Phenotypes of Metabolic Dysfunction-Associated Steatotic Liver Disease-Associated Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) typically develops as a consequence of liver cirrhosis, but HCC epidemiology has evolved drastically in recent years. Metabolic dysfunction-associated steatotic liver disease (MASLD), including metabolic dysfunction-associated steatohepatitis, has emerged as the most common chronic liver disease worldwide and a leading cause of HCC. A substantial proportion of MASLD-associated HCC (MASLD-HCC) also can develop in patients without cirrhosis. The specific pathways that trigger carcinogenesis in this context are not elucidated completely, and recommendations for HCC surveillance in MASLD patients are challenging. In the era of precision medicine, it is critical to understand the processes that define the profiles of patients at increased risk of HCC in the MASLD setting, including cardiometabolic risk factors and the molecular targets that could be tackled effectively. Ideally, defining categories that encompass key pathophysiological features, associated with tailored diagnostic and treatment strategies, should facilitate the identification of specific MASLD-HCC phenotypes. In this review, we discuss MASLD-HCC, including its epidemiology and health care burden, the mechanistic data promoting MASLD, metabolic dysfunction-associated steatohepatitis, and MASLD-HCC. Its natural history, prognosis, and treatment are addressed specifically, as the role of metabolic phenotypes of MASLD-HCC as a potential strategy for risk stratification. The challenges in identifying high-risk patients and screening strategies also are discussed, as well as the potential approaches for MASLD-HCC prevention and treatment.

An Immune Gene Expression Signature Associated With Development of Human Hepatocellular Carcinoma Identifies Mice That Respond to Chemopreventive Agents.

BACKGROUND & AIMS: Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis. METHODS: We analyzed gene expression profiles of nontumor liver tissues from 392 patients with early-stage HCC (training set, N = 167 and validation set, N = 225) and liver tissue from patients with cirrhosis without HCC (N = 216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up, 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of diethylnitrosamine (DEN) followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then orally given the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, orally given aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet. RESULTS: We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of nontumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor ß signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% confidence interval, 1.21-4.80). Mice with chemically induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel down-regulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle. CONCLUSIONS: We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that is associated with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and development of fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis.

G protein-membrane interactions I: Gαi1 myristoyl and palmitoyl modifications in protein-lipid interactions and its implications in membrane microdomain localization.

G proteins are fundamental elements in signal transduction involved in key cell responses, and their interactions with cell membrane lipids are critical events whose nature is not fully understood. Here, we have studied how the presence of myristic and palmitic acid moieties affects the interaction of the Gαi1 protein with model and biological membranes. For this purpose, we quantified the binding of purified Gαi1 protein and Gαi1 protein acylation mutants to model membranes, with lipid compositions that resemble different membrane microdomains. We observed that myristic and palmitic acids not only act as membrane anchors but also regulate Gαi1 subunit interaction with lipids characteristics of certain membrane microdomains. Thus, when the Gαi1 subunit contains both fatty acids it prefers raft-like lamellar membranes, with a high sphingomyelin and cholesterol content and little phosphatidylserine and phosphatidylethanolamine. By contrast, the myristoylated and non-palmitoylated Gαi1 subunit prefers other types of ordered lipid microdomains with higher phosphatidylserine content. These results in part explain the mobility of Gαi1 protein upon reversible palmitoylation to meet one or another type of signaling protein partner. These results also serve as an example of how membrane lipid alterations can change membrane signaling or how membrane lipid therapy can regulate the cell's physiology.

Optimized Protocol To Analyze Changes in the Lipidome of Xenografts after Treatment with 2-Hydroxyoleic Acid.

Xenografts are a popular model for the study of the action of new antitumor drugs. However, xenografts are highly heterogeneous structures, and therefore it is sometimes difficult to evaluate the effects of the compounds on tumor metabolism. In this context, imaging mass spectrometry (IMS) may yield the required information, due to its inherent characteristics of sensitivity and spatial resolution. To the best of our knowledge, there is still no clear analysis protocol to properly evaluate the changes between samples due to the treatment. Here we present a protocol for the evaluation of the effect of 2-hydroxyoleic acid (2-OHOA), an antitumor compound, on xenografts lipidome based on IMS. Direct treated/control comparison did not show conclusive results. As we will demonstrate, a more sophisticated protocol was required to evaluate these changes including the following: (1) identification of different areas in the xenograft, (2) classification of these areas (necrotic/viable) to compare similar types of tissues, (3) suppression of the effect of the variation of adduct formation between samples, and (4) normalization of the variables using the standard deviation to eliminate the excessive impact of the stronger peaks in the statistical analysis. In this way, the 36 lipid species that experienced the largest changes between treated and control were identified. Furthermore, incorporation of 2-hydroxyoleic acid to a sphinganine base was also confirmed by MS/MS. Comparison of the changes observed here with previous results obtained with different techniques demonstrates the validity of the protocol.

