wide analysis of 10 indigenous cattle populations from continental Greece and the Greek islands, and one from Cyprus, and compares them with 104 international breeds using more than 46,000 single nucleotide polymorphisms (SNPs). RESULTS: We estimated several parameters of genetic diversity (e.g. heterozygosity and allelic diversity) that indicated a severe loss of genetic diversity for the island populations compared to the mainland populations, which is mainly due to the declining size of their population in recent years and subsequent inbreeding. This high inbreeding status also resulted in higher genetic differentiation within the Greek and Cyprus cattle group compared to the remaining geographical breed groups. Supervised and unsupervised cluster analyses revealed that the phylogenetic patterns in the indigenous Greek breeds were consistent with their geographical origin and historical information regarding crosses with breeds of Anatolian or Balkan origin. Cyprus cattle showed a relatively high indicine ancestry. Greek island populations are placed close to the root of the tree as defined by Gir and the outgroup Yak, whereas the mainland breeds share a common historical origin with Busa. Unsupervised clustering and D-statistics analyses provided strong support for Bos indicus introgression in almost all the investigated local cattle breeds along the route from Anatolia up to the southern foothills of the Alps, as well as in most cattle breeds along the Apennine peninsula to the southern foothills of the Alps. CONCLUSIONS: All investigated Cyprus and Greek breeds present complex mosaic genomes as a result of historical and recent admixture events between neighbor and well-separated breeds. While the contribution of some mainland breeds to the genetic diversity pool seems important, some island and fragmented mainland breeds suffer from a severe decline of population size and loss of alleles due to genetic drift. Conservation programs that are a compromise between what is feasible and what is desirable should focus not only on the still highly diverse mainland breeds but also promote and explore the conservation possibilities for island breeds.
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Bovinos/genética , Polimorfismo de Nucleotídeo Único , Animais , Chipre , Frequência do Gene , Introgressão Genética , Grécia , Isolamento ReprodutivoRESUMO
BACKGROUND: Sheep's reproductive physiology in temperate latitudes (such as Greece), is characterized by seasonality and is also regulated by photoperiodic exposure. Melatonin is the key hormone involved in this regulation. However, the melatonin secretion and therefore the ewes reproductive activity underlies variation, proposed to be linked with the melatonin receptor subtype 1A (MNTR1A) gene structure. This study was designed to investigate the polymorphism of the MNTR1A gene in a local Greek sheep breed and to determine its potential association with reproductive seasonality. RESULTS: Two groups of farmed ewes, each consisted of 30 individuals, were chosen. Males were introduced in both groups in spring (April). The first group consisted of ewes that showed reproductive activity in spring (May), while the second of ewes that showed reproductive activity 3 months later, in summer. The PCR-RFLP methodology was carried out on a 824-bp DNA fragment of the MTNR1A exon 2 using the RsaI restriction endonuclease. The electrophoretic procedure revealed three genotypes, C/C, C/T and T/T. Specifically, 44 animals showed the C/C genotype (28 from the first group and 16 from the second), 14 the C/T genotype (2 from the first and 12 from the second) and 2 animals had the T/T genotype (both from the second group). CONCLUSIONS: Statistical analysis indicated a positive correlation between genotype and reproductive seasonality, with C/C genotype playing a crucial role in out-of-season reproduction activity.
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Toll-like receptors (TLRs) and b-defensins (BD) molecules are group of molecules that recognize various microbial components and play a crucial role in the activation of the innate immune system in vertebrate species. Although TLRs gene expression has been studied in various pig tissues, little is known about their expression in porcine reproductive tract. Concerning b-defensins genes, only BD1, 2 and 3 counterparts have been well studied in pigs' reproductive organs. The aim of this study was to investigate the expression pattern of both gene families in pigs' male and female reproductive organs, and embryos, as potential tool for further association studies in respect to immunity and disease resistance. RT-PCR analysis revealed that all of the examined TLR genes were expressed in the reproductive organs of male and female pigs, with TLR3 and TLR5 showing the higher levels and TLR9 the lowest, in all analyzed tissues. BD genes showed a different expression pattern in respect to the examined tissue. In embryos, TLR1 revealed high expression levels, while only BD3, BD108, and BD123 were found to be expressed.
