Protocol for the assessment of mTOR activity in mouse primary hepatocytes.

We present a protocol for measuring the activity of the mechanistic target of rapamycin (mTOR) pathway in ex vivo isolated mouse primary hepatocytes. It can be used as a tool for genetic, pharmacological, metabolomic, and signal transduction procedures. We discuss critical aspects for improving yield, viability, and modulation of the mTOR pathway. This protocol can be adapted to other signaling cascades and is compatible with multiple readouts. For complete details on the use and execution of this protocol, please refer to Ortega-Molina et al. (2021).

The effectiveness of monotherapy with PI3K/AKT/mTOR pathway inhibitors in ovarian cancer: A meta-analysis.

OBJECTIVE: To determine the clinical benefit of monotherapy with PI3K/AKT/mTOR inhibitors in patients diagnosed with advanced or recurrent ovarian cancer and to investigate the predictive value of current PI3K/AKT/mTOR biomarkers on therapy response. METHODS: A systematic search was conducted in PubMed, Embase and the Cochrane Library for articles reporting on treatment with PI3K/AKT/mTOR inhibitors in ovarian cancer. The primary endpoint was defined as the clinical benefit rate (CBR), including the proportion of patients with complete (CR) and partial response (PR) and stable disease (SD). Secondary endpoints included the overall response rate (ORR, including CR and PR) and drug-related grade 3 and 4 adverse events. RESULTS: We included 233 patients from 19 studies and observed a pooled CBR of 32% (95% CI 20-44%) and ORR of 3% (95% CI 0-6%) in advanced or recurrent ovarian cancer patients treated with PI3K/AKT/mTOR inhibitors. Subgroup analysis tended to favor the studies who selected patients based on current PI3K/AKT/mTOR biomarker criteria (e.g. genomic alterations or loss of PTEN protein expression), but the difference in CBR was not statistically significant from studies with unselected populations (respectively, CBR of 42% (95% CI 23-62%) and 27% (95% CI 14-42%), P = 0.217). To better reflect true patient benefit, we excluded SD <6 months as a beneficial outcome which resulted in a pooled CBR of 7% (95% CI 2-13%). The overall proportion of patients with drug-related grade 3 and 4 adverse events was 36%. CONCLUSIONS: The efficacy of monotherapy with PI3K/AKT/mTOR inhibitors in advanced recurrent ovarian cancer patients is limited to a small subgroup and selection of patients with the use of current biomarkers did not improved the CBR significantly. Given the toxicity profile, we suggest that current treatment with PI3K/AKT/mTOR inhibitors should not be initiated unless in clinical trials. Furthermore, improved biomarkers to measure functional PI3K/AKT/mTOR pathway activity are needed to optimize patient selection.

Role of PRPS2 as a prognostic and therapeutic target in osteosarcoma.

AIMS: Osteosarcoma (OS) is the most common primary malignant tumour of the bone. However, further improvement in survival has not been achieved due to a lack of well-validated prognostic markers and more effective therapeutic agents. Recently, the c-Myc-phosphoribosyl pyrophosphate synthetase 2 (PRPS2) pathway has been shown to promote nucleic acid metabolism and cancer cell proliferation in malignant melanoma; phosphorylated mammalian target of rapamycin (p-mTOR) has been upregulated and an effective therapeutic target in OS. However, the p-mTOR-PRPS2 pathway has not been evaluated in OS. METHODS: In this study, the expression level of PRPS2, p-mTOR and marker of proliferation (MKI-67) was observed in a cohort of specimens (including 236 OS cases and 56 control samples) using immunohistochemistry, and the association between expression level and clinicopathological characteristics of patients with OS was analysed. RESULTS: PRPS2 protein level, which is related to tumour proliferation, was higher in OS cells (p=0.003) than in fibrous dysplasia, and the higher PRPS2 protein level was associated with a higher tumour recurrence (p=0.001). In addition, our statistical analysis confirmed that PRPS2 is a novel, independent prognostic indicator of OS. Finally, we found that the expression of p-mTOR was associated with the poor prognosis of patients with OS (p<0.05). CONCLUSIONS: PRPS2 is an independent prognostic marker and a potential therapeutic target for OS.