2-Hydroxyoleate, a nontoxic membrane binding anticancer drug, induces glioma cell differentiation and autophagy.

Despite recent advances in the development of new cancer therapies, the treatment options for glioma remain limited, and the survival rate of patients has changed little over the past three decades. Here, we show that 2-hydroxyoleic acid (2OHOA) induces differentiation and autophagy of human glioma cells. Compared to the current reference drug for this condition, temozolomide (TMZ), 2OHOA combated glioma more efficiently and, unlike TMZ, tumor relapse was not observed following 2OHOA treatment. The novel mechanism of action of 2OHOA is associated with important changes in membrane-lipid composition, primarily a recovery of sphingomyelin (SM) levels, which is markedly low in glioma cells before treatment. Parallel to membrane-lipid regulation, treatment with 2OHOA induced a dramatic translocation of Ras from the membrane to the cytoplasm, which inhibited the MAP kinase pathway, reduced activity of the PI3K/Akt pathway, and downregulated Cyclin D-CDK4/6 proteins followed by hypophosphorylation of the retinoblastoma protein (RB). These regulatory effects were associated with induction of glioma cell differentiation into mature glial cells followed by autophagic cell death. Given its high efficacy, low toxicity, ease of oral administration, and good distribution to the brain, 2OHOA constitutes a new and potentially valuable therapeutic tool for glioma patients.

The role of membrane fatty acid remodeling in the antitumor mechanism of action of 2-hydroxyoleic acid.

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is a potent antitumor drug that we rationally designed to regulate the membrane lipid composition and structure. The lipid modifications caused by 2OHOA treatments induce important signaling changes that end up with cell death (Terés et al., 2012 [1]). One of these regulatory effects is restoration of sphingomyelin levels, which are markedly lower in cancer cells compared to normal cells (Barceló-Coblijn et al., 2011 [2]). In this study, we report another important regulatory effect of 2OHOA on cancer cell membrane composition: a large increase in 2OHOA levels, accounting for ~15% of the fatty acids present in membrane phospholipids, in human glioma (SF767 and U118) and lung cancer (A549) cells. Concomitantly, we observed marked reductions in oleic acid levels and inhibition of stearoyl-CoA desaturase. The impact of these changes on the biophysical properties of the lipid bilayer was evaluated in liposomes reconstituted from cancer cell membrane lipid extracts. Thus, 2OHOA increased the packing of ordered domains and decreased the global order of the membrane. The present results further support and extend the knowledge about the mechanism of action for 2OHOA, based on the regulation of the membrane lipid composition and structure and subsequent modulation of membrane protein-associated signaling.

Hepatitis-C-Related Hepatocellular Carcinoma, Still a Relevant Etiology beyond a Hepatitis C Infection Cure.

BACKGROUND: In the past decades, global changes, including hepatitis B vaccination, hepatitis B and C antiviral therapies, and the increasing prevalence of steatotic liver disease, have influenced the landscape of liver cancer etiologies. METHODS: We performed a retrospective study focused on the etiological factors of de novo hepatocellular carcinoma (HCC) diagnoses in an academic center between 2019 and 2022. RESULTS: Among 352 consecutive patients with HCC, alcohol-related liver disease was the predominant etiology (33.3%), followed by hepatitis C (HCV) infection (30.7%). Significant associations were found between HCC etiology and patient demographics, BCLC stage at diagnosis, and cirrhosis prevalence. CONCLUSIONS: Whereas accessibility to antiviral therapy is granted, HCV infection remains as one of the main HCC etiologies. MASLD-related HCC, although growing globally, is not as relevant in our area. Strong public policies need to be implemented to prevent alcohol consumption, the main etiology of liver disease and liver cancer.

