Collagen release by human hepatic stellate cells requires vitamin C and is efficiently blocked by hydroxylase inhibition.

Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFß) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFß. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.

Unraveling the complexity of autophagy: Potential therapeutic applications in Pancreatic Ductal Adenocarcinoma.

Autophagy is a highly dynamic, evolutionary conserved cellular homeostatic process that occurs at baseline levels in most cells. It exerts predominantly cytoprotective effects by removing damaged organelles and protein aggregates. In cancer, however, autophagy acts as both a tumor suppressor by preventing ROS-induced tumorigenesis and as a tumor inducer by providing nutrients to tumor cells under hypoxic, low-energy conditions and protecting them against therapeutically induced stress. Pancreatic Ductal Adenocarcinoma is an extremely lethal and aggressive neoplasm with a 5 year-survival rate between 1% and 5%. One of the most important factors affecting its poor prognosis is its high resistance to most of the existing chemotherapeutic regimens. The role of autophagy in PDAC has been investigated by different research groups and the results are quite divergent; some research lines point at autophagy as a tumor promoting mechanism, whereas other studies assign oncosuppressive functions to it. Nevertheless, several distinct preclinical studies and clinical trials have evaluated the efficacy of both autophagy inducers and autophagy inhibitors as therapeutic compounds against PDAC, many of them providing promising results. Although a better understanding of the complexity of autophagy is needed, the modulation of this process opens new opportunities for prognostic and therapeutic purposes.

Tobacco consumption and quality of life among teachers: a bidirectional problem.

Objective: This study aimed to assess a bidirectional relationship between tobacco consumption and quality of life among Chilean teachers. Participants and methods: A total sample of 647 Chilean teachers was included in a cross-sectional study (71.8% female). Teachers completed a socio-demographic questionnaire, tobacco consumption habits, and the SF-36 questionnaire to assess quality of life. Logistic regression models were employed for statistical analysis of quality of life (physical component summary; mental component summary), and tobacco consumption habits, adjusted for socio-demographic characteristics. Results: A total of 34.2% of teachers were smokers, with the majority (68.7%) being under 45 years old. Smoking teachers demonstrated lower quality of life scores, particularly mental health and emotional problems dimensions, and mental component summary (p < 0.05) versus nonsmoking teachers. Teachers with tobacco consumption had a higher risk of low mental component summary (OR: 1.74; p < 0.001), and those with low mental component summary were more likely to be smokers (OR: 1.77; p < 0.002). Conclusion: These findings indicate that tobacco consumption adversely affects the quality of life of Chilean teachers, especially their mental health. Psychological support should be provided to help teachers cope with work stress and tobacco consumption.

Competing endogenous RNAs in human astrocytes: crosstalk and interacting networks in response to lipotoxicity.

Neurodegenerative diseases (NDs) are characterized by a progressive deterioration of neuronal function, leading to motor and cognitive damage in patients. Astrocytes are essential for maintaining brain homeostasis, and their functional impairment is increasingly recognized as central to the etiology of various NDs. Such impairment can be induced by toxic insults with palmitic acid (PA), a common fatty acid, that disrupts autophagy, increases reactive oxygen species, and triggers inflammation. Although the effects of PA on astrocytes have been addressed, most aspects of the dynamics of this fatty acid remain unknown. Additionally, there is still no model that satisfactorily explains how astroglia goes from being neuroprotective to neurotoxic. Current incomplete knowledge needs to be improved by the growing field of non-coding RNAs (ncRNAs), which is proven to be related to NDs, where the complexity of the interactions among these molecules and how they control other RNA expressions need to be addressed. In the present study, we present an extensive competing endogenous RNA (ceRNA) network using transcriptomic data from normal human astrocyte (NHA) cells exposed to PA lipotoxic conditions and experimentally validated data on ncRNA interaction. The obtained network contains 7 lncRNA transcripts, 38 miRNAs, and 239 mRNAs that showed enrichment in ND-related processes, such as fatty acid metabolism and biosynthesis, FoxO and TGF-ß signaling pathways, prion diseases, apoptosis, and immune-related pathways. In addition, the transcriptomic profile was used to propose 22 potential key controllers lncRNA/miRNA/mRNA axes in ND mechanisms. The relevance of five of these axes was corroborated by the miRNA expression data obtained in other studies. MEG3 (ENST00000398461)/hsa-let-7d-5p/ATF6B axis showed importance in Parkinson's and late Alzheimer's diseases, while AC092687.3/hsa-let-7e-5p/[SREBF2, FNIP1, PMAIP1] and SDCBP2-AS1 (ENST00000446423)/hsa-miR-101-3p/MAPK6 axes are probably related to Alzheimer's disease development and pathology. The presented network and axes will help to understand the PA-induced mechanisms in astrocytes, leading to protection or injury in the CNS under lipotoxic conditions as part of the intricated cellular regulation influencing the pathology of different NDs. Furthermore, the five corroborated axes could be considered study targets for new pharmacologic treatments or as possible diagnostic molecules, contributing to improving the quality of life of millions worldwide.