Vías moleculares de regulación de la autofagia por BRCA1: implicaciones en cáncer / Molecular pathways of autophagy regulation by BRCA1: implications in cancer

La proteína BRCA1 contribuye a mantener la integridad genómica, a través de la regulación transcripcional de proteínas que controlan el ciclo celular y la reparación del ADN o por interacción directa con estas proteínas. La inestabilidad genética generada por mutaciones que dan lugar a un déficit de la actividad de BRCA1 confiere un riesgo incrementado de padecer, fundamentalmente, cánceres de mama y ovario. En los últimos años, se ha demostrado que la autofagia tiene un papel dual en el desarrollo tumoral, y agentes químicos como lucanthone, cloroquina, Z-ligustilide, spautin-1, tunicamicina, T-12, y olaparib, regulan la supervivencia/muerte del tumor dependiente de autofagia. Aquí revisamos también las diferentes vías moleculares por las que BRCA1 regula (mayoritariamente de forma negativa) la autofagia, fundamentalmente en cánceres de mama y ovario, y donde el estado rédox celular (ROS, GSH) y las proteínas mTOR, p53-Mdm2, STAT3, y Parkin, se ha demostrado que tienen un papel esencial

The BRCA1 protein contributes to maintain genomic integrity, through transcriptional regulation of proteins that control the cell cycle and DNA repair or by direct interaction with these proteins. The genetic instability caused by mutations that result in a deficit of BRCA1 activity, confers an increased risk of mainly breast and ovarian cancers. In recent years, it has been shown that autophagy has a dual role in tumor development, and chemical agents such as lucanthone, chloroquine, Z-ligustilide, spautin-1, tunicamycin, T-12, and olaparib, regulate tumor survival/death autophagy-dependent. Here we also review the different molecular pathways by which BRCA1 regulates (mostly negatively) autophagy, mainly in breast and ovarian cancers, and where the cellular redox state (ROS, GSH) and proteins mTOR, p53-Mdm2, STAT3, and Parkin, have been shown to play an essential role

The incidence of mTOR marker in tracheal adenoid cystic carcinoma by immunohistochemical staining.

INTRODUCTION: There is an association between the activation of mammalian target of rapamycin (mTOR) signaling and aggressive tumor growth in multiple forms of cancer,including adenoid cystic carcinoma (ACC). ACCs are uncommon yet a malignant form of neoplasms that arises within the secretory glands. Therefore, the aim of this study was to investigate the increase of mTOR in the ACC tumors in order to survey the possibility of treating these tumors with mTOR inhibitors. MATERIAL AND METHODS: Samples from known cases of the lung and tracheal ACC were retrievedfrom the archives of the pa-thology department of Masih Daneshvari hospital, and immunohistochemical (IHC) staining for mTOR was performed on them. After preparation of the blocks with specific antibodies, tumor cells with cytoplasmic and/or nuclear expression of mTOR were considered as positive cells by applying a specific scoring method introduced in this study. RESULTS: The paraffin blocks of 26 patients were surveyed and the IHC marker of mTOR was positive in the tumors of 10 patients (38.5%). Out of 10 mTOR positive cases, 5 were females and 5 were males. The primary site of the surveyed tumors was the trachea and bronchus in 12 cases (46%), salivary glands in 7 individuals (27%), and lung tissue in 7 cases (27%), and there was no significant correlation between the primary site of the ACC tumors and the existence of the mTOR markers in them (P = 0.67). From all cases, 13 patients (50%) had cribriform and tubular cells without solid components, 9 cases (34.6%) had cribriform and tubular with less than 30% of solid components, and 4 cases (15.4%) had cribriform and tubular cells with more than 30% of solid com-ponents. There was no significant difference between the morphologies and the existence of mTOR markers in them (P = 0.741). CONCLUSIONS: As the incidence of mTOR markers is seen in patients with tracheal ACC, evaluation and scoring of mTOR in these persons can be helpful as further studies can distinguish the use of it in the treatment of the disease. .

mTOR Signaling Combined with Cancer Stem Cell Markers as a Survival Predictor in Stage II Colorectal Cancer.