Pivotal role of dihydrofolate reductase knockdown in the anticancer activity of 2-hydroxyoleic acid.

alpha-Hydroxy-9-cis-octadecenoic acid, a synthetic fatty acid that modifies the composition and structure of lipid membranes. 2-Hydroxyoleic acid (HOA) generated interest due to its potent, yet nontoxic, anticancer activity. It induces cell cycle arrest in human lung cancer (A549) cells and apoptosis in human leukemia (Jurkat) cells. These two pathways may explain how HOA induces regression of a variety of cancers. We showed that HOA repressed the expression of dihydrofolate reductase (DHFR), the enzyme responsible for tetrahydrofolate (THF) synthesis. Folinic acid, which readily produces THF without the participation of DHFR, reverses the antitumor effects of HOA in A549 and Jurkat cells, as well as the inhibitory influence on cyclin D and cdk2 in A549 cells, and on DNA and PARP degradation in Jurkat cells. This effect was very specific, because either elaidic acid (an analog of HOA) or other lipids, failed to alter A549 or Jurkat cell growth. THF is a cofactor necessary for DNA synthesis. Thus, impairment of DNA synthesis appears to be a common mechanism involved in the different responses elicited by cancer cells following treatment with HOA, namely cell cycle arrest or apoptosis. Compared with other antifolates, such as methotrexate, HOA did not directly inhibit DHFR but rather, it repressed its expression, a mode of action that offers certain therapeutic advantages. These results not only demonstrate the effect of a fatty acid on the expression of DHFR, but also emphasize the potential of HOA to be used as a wide-spectrum drug against cancer.

Ultra Deep Sequencing of Circulating Cell-Free DNA as a Potential Tool for Hepatocellular Carcinoma Management.

BACKGROUND: Cell-free DNA (cfDNA) concentrations have been described to be inversely correlated with prognosis in cancer. Mutations in HCC-associated driver genes in cfDNA have been reported, but their relation with patient's outcome has not been described. Our aim was to elucidate whether mutations found in cfDNA could be representative from those present in HCC tissue, providing the rationale to use the cfDNA to monitor HCC. METHODS: Tumoral tissue, paired nontumor adjacent tissue and blood samples were collected from 30 HCC patients undergoing curative therapies. Deep sequencing targeting HCC driver genes was performed. RESULTS: Patients with more than 2 ng/µL of cfDNA at diagnosis had higher mortality (mean OS 24.6 vs. 31.87 months, p = 0.01) (AUC = 0.782). Subjects who died during follow-up, had a significantly higher number of mutated genes (p = 0.015) and number of mutations (p = 0.015) on cfDNA. Number of mutated genes (p = 0.001), detected mutations (p = 0.001) in cfDNA and ratio (number of mutations/cfDNA) (p = 0.003) were significantly associated with recurrence. However, patients with a ratio (number of mutations/cfDNA) above 6 (long-rank p = 0.0003) presented a higher risk of recurrence than those with a ratio under 6. Detection of more than four mutations in cfDNA correlated with higher risk of death (long-rank p = 0.042). CONCLUSIONS: In summary, cfDNA and detection of prevalent HCC mutations could have prognostic implications in early-stage HCC patients.

Clinical course and seroprevalence of COVID-19 in children with rheumatic diseases-cross-sectional study from a reference centre in Spain.

SARS-CoV-2 infections in children are frequently asymptomatic or mild and can go unnoticed. This study aimed to describe the seroprevalence and clinical course of SARS-CoV-2 in a cohort of children with rheumatic diseases in a real-life setting and assess possible risk factors. A cross-sectional study was performed in a paediatric rheumatology unit (September 2020 to February 2021). At inclusion, a specific questionnaire was completed and SARS-CoV-2 serology was performed. Demographics, treatment and disease activity of patients with and without laboratory-confirmed SARS-CoV-2 infection were compared. A total of 105 children were included. SARS-CoV-2 infection was demonstrated in 27 patients (25.7%). The mean age was 11.8 years, and most patients were females (72.4%). The most frequent underlying condition was juvenile idiopathic arthritis (70.3%; 19/27). Patients received immunosuppressive treatment in 78% of cases (21/27). Overall, 44.4% (12/27) of infected patients were asymptomatic. A total of 66.7% (18/27) of patients did not require medical assistance. Three patients required hospital admission because of COVID-19. Children with confirmed SARS-CoV-2 infection were less frequently in remission (52% vs 72%; p 0.014). Moderate disease activity and treatment with oral corticosteroids were associated with higher risk for SARS-CoV-2 (OR 5.05; CI 95%: 1.56-16.3 and OR 4.2; CI 95%: 1.26-13.9, respectively). In a cohort of Spanish paediatric patients with rheumatic diseases, clinical course of COVID-19 was mild, with more than one third of asymptomatic cases. Higher disease activity and oral corticosteroids appear to be risk factors for SARS-CoV-2 infection. Key Points • We aimed to investigate the seroprevalence of SARS-CoV-2 infection in a cohort of Spanish paediatric patients with RD, testing both symptomatic and asymptomatic patients. We also compared treatment and disease activity of patients with and without laboratory-confirmed SARS-CoV-2 infection. • In our cohort of 105 paediatric patients with rheumatic diseases, the clinical course of COVID-19 was mild and 44% of cases were asymptomatic. Three cases required hospital admission with no complications. Seroprevalence was 20%. • No association was found between disease activity or treatment with corticosteroids and symptomatic or asymptomatic infection. Higher disease activity and treatment with oral corticosteroids appeared to be risk factors for laboratory-confirmed SARS-CoV-2 infection.