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Defensinas/genética , Defensinas/metabolismo , Genitália Feminina/metabolismo , Genitália Masculina/metabolismo , Suínos/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Animais , Defensinas/análise , Embrião de Mamíferos , Feminino , Genitália Feminina/química , Genitália Masculina/química , Masculino , Especificidade de Órgãos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Toll-Like/análiseRESUMO
G6PDH is the rate-limiting enzyme of the pentose phosphate pathway and one of the principal source of NADPH, a major cellular reductant. Importantly, in ruminant's metabolism the aforementioned NADPH provided, is utilized for de novo fatty acid synthesis. Previous work of cloning the ovine (Ovis aries) og6pdh gene has revealed the presence of two cDNA transcripts (og6pda and og6pdb), og6pdb being a product of alternative splicing not similar to any other previously reported.(1) In the current study the effect of energy balance in the ovine G6PDH protein expression was investigated, shedding light on the biochemical features and potential physiological role of the oG6PDB isoform. Changes in energy balance leads to protein expression changes in both transcripts, to the opposite direction and not in a proportional way. Negative energy balance was not in favor of the presence of any particular isoform, while both protein expression levels were not significantly different (P > 0.05). In contrast, at the transition point from negative to positive and on the positive energy balance, there is a significant increase of oG6PDA compared with oG6PDB protein expression (P < 0.001). Both oG6PDH protein isoforms changed significantly toward the positive energy balance. oG6PDA is escalating, while oG6PDB is falling, under the same stimulus (positive energy balance alteration). This change is also positively associated with increasing levels in enzyme activity, 4 weeks post-weaning in ewes' adipose tissue. Furthermore, regression analysis clearly demonstrated the linear correlation of both proteins in response to the WPW, while energy balance, enzyme activity, and oG6PDA relative protein expression follow the same escalating trend; in contrast, oG6PDB relative protein expression falls in time, similar to both transcripts accumulation pattern, as reported previously.(2.)
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The objective of the present study was to examine changes observed in the expression of cytosolic NADP isocitrate dehydrogenase (ICDH) and glucose 6-phosphate dehydrogenase (G6PD) genes, the major implicated genes in ruminant lipogenesis in terms of produce NADPH, during the early post-weaning period in dairy ewes in respect to energy intake, and to further correlate the noted changes with their respective enzymatic activities. A total of 21 subcutaneous adipose tissue samples were obtained from seven lactating (2nd lactation period) dairy ewes of the Chios breed. Adipose tissue samples were taken from the tail head region at weeks 1, 2, and 4 after weaning (45 days after parturition). Dairy ewes were in negative energy balance during weeks 1 and 2 after weaning and they moved into a strong positive energy balance at week 4 after weaning. Expression of ICDH and G6PD genes and their respective enzymatic activity was determined. Results showed that both genes' expression and enzymatic activities were significantly minimal at week 1 after weaning, reaching a maximum level at week 4 after weaning (P < 0.05). A 3.5-fold and a 5-fold increase of G6PD and ICDH mRNA levels were observed, respectively. Concerning their respective enzymatic activities, a 5.5-fold and 2-fold increase was noted, respectively. A positive correlation was found between ICDH and G6PD gene expression (P < 0.001) indicating a synchronized response to energy intake changes. Almost similar changes were observed for enzymatic activities, rendering these enzymes as potential biochemical markers of ovine lipogenesis.
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Ingestão de Energia/fisiologia , Lactação/genética , Lipogênese/genética , Carneiro Doméstico/fisiologia , Animais , Metabolismo Energético/genética , Feminino , Perfilação da Expressão Gênica , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Reação em Cadeia da Polimerase , Carneiro Doméstico/genética , Carneiro Doméstico/metabolismo , Gordura Subcutânea/enzimologiaRESUMO
Ovine 6-phosphogluconate dehydrogenase (6PGD) is an enzyme of the pentose phosphate pathway, providing the necessary compounds of NADPH for the synthesis of fatty acids. Much of research has been conducted both on enzymatic level and on molecular level. However, to our knowledge, any correlation between enzymatic activity and 6PGD gene expression pattern related to different physiological stages has not been yet reported. With this report, we tried to highlight if any correlation between enzymatic activity and expression of ovine 6PGD gene exists, in respect to different milk yield. According to the determined enzymatic activities and adipocytes characteristics, ewes with low milk production possessed a greater (P ≤ .001) 6PGD activity and larger adipocytes than the highly productive ewes. Although 6PGD expression pattern was higher in low milk yield ewes than in ewes with high milk production, this difference was not found statistically significant. Thus, 6PGD gene expression pattern was not followed by so rapid and great/sizeable changes as it was observed for its respective enzymatic activity, suggesting that other mechanisms such as post translation regulation may be involved in the regulation of the respective gene.
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Ovine 6-phosphogluconate dehydrogenase (6PGD), an enzyme of the pentose phosphate pathway, provides the necessary compounds of NADPH for the synthesis of fatty acids. Much of research has been conducted not only on the enzymatic level, but also on molecular level elucidating its cDNA sequence. Herein, we tried to elucidate if any correlation between enzymatic activity and expression of ovine 6PGD gene exists, in respect to two different weights from weaning to 4 months old. 18 male and 16 female lambs of Chios breed were randomly selected after weaning and assigned to two groups based on sex in a different experimental open-plan shed. Two subgroups were defined in each sex and they were slaughtered at 25 kg and 30 kg, respectively. Samples of adipose tissue (tail, perirenal and shoulder site) were collected and 6PGD enzymatic activity, gene expression, and characteristics of adipocytes were determined. According to the determined data, tail subcutaneous adipose tissue matures later than the others examined tissues and has a diminished lipogenic activity. A 6PGD gene expression pattern was not followed by analogous changes of its enzymatic activity, suggesting that other mechanisms such as post transcription or/and post translation regulation may be involved.