PURPOSE: Wnt and mammalian target of rapamycin (mTOR) are major molecular signaling pathways associated with the development and progression of tumor, as well as the maintenance and proliferation of cancer stem cells (CSCs), in colorectal cancer (CRC). Identifying patients at risk of poor prognosis is important to determining whether to add adjuvant treatment in stage II CRC and thus improve survival. In the present study, we evaluated the prognostic value of Wnt, mTOR, and CSC markers as survival predictors in stage II CRC. MATERIALS AND METHODS: We identified 148 cases of stage II CRC and acquired their tumor tissue. Tissue microarrays for immunohistochemical staining were constructed, and the expressions of CD166, CD44, EphB2, ß-catenin, pS6 were evaluated using immunohistochemical staining. RESULTS: The expressions of CD166 (p=0.045) and pS6 (p=0.045) and co-expression of pS6/CD166 (p=0.005), pS6/CD44 (p=0.042), and pS6/CD44/CD166 (p=0.013) were negatively correlated with cancer-specific survival. Cox proportional hazard analysis showed the combination of CD166/pS6 [hazard ratio, 9.42; 95% confidence interval, 2.36-37.59; p=0.002] to be the most significant predictor related with decreased cancer-specific survival. In addition, co-expression of CD44/CD166 (p=0.017), CD166/ß-catenin (p=0.036), CD44/ß-catenin (p=0.001), and CD44/CD166/ß-catenin (p=0.001) were significant factors associated with liver metastasis. CONCLUSION: Specific combinations of CSC markers and ß-catenin/mTOR signaling could be a significant predictor of poor survival in stage II CRC.

Distinct Roles of mTOR Targets S6K1 and S6K2 in Breast Cancer.

The mechanistic target of rapamycin (mTOR) is a master regulator of protein translation, metabolism, cell growth and proliferation. It forms two complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). mTORC1 is frequently deregulated in many cancers, including breast cancer, and is an important target for cancer therapy. The immunosuppressant drug rapamycin and its analogs that inhibit mTOR are currently being evaluated for their potential as anti-cancer agents, albeit with limited efficacy. mTORC1 mediates its function via its downstream targets 40S ribosomal S6 kinases (S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). There are two homologs of S6K: S6K1 and S6K2. Most of the earlier studies focused on S6K1 rather than S6K2. Because of their high degree of structural homology, it was generally believed that they behave similarly. Recent studies suggest that while they may share some functions, they may also exhibit distinct or even opposite functions. Both homologs have been implicated in breast cancer, although how they contribute to breast cancer may differ. The purpose of this review article is to compare and contrast the expression, structure, regulation and function of these two S6K homologs in breast cancer.

Spa-RQ: an Image Analysis Tool to Visualise and Quantify Spatial Phenotypes Applied to Non-Small Cell Lung Cancer.

To facilitate analysis of spatial tissue phenotypes, we created an open-source tool package named 'Spa-RQ' for 'Spatial tissue analysis: image Registration & Quantification'. Spa-RQ contains software for image registration (Spa-R) and quantitative analysis of DAB staining overlap (Spa-Q). It provides an easy-to-implement workflow for serial sectioning and staining as an alternative to multiplexed techniques. To demonstrate Spa-RQ's applicability, we analysed the spatial aspects of oncogenic KRAS-related signalling activities in non-small cell lung cancer (NSCLC). Using Spa-R in conjunction with ImageJ/Fiji, we first performed annotation-guided tumour-by-tumour phenotyping using multiple signalling markers. This analysis showed histopathology-selective activation of PI3K/AKT and MAPK signalling in Kras mutant murine tumours, as well as high p38MAPK stress signalling in p53 null murine NSCLC. Subsequently, Spa-RQ was applied to measure the co-activation of MAPK, AKT, and their mutual effector mTOR pathway in individual tumours. Both murine and clinical NSCLC samples could be stratified into 'MAPK/mTOR', 'AKT/mTOR', and 'Null' signature subclasses, suggesting mutually exclusive MAPK and AKT signalling activities. Spa-RQ thus provides a robust and easy to use tool that can be employed to identify spatially-distributed tissue phenotypes.

The mTOR Signaling Pathway Is Associated With the Prognosis of Malignant Pleural Mesothelioma After Multimodality Therapy.

BACKGROUND/AIM: We performed multimodality therapy comprising preoperative chemotherapy, extrapleural pneumonectomy (EPP), and radiation therapy for patients with malignant pleural mesothelioma (MPM). Although multimodality therapy resulted in good prognosis, further improvement is required. Therefore, herein, we analysed the prognostic factors using surgical specimens and searched for suitable molecular targets to improve the prognosis after multidisciplinary treatment. PATIENTS AND METHODS: Forty-six patients with MPM underwent multimodality therapy. Paraffin-embedded surgical samples were used for immunohistochemistry to evaluate the expression of phosphorylated (p-) AKT, extracellular signal-regulated kinase (ERK), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and S6 ribosomal protein (S6RP). RESULTS: On univariate and multivariate analyses, significant differences were observed according to the histological type, pathological stage, and p-mTOR expression rate. CONCLUSION: The prognosis of MPM is affected by p-mTOR expression, suggesting that molecular-targeted treatment might be used during multimodal therapy for MPM.

mTOR-mediated podocyte hypertrophy regulates glomerular integrity in mice and humans.