Nathan H. Azrin: A Case Study in Research Translation in Behavior Science.

Nathan H. Azrin (1930-2013) contributed extensively to the fields of experimental and applied behavior analysis. His creative and prolific research programs covered a wide range of experimental and applied areas that resulted in 160 articles and several books published over a period of almost 6 decades. As a result, his career illustrates an unparalleled example of translational work in behavior analysis, which has had a major impact not only within our field, but across disciplines and outside academia. In the current article we present a summary of Azrin's wide ranging contributions in the areas of punishment, behavioral engineering, conditioned reinforcement and token economies, feeding disorders, toilet training, overcorrection, habit disorders, in-class behavior, job finding, marital therapy, and substance abuse. In addition, we use scientometric evidence to gain an insight on Azrin's general approach to treatment evaluation and programmatic research. The analysis of Azrin's approach to research, we believe, holds important lessons to behavior analysts today with an interest in the applied and translational sectors of our science. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40614-020-00278-4.

ARMCX3 Mediates Susceptibility to Hepatic Tumorigenesis Promoted by Dietary Lipotoxicity.

ARMCX3 is encoded by a member of the Armcx gene family and is known to be involved in nervous system development and function. We found that ARMCX3 is markedly upregulated in mouse liver in response to high lipid availability, and that hepatic ARMCX3 is upregulated in patients with NAFLD and hepatocellular carcinoma (HCC). Mice were subjected to ARMCX3 invalidation (inducible ARMCX3 knockout) and then exposed to a high-fat diet and diethylnitrosamine-induced hepatocarcinogenesis. The effects of experimental ARMCX3 knockdown or overexpression in HCC cell lines were also analyzed. ARMCX3 invalidation protected mice against high-fat-diet-induced NAFLD and chemically induced hepatocarcinogenesis. ARMCX3 invalidation promoted apoptotic cell death and macrophage infiltration in livers of diethylnitrosamine-treated mice maintained on a high-fat diet. ARMCX3 downregulation reduced the viability, clonality and migration of HCC cell lines, whereas ARMCX3 overexpression caused the reciprocal effects. SOX9 was found to mediate the effects of ARMCX3 in hepatic cells, with the SOX9 interaction required for the effects of ARMCX3 on hepatic cell proliferation. In conclusion, ARMCX3 is identified as a novel molecular actor in liver physiopathology and carcinogenesis. ARMCX3 downregulation appears to protect against hepatocarcinogenesis, especially under conditions of high dietary lipid-mediated hepatic insult.

Minerval induces apoptosis in Jurkat and other cancer cells.

Minerval is an oleic acid synthetic analogue that impairs lung cancer (A549) cell proliferation upon modulation of the plasma membrane lipid structure and subsequent regulation of protein kinase C localization and activity. However, this mechanism does not fully explain the regression of tumours induced by this drug in animal models of cancer. Here we show that Minerval also induced apoptosis in Jurkat T-lymphoblastic leukaemia and other cancer cells. Minerval inhibited proliferation of Jurkat cells, concomitant with a decrease of cyclin D3 and cdk2 (cyclin-dependent kinase2). In addition, the changes that induced on Jurkat cell membrane organization caused clustering (capping) of the death receptor Fas (CD95), caspase-8 activation and initiation of the extrinsic apoptosis pathway, which finally resulted in programmed cell death. The present results suggest that the intrinsic pathway (associated with caspase-9 function) was activated downstream by caspase-8. In a xenograft model of human leukaemia, Minerval also inhibited tumour progression and induced tumour cell death. Studies carried out in a wide variety of cancer cell types demonstrated that apoptosis was the main molecular mechanism triggered by Minerval. This is the first report on the pro-apoptotic activity of Minerval, and in part explains the effectiveness of this non-toxic anticancer drug and its wide spectrum against different types of cancer.