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Adipócitos/enzimologia , Peso Corporal/fisiologia , Fosfogluconato Desidrogenase/metabolismo , RNA Mensageiro/metabolismo , Ovinos/metabolismo , Gordura Subcutânea/enzimologia , Adipócitos/citologia , Animais , Feminino , Regulação Enzimológica da Expressão Gênica , Análise dos Mínimos Quadrados , Masculino , Fosfogluconato Desidrogenase/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Ovinos/genética , Gordura Subcutânea/citologiaRESUMO
Glucose 6-phosphate dehydrogenase (G6PD) plays an important role in a ruminant's metabolism catalyzing the first committed reaction in the pentose phosphate pathway as it provides necessary compounds of NADPH for the synthesis of fatty acids. The cloning of ovine (Ovis aries) G6PD gene revealed the presence of two cDNA transcripts (oG6PD(A) and oG6PD(B)), with oG6PD(B) being a product of alternative splicing and with no similarity to any other previously reported G6PD transcript. Here, we attempt to study the effect of energy balance in ovine G6PD transcript expression, trying simultaneously to find out any potential physiological role of the oG6PD(B) transcript. Changes of energy balance that lead to synergistic changes in the expression of both transcripts, but in opposite directions and not in a proportional way. Negative energy balance favours the presence of the oG6PD(B) transcript leading to a significant increase of its expression, compared to oG6PD(A) expression (P<0.05). In contrast, positive energy balance leads to a significant increase of oG6PD(A) compared to oG6PD(B) expression (P<0.05). In either condition oG6PD(B) expression is unchanged. Regression analysis showed that there is an energy balance threshold where the expression of both transcripts shows no change.
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Ingestão de Energia , Regulação da Expressão Gênica , Glucosefosfato Desidrogenase/genética , Carneiro Doméstico/genética , Animais , Perfilação da Expressão Gênica , Glucosefosfato Desidrogenase/metabolismo , Carneiro Doméstico/metabolismoRESUMO
To better understand the structure and the function of ovine glucose 6-phosphate dehydrogenase (G6PD) promoter region, a genome-walking procedure was followed to isolate and sequence a 1628 bp fragment, containing the 5' regulatory region of the G6PD gene. In silico analysis of the sequence showed many conserved blocks and features with other known mammalian G6PD promoter regions. The analysis also revealed the presence of one TATA box, three GC boxes, two E-boxes and several binding sites for Stimulating Protein 1 (Sp1) and Activator Protein 2 (AP2). Moreover, elements involved in the regulation of lipogenesis like USF (Upstream stimulating factor), HSF (Heat Shock Factor), F2F (Prolactin receptor), RAR (Retinoid Acid Receptor), STRE (STress Response Element), RORa (Retinoid related Orphan Receptor alpha), GATA (GATA binding factor), RFX (Regulatory Factor X), SREBP (Sterol Regulatory Element Binding Protein), MEP (Metal Element Protein), CREB (insulin receptor), PRE (Progesterone receptor), and HNF4 (Hepatic Nuclear Factor 4) were detected. The most important regulatory motifs were found to be conserved as compared to those in human and mouse counterparts. However, some differences were noted, likely indicating differences in the transcription regulation of G6PD gene between ruminant and non-ruminant species.
Assuntos
Região 5'-Flanqueadora , Glucosefosfato Desidrogenase/genética , Sequências Reguladoras de Ácido Nucleico , Ovinos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Biologia Computacional , Dados de Sequência Molecular , Análise de Sequência de DNA/métodos , Homologia de Sequência do Ácido NucleicoRESUMO
Glucose 6-phosphate dehydrogenase (G6PD) plays an important role in ruminant's lipogenesis, as it provides necessary compounds of NADPH for the synthesis of fatty acids catalyzing the first committed reaction in the pentose phosphate pathway. In this work the full length ovine glucose 6-phosphate dehydrogenase cDNA was isolated using a polymerase chain reaction based strategy. Two isoforms (OG6PDA and OG6PDB) were detected encoding a protein of 515 and 524 amino acids, respectively. Both deduced amino acid sequences reveal a well conserved protein containing all the important residues for its catalytic role. The extra nine amino acids encoded by OG6PDB cause a frameshift in the polypeptide chain resulting in changes around the area of the potential substrate binding site. A three-dimensional model of ovine G6PD protein shows that this frameshift cause structural changes in the catalytic binding "pocket" of the molecule. Southern blot and RT analysis revealed that ovine G6PD appears as a single copy gene while it is expressed, with slight variability, in all tissues analyzed. Moreover, expression analysis of the ovine G6PD isoforms showed that OG6PDB is expressed only in tissues where lipogenesis is high in ruminants. Thus, we hypothesize that in ruminants G6PD may be regulated by the ratio of the two transcripts, according to the existence stimulus.