The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin-mediated (mTOR-mediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation-related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis - all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.

Effect of lncRNA HULC knockdown on rat secreting pituitary adenoma GH3 cells.

Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.

Prevention of kidney cell damage in hyperglycaemia condition by adiponectin.

Adiponectin (APN) is an adipocytokine, secreted from adipose tissue and has anti-inflammatory, anti-ageing, and antidiabetic properties. Hyperglycaemia can damage the renal cells, and mammalian target of rapamycin (mTOR), along with Sirtuin 1 (SIRT1), have an important role in kidney cell response to hyperglycaemia. Therefore, understanding the relationship between adiponectin, mTOR, and SIRT1 proteins is beneficial for deciphering the mechanism of adiponectin function. In this study, Human Embryonic Kidney-293 (HEK-293) cells were cultured under normal and high-glucose condition, with and without APN (1, 10, and 100 ng/mL) for 48 hours. mTOR protein expression was evaluated by western blot analysis, and SIRT1 protein was assessed using ELISA method. To evaluate hyperglycaemia-mediated cytotoxicity, cell viability was determined using MTT assay. Data showed that APN in high dose (100 ng/mL) significantly reduced the expression of mTOR and p-mTOR, increased SIRT1 protein, and also improved cell viability compared with the control high glucose (p ≤ 0.05). According to this results, APN can be useful in preventing renal cell damage, by affecting on the expression of mTOR and SIRT1 proteins, as well as increasing the survival of kidney cells in hyperglycaemia conditions. SIGNIFICANCE OF THE STUDY: Adiponectin triggered mTOR/p-mTOR/SIRT1 pathway and decreased cell death in human kidney cells. Our findings provide preliminary experimental data that support further studies on the potential therapeutic role of adiponectin in diabetes and diabetic-induced metabolic complications.

Roles of Autophagy and Protein Kinase C-epsilon in Lipid Metabolism of Nonalcoholic Fatty Liver Cell Models.

OBJECTIVE: To investigate interaction between autophagy and PKC-ε in lipid metabolism of NAFLD cell models. METHODS: HL-7702 cells and SK-HEP-1 cells were cultured in vitro as NAFLD cell models and treated with RAPA to induce autophagy. 3-MA was used to inhibit cell autophagy. And HL-7702 and SK-HEP-1 cell were ordinary cultured as control groups. Cell viability was determined by MTT colorimetric assay. The levels of TG, TC and PKC-ε were detected by ELISA. PKC-ε was detected by IF. LC3-II/LC3-I, P62, IRS-1, IRS-2, PI3Kp85, mTOR were detected by Western-blot. SPSS 20 software was used for statistical analysis. RESULTS: The values of PKC-ε were the highest in the steatosis groups (HL-7702 cells were 91.10%, SK-HEP-1 cells were 98.20%). Compared with the steatosis groups, the LC3-II/LC3-I ratio in the induced autophagy groups increased obviously (p <0.05). P62/ß-actin grayscale ratio of the induced autophagy groups decreased significantly compared with the steatosis group (p <0.05). MTOR/ß-actin grayscale ratio in the induced autophagy groups were significantly lower than those in the steatosis groups (p <0.05). PI3Kp85, IRS-1 and IRS-2/ß-actin grayscale ratio of the induced autophagy groups increased significantly compared with the steatosis group (p <0.05). CONCLUSION: Up-regulation of autophagy can promote the elimination of liver fat; while down-regulation can promote lipid accumulation. The expression of PKC-ε is positively related to the degree of hepatic steatosis. PI3K was involved in both autophagy and IR induced by PKC-ε. PKC-ε might participate in hepatocyte autophagy by regulating PI3K.

The low protein diet affects the nonspecific inflammatory response of middle-aged and old mice through mTOR.