The triacylglycerol, hydroxytriolein, inhibits triple negative mammary breast cancer cell proliferation through a mechanism dependent on dihydroceramide and Akt.

The plasma membrane is an attractive target for new anticancer drugs, not least because regulating its lipid structure can control multiple signaling pathways involved in cancer cell proliferation, differentiation and survival. Accordingly, the novel anticancer drug hydroxytriolein (HTO) was designed to interact with and regulate the composition and structure of the membrane, which in turn controls the interaction of amphitropic signaling membrane proteins with the lipid bilayer. Changes in signaling provoked by HTO impair the growth of triple negative breast cancer (TNBC) cells, aggressive breast tumor cells that have a worse prognosis than other types of breast cancers and for which there is as yet no effective targeted therapy. HTO alters the lipid composition and structure of cancer cell membranes, inhibiting the growth of MDA-MB-231 and BT-549 TNBC cells in vitro. Depending on the cellular context, HTO could regulate two pathways involved in TNBC cell proliferation. On the one hand, HTO might stimulate ERK signaling and induce TNBC cell autophagy, while on the other, it could increase dihydroceramide and ceramide production, which would inhibit Akt independently of EGFR activation and provoke cell death. In vivo studies using a model of human TNBC show that HTO and its fatty acid constituent (2-hydroxyoleic acid) impair tumor growth, with no undesired side effects. For these reasons, HTO appears to be a promising anticancer molecule that targets the lipid bilayer (membrane-lipid therapy). By regulating membrane lipids, HTO controls important signaling pathways involved in cancer cell growth, the basis of its pharmacological efficacy and safety.

Erythropoietin is expressed in the human retina and it is highly elevated in the vitreous fluid of patients with diabetic macular edema.

OBJECTIVE: Erythropoietin has been recently found to be increased in the vitreous fluid from ischemic retinal diseases such as proliferative diabetic retinopathy (PDR). The aims of the present study were 1) to measure erythropoietin levels in the vitreous fluid from patients with diabetic macular edema (DME), a condition in which the ischemia is not a predominant event, and 2) to compare erythropoietin mRNA expression between human retinas from nondiabetic and diabetic donors without retinopathy. RESEARCH DESIGN AND METHODS: Vitreous samples from 12 type 2 diabetic patients with DME without significant retinal ischemia and 12 PDR patients were prospectively analyzed. Ten nondiabetic patients with macular holes served as the control group. Erythropoietin was assessed by radioimmunoassay (milliunits per milliliter). Erythropoietin mRNA expression was measured by quantitative real-time RT-PCR analysis in the retina from eight nondiabetic and eight age-matched diabetic donors without diabetic retinopathy RESULTS: Intravitreal erythropoietin concentration was higher in both PDR and DME patients than in nondiabetic control subjects (PDR vs. control subjects: median 302 [range 117-1,850] vs. 30 mU/ml [10-75], P < 0.01; DME vs. control subjects: 430 [41-3,000] vs. 30 mU/ml [10-75], P < 0.01). However, no significant differences were found between DME and PDR patients. Erythropoietin mRNA expression was detected in the human retina, and it was higher in the retina from diabetic than from nondiabetic donors. CONCLUSIONS: As occurs in PDR, intravitreous erythropoietin concentrations are strikingly higher in DME. Erythropoietin is expressed in the human retina, and it is upregulated in diabetic patients even without retinopathy. These findings suggest that other factors apart from ischemia are involved in the overexpression of erythropoietin in diabetic retinopathy.

Assessing Mongolian gerbil emotional behavior: effects of two shock intensities and response-independent shocks during an extended inhibitory-avoidance task.