Correction: Lactate dehydrogenase A inhibition by small molecular entities: steps in the right direction.

[This corrects the article DOI: 10.18632/oncoscience.519.].

Lactate dehydrogenase A inhibition by small molecular entities: steps in the right direction.

Direct targeting of energy metabolism to defeat cancer is not a recent strategy. Although quite a few drugs use cellular metabolism for their antitumor effect, no direct inhibitors of energy metabolism have been approved by the FDA. Currently, several inhibitors of lactate dehydrogenase A (LDH-A), a key player in glycolysis, are in development. Earlier, we demonstrated the efficacy of N-hydroxyindole-based LDH-A inhibitors in different cancer types. In this study we describe the efficacy of NHI-Glc-2, which is designed to dual target cancer cells, by exploiting a simultaneous enhanced glucose uptake by overexpressed glucose transporter 1 (GLUT1) and by inhibition of LDH-A. NHI-Glc-2 inhibits LDH-A enzyme activity, PANC-1 cell growth and disrupts spheroid integrity, with an overall effect that is more pronounced when combined with gemcitabine.

Uso de Hidróxido de Sodio como Alternativa en Técnica de Diafanización de Diente / Use of Sodium Hydroxide as an Alternative in Tooth Clearing Technique

Con el objetivo de conservar material cadavérico, se han creado diferentes técnicas y/o soluciones donde una técnica es la diafanización dental para estudiar la morfología interna del diente. Esta técnica consta en trasparentar el tejido calcificado del diente haciendo visible los conductos radiculares al inyectar una mezcla colorante en ellos. Se han descrito diferentes variantes de la técnica de diafanización como la técnica de Okumura y la técnica de Robertson, pero ambas técnicas utilizan reactivos tóxicos o de difícil acceso, por lo que se ha realizado una búsqueda de reactivos de bajo costo y fácil acceso para realizar la técnica de diafanización, reportándose que la técnica de diafanización por maceración con KOH es válida para diafanizar dientes. El objetivo del presente estudio fue utilizar NaOH en la técnica de diafanización dental por maceración, como una variante de KOH al ser una base de similar característica que el KOH. Se utilizaron 13 dientes (siete tercer molares, cinco premolares y un canino) para realizar tres variantes de la técnica de diafanización: técnica de Robertson, por maceración con KOH y por maceración con NaOH utilizando barra agitadora y agitador magnético en los dientes. Con la técnica de Robertson se obtuvo un diente completamente transparentado, mientras que los dientes diafanizados por maceración, tanto con KOH y NaOH, se transparentaron menos, aunque se hicieron visibles los conductos radiculares, por lo que el uso de NaOH en la técnica de diafanización por maceración es válida, aunque requiere más tiempo que la maceración por KOH.

SUMMARY: To preserve cadaveric material, different techniques, and solutions have been created where one technique is dental diaphanization to study the internal morphology of the tooth. This technique consists of making the calcified tooth tissue transparent and making the root canals visible by injecting a dye mixture into them. Different variants of the diaphanization technique have been described, such as the Okumura and the Robertson techniques. However, both techniques use toxic or difficult-to-access reagents, so a search has been made for low- cost and easily accessible reagents to perform the diaphanization technique, reporting that the diaphanization technique by maceration with KOH is valid for the diaphanization of teeth. This study aimed to use NaOH in the dental clearing technique by maceration as a variant of KOH since it is a base with similar characteristics to KOH. Thirteen teeth (seven third molars, five premolars, and one canine) were used to perform three variants of the diaphanization technique: Robertson technique, KOH maceration, and NaOH maceration using a stirring bar and magnetic stirrer on the teeth. With the Robertson technique, a completely transparent tooth was obtained, while the teeth cleared by maceration, with both KOH and NaOH, were less transparent, although the root canals became visible. Therefore, using NaOH in the diaphanization technique by maceration is valid, although it requires more time than KOH maceration.

Development of bioluminescent chick chorioallantoic membrane (CAM) models for primary pancreatic cancer cells: a platform for drug testing.