OBJECTIVE: To explore the effect of low protein diet on nonspecific inflammatory changes in mice during aging and related mechanisms. MATERIALS AND METHODS: Thirty-two 14-month-old female KM mice were randomly divided into 4 groups: control group, low protein group, high protein group, high protein + rapamycin group. Hematoxylin-eosin (HE) staining was performed to observe the pathological changes of the liver. Immunohistochemistry of liver sections was performed to detect the expression of CD68 protein. HE staining of colon sections was performed to observe intestinal lymphocyte infiltration. The percentage of spleen CD4+ T and CD8+ T cells was detected by flow cytometry. The mTOR expression in the liver was detected by Western blot and immunohistochemistry. RESULTS: Compared with the control group, HE staining of liver tissue sections in high-protein group showed the cytoplasm of hepatocytes was loose and disordered, and the hepatic sinus was significantly expanded. Immunohistochemistry of the liver showed a significant increase in CD68 protein expression. Colorectal HE staining showed extensive lymphocyte infiltration. The number of CD4+ T and CD8+ T cells in spleen flow cytometry was significantly decreased (*p < 0.05). Western blot and immunohistochemistry detected a significant increase in mTOR expression in the liver (*p < 0.05, *p < 0.05). In the High Protein+Rapamycin group and Low-protein group, the time-dependent changes were reduced, the numbers of CD4+ T cell and CD8+ T cell in the spleen were significantly increased (*p < 0.05) and the expression of mTOR was significantly reduced (*p < 0.05). CONCLUSIONS: Low-protein diet is beneficial for delaying the non-specific inflammatory changes of liver and intestines in middle-aged and aged mice, and this effect may be achieved through down-regulation of mTOR.

Induction of ER Stress-Mediated Apoptosis by the Major Component 5,7,4'-Trimethoxyflavone Isolated from Kaempferia parviflora Tea Infusion.

Kaempferia parviflora (KP) is a famous medicinal plant from Thailand, and is a rich source of various kinds of methoxyflavones (MFs). Many kinds of food products such as tea, capsule, and liquor are manufactured from the rhizomes of KP. In this study, KP infusions were prepared with different brewing conditions, and the amounts of three major methoxylflavones, 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF), and 3,5,7,3',4'-pentamethoxyflavone (PMF), were analyzed. The antiproliferative activities of DMF, TMF, and PMF isolated from the brewed tea samples were evaluated. TMF was discovered to be significantly effective at inhibiting proliferation of SNU-16 human gastric cancer cells in a concentration dependent manner. TMF induced apoptosis, as evidenced by increments of sub-G1 phase, DNA fragmentation, annexin-V/PI staining, the Bax/Bcl-xL ratio, proteolytic activation of caspase-3,-7,-8, and degradation of poly (ADP-ribose) polymerase (PARP) protein. Furthermore, it was found that TMF induced apoptosis via ER stress, verified by an increase in the level of C/EBP homologous protein (CHOP), glucose regulated protein 78 (GRP78), inositol-requiring enzyme 1 α (IRE1α), activating transcription factor-4 (ATF-4), and the splice isoform of X-box-binding protein-1 (XBP-1) mRNA.

mTOR-Related Brain Dysfunctions in Neuropsychiatric Disorders.

The mammalian target of rapamycin (mTOR) is an ubiquitously expressed serine-threonine kinase, which senses and integrates several intracellular and environmental cues to orchestrate major processes such as cell growth and metabolism. Altered mTOR signalling is associated with brain malformation and neurological disorders. Emerging evidence indicates that even subtle defects in the mTOR pathway may produce severe effects, which are evident as neurological and psychiatric disorders. On the other hand, administration of mTOR inhibitors may be beneficial for a variety of neuropsychiatric alterations encompassing neurodegeneration, brain tumors, brain ischemia, epilepsy, autism, mood disorders, drugs of abuse, and schizophrenia. mTOR has been widely implicated in synaptic plasticity and autophagy activation. This review addresses the role of mTOR-dependent autophagy dysfunction in a variety of neuropsychiatric disorders, to focus mainly on psychiatric syndromes including schizophrenia and drug addiction. For instance, amphetamines-induced addiction fairly overlaps with some neuropsychiatric disorders including neurodegeneration and schizophrenia. For this reason, in the present review, a special emphasis is placed on the role of mTOR on methamphetamine-induced brain alterations.

Comparación del perfil de expresión de la vía PI3K-AKT-mTOR en muestras de tejido tumoral y no tumoral de pacientes sometidos a trasplante cardiaco / Comparison of the PI3K-AKT-mTOR pathway expression profile in tumoral versus nontumoral tissue samples from heart transplant recipients

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Evaluation of miRNAs expression in medullary thyroid carcinoma tissue samples: miR-34a and miR-144 as promising overexpressed markers in MTC.