The aim of the present study was to develop chick-embryo chorioallantoic membrane (CAM) bioluminescent tumor models employing low passage cell cultures obtained from primary pancreatic ductal adenocarcinoma (PDAC) cells. Primary PDAC cells transduced with lentivirus expressing Firefly-luciferase (Fluc) were established and inoculated onto the CAM membrane, with >80% engraftment. Fluc signal reliably correlated with tumor growth. Tumor features were evaluated by immunohistochemistry and genetic analyses, including analysis of mutations and mRNA expression of PDAC pivotal genes, as well as microRNA (miRNA) profiling. These studies showed that CAM tumors had histopathological and genetic characteristic comparable to the original tumors. We subsequently tested the modulation of key miRNAs and the activity of gemcitabine and crizotinib on CAM tumors, showing that combination treatment resulted in 63% inhibition of tumor growth as compared to control (p < 0.01). These results were associated with reduced expression of miR-21 and increased expression of miR-155. Our study provides the first evidence that transduced primary PDAC cells can form tumors on the CAM, retaining several histopathological and (epi)genetic characteristics of original tumors. Moreover, our results support the use of these models for drug testing, providing insights on molecular mechanisms underlying antitumor activity of new drugs/combinations.

A specific inhibitor of lactate dehydrogenase overcame the resistance toward gemcitabine in hypoxic mesothelioma cells, and modulated the expression of the human equilibrative transporter-1.

Malignant pleural mesothelioma (MPM) is a very hypoxic malignancy, and hypoxia has been associated with resistance towards gemcitabine. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis. Therefore we investigated the combination of a new LDH-A inhibitor (NHI-1) with gemcitabine in MPM cell lines. Under hypoxia (O2 tension of 1%) the cell growth inhibitory effects of gemcitabine, were reduced, as demonstrated by a 5- to 10-fold increase in IC50s. However, the simultaneous addition of NHI-1 was synergistic (combination index < 1). Flow cytometry demonstrated that hypoxia caused a G1 arrest, whereas the combination of NHI-1 significantly increased gemcitabine-induced cell death. Finally, the mRNA expression levels of the human equilibrative transporter-1 (hENT1) were significantly down-regulated under hypoxia, but treatment with NHI-1 was associated with a recovery of hENT1 expression. In conclusion, our data show that hypoxia increased MPM resistance to gemcitabine. However, cell death induction and modulation of the key transporter in gemcitabine uptake may contribute to the synergistic interaction of gemcitabine with the LDH-A inhibitor NHI-1 and support further studies for the rational development of this combination.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Evaluación del canal cervical mediante histerosalpingografía virtual y su comparación con la prueba de transferencia embrionaria / Cervical Evaluation by Virtual Hysterosalpingography and its Comparison with the Embryo Transfer Test

Objetivo: Comparar la prueba del catéter cervical con la histerosalpingografía virtual (HSGV) en la evaluación del cuello uterino antes de la transferencia embrionaria. Material y métodos: Fueron evaluadas 100 pacientes con antecedentes de infertilidad. El día del examen, un ginecólogo realizó la prueba del canal cervical con un catéter de Wallace. Luego se realizó una HSGV con un tomógrafo de 256 cortes. Las imágenes fueron evaluadas por un radiólogo, y se determinaron la permeabilidad del cuello uterino, el ángulo cérvico-uterino y la presencia de patología cervical. Resultados: Se observó una buena correlación (r=0,92) en la evaluación de la permeabilidad del cuello uterino entre ambos métodos. En el 35 % de las pacientes la prueba del catéter cervical no fue exitosa, y la HSGV detectó pólipos, sinequias y trayectos sinuosos del canal cervical; mientras que en 23 pacientes el cuello uterino fue normal, pero el ángulo cérvico-uterino fue < 90°. Conclusiones: Hubo una buena correlación entre la HSGV y la prueba de catéter cervical en la evaluación de la permeabilidad del cuello uterino. La HSGV proporciona, además, información anatómica útil para identificar la causa probable del fracaso de la transferencia de embriones.

Objective: To compare cervical catheter test and virtual HSG in the evaluation of cervix before embryo transfer. Methods: We evaluated 100 patients with history of infertility. On the day of examination, a gynaecologist performed a cervical test with a Wallace catheter. Then, patients underwent Virtual HSG performed with a 256-slice CT scanner. CT images were evaluated by a radiologist, and the cervical patency, utero-cervical angle and the presence of cervical pathology were determined. Results: There was a good correlation (r=0,92) in cervical patency evaluation between both methods. Unsuccessful cervical catheter test was observed in 35% of patients. In these patients, Virtual HSG detected polyps, adhesions and sinuous cervical canal, while cervix was normal in 23 patients, but the utero-cervical angle was < 90°. Conclusions: There was a good correlation between HSG findings and the cervical catheter test in the evaluation of cervical patency. Moreover virtual HSG provides anatomic information useful to identify the probable causes of failure of embryo transfers.