Medullary thyroid carcinoma (MTC) is a rare neoplasia derived from neural parafollicular C cells. MicroRNAs (miRNAs) are small regulatory RNAs with essential roles in the biology of cancers such as MTC and can be applied as diagnostic markers. According to previous studies, miR-144 and miR-34 and their two oncogenes target, mammalian target of rapamycin (mTOR) and AXL receptor tyrosine kinase (AXL), were selected for further investigations in our study. Thirty MTC samples as well as thirty adjacent normal thyroid tissues were applied in this study including 28 formalin-fixed, paraffin-embedded (FFPE) and 2 fresh-frozen MTC samples. RNA extraction and complementary DNA (cDNA) synthesis were performed for all samples. After primer pairs and probes were designed, real-time polymerase chain reaction (real-time PCR) method was used, and the results were analyzed using 2-ΔΔCt method. Receiver operating characteristic (ROC) curve analysis was applied to assess the diagnostic value of the two miRNAs. AXL protein level was measured in all clinical samples using enzyme-linked immunosorbent assay (ELISA) method. Both miRNAs were up-regulated in all clinical samples compared to the normal tissues. AXL was up-regulated in most clinical samples while mTOR was down-regulated in most samples. Furthermore, the level of AXL protein increased. ROC curve analysis demonstrated that increased expression of miR-34a and miR-144 in MTC patients had significant predictive value. The results demonstrated that high expression of miR-144 and miR-34a can be considered as biomarkers of MTC. However, there was no statistically significant correlation between the expression of these miRNAs and target genes in MTC clinical samples.

Immunohistological features related to functional impairment in lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a low-grade neoplasm characterized by the pulmonary infiltration of smooth muscle-like cells (LAM cells) and cystic destruction. Patients usually present with airway obstruction in pulmonary function tests (PFTs). Previous studies have shown correlations among histological parameters, lung function abnormalities and prognosis in LAM. We investigated the lung tissue expression of proteins related to the mTOR pathway, angiogenesis and enzymatic activity and its correlation with functional parameters in LAM patients. METHODS: We analyzed morphological and functional parameters of thirty-three patients. Two groups of disease severity were identified according to FEV1 values. Lung tissue from open biopsies or lung transplants was immunostained for SMA, HMB-45, mTOR, VEGF-D, MMP-9 and D2-40. Density of cysts, density of nodules and protein expression were measured by image analysis and correlated with PFT parameters. RESULTS: There was no difference in the expression of D2-40 between the more severe and the less severe groups. All other immunohistological parameters showed significantly higher values in the more severe group (p ≤ 0.002). The expression of VEGF-D, MMP-9 and mTOR in LAM cells was associated with the density of both cysts and nodules. The density of cysts and nodules as well as the expression of MMP-9 and VEGF-D were associated with the impairment of PFT parameters. CONCLUSIONS: Severe LAM represents an active phase of the disease with high expression of VEGF-D, mTOR, and MMP-9, as well as LAM cell infiltration. Our findings suggest that the tissue expression levels of VEGF-D and MMP-9 are important parameters associated with the loss of pulmonary function and could be considered as potential severity markers in open lung biopsies of LAM patients.

Ketogenic diet enhances neurovascular function with altered gut microbiome in young healthy mice.

Neurovascular integrity, including cerebral blood flow (CBF) and blood-brain barrier (BBB) function, plays a major role in determining cognitive capability. Recent studies suggest that neurovascular integrity could be regulated by the gut microbiome. The purpose of the study was to identify if ketogenic diet (KD) intervention would alter gut microbiome and enhance neurovascular functions, and thus reduce risk for neurodegeneration in young healthy mice (12-14 weeks old). Here we show that with 16 weeks of KD, mice had significant increases in CBF and P-glycoprotein transports on BBB to facilitate clearance of amyloid-beta, a hallmark of Alzheimer's disease (AD). These neurovascular enhancements were associated with reduced mechanistic target of rapamycin (mTOR) and increased endothelial nitric oxide synthase (eNOS) protein expressions. KD also increased the relative abundance of putatively beneficial gut microbiota (Akkermansia muciniphila and Lactobacillus), and reduced that of putatively pro-inflammatory taxa (Desulfovibrio and Turicibacter). We also observed that KD reduced blood glucose levels and body weight, and increased blood ketone levels, which might be associated with gut microbiome alteration. Our findings suggest that KD intervention started in the early stage may enhance brain vascular function, increase beneficial gut microbiota, improve metabolic profile, and reduce risk for AD.