Despite step-down inhibitory avoidance procedures that have been widely implemented in rats and mice to study learning and emotion phenomena, performance of other species in these tasks has received less attention. The case of the Mongolian gerbil is of relevance considering the discrepancies in the parameters of the step-down protocols implemented, especially the wide range of foot-shock intensities (i.e., 0.4-4.0 mA), and the lack of information on long-term performance, extinction effects, and behavioral patterning during these tasks. Experiment 1 aimed to (a) characterize gerbils' acquisition, extinction, and steady-state performance during a multisession (i.e., extended) step-down protocol adapted for implementation in a commercially-available behavioral package (Video Fear Conditioning System-MED Associates Fairfax, VT, USA), and (b) compare gerbils' performance in this task with two shock intensities - 0.5 vs. 1.0 mA-considered in the low-to-mid range. Results indicated that the 1.0 mA protocol produced more reliable and clear evidence of avoidance learning, extinction, and reacquisition in terms of increments in freezing and on-platform time as well as suppression of platform descent. Experiment 2 aimed to (a) assess whether an alternate protocol consisting of a random delivery of foot shocks could replicate the effects of Experiment 1 and (b) characterize gerbils' exploratory behavior during the step-down task (jumping, digging, rearing, and probing). Random shocks did not reproduce the effects observed with the first protocol. The data also indicated that a change from random to response-dependent shocks affects (a) the length of each visit to the platform, but not the frequency of platform descends or freezing time, and (b) the patterns of exploratory behavior, namely, suppression of digging and rearing, as well as increments in probing and jumping. Overall, the study demonstrated the feasibility of the extended step-down protocol for studying steady performance, extinction, and reacquisition of avoidance behavior in gerbils, which could be easily implemented in a commercially available system. The observation that 1.0 mA shocks produced a clear and consistent avoidance behavior suggests that implementation of higher intensities is unnecessary for reproducing aversive-conditioning effects in this species. The observed patterning of freezing, platform descents, and exploratory responses produced by the change from random to periodic shocks may relate to the active defensive system of the gerbil. Of special interest is the probing behavior, which could be interpreted as risk assessment and has not been reported in other rodent species exposed to step-down and similar tasks.

Identification of Biomarkers of Necrosis in Xenografts Using Imaging Mass Spectrometry.

Xenografts are commonly used to test the effect of new drugs on human cancer. However, because of their heterogeneity, analysis of the results is often controversial. Part of the problem originates in the existence of tumor cells at different metabolic stages: from metastatic to necrotic cells, as it happens in real tumors. Imaging mass spectrometry is an excellent solution for the analysis of the results as it yields detailed information not only on the composition of the tissue but also on the distribution of the biomolecules within the tissue. Here, we use imaging mass spectrometry to determine the distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and their plasmanyl- and plasmenylether derivatives (PC-P/O and PE-P/O) in xenografts of five different tumor cell lines: A-549, NCI-H1975, BX-PC3, HT29, and U-87 MG. The results demonstrate that the necrotic areas showed a higher abundance of Na(+) adducts and of PC-P/O species, whereas a large abundance of PE-P/O species was found in all the xenografts. Thus, the PC/PC-ether and Na(+)/K(+) ratios may highlight the necrotic areas while an increase on the number of PE-ether species may be pointing to the existence of viable tumor tissues. Furthermore, the existence of important changes in the concentration of Na(+) and K(+) adducts between different tissues has to be taken into account while interpreting the imaging mass spectrometry results. Graphical Abstract ᅟ.

Normalization of sphingomyelin levels by 2-hydroxyoleic acid induces autophagic cell death of SF767 cancer cells.

The very high mortality rate of gliomas reflects the unmet therapeutic need associated with this type of brain tumor. We have discovered that the plasma membrane fulfills a critical role in the propagation of tumorigenic signals, whereby changes in membrane lipid content can either activate or silence relevant pathways. We have designed a synthetic fatty acid, 2-hydroxyoleic acid (2OHOA), that specifically activates sphingomyelin synthase (SGMS), thereby modifying the lipid content of cancer cell membranes and restoring lipid levels to those found in normal cells. In reverting, the structure of the membrane by activating SGMS, 2OHOA inhibits the RAS-MAPK pathway, which in turn fails to activate the CCND (Cyclin D)-CDK4/CDK6 and PI3K-AKT1 pathways. The overall result in SF767 cancer cells, a line that is resistant to apoptosis, is the sequential induction of cell cycle arrest, cell differentiation and autophagy. Such effects are not observed in normal cells (MRC-5) and thus, this specific activation of programmed cell death infers greater efficacy and lower toxicity to 2OHOA than that associated with temozolomide (TMZ), the reference drug for the treatment of glioma.

Apolipoprotein A1 is overexpressed in the retina of diabetic patients.

PURPOSE: To compare apolipoprotein A1 (ApoA1) expression in the retina from diabetic and nondiabetic donors. DESIGN: Case-control study. METHODS: Diabetic postmortem eyes (n = 8) were compared with eyes (n = 8) from nondiabetic donors matched by age. Messenger ribonucleic acid (mRNA) of ApoA1 (quantitative reverse-transcriptase polymerase chain reaction) was measured separately in the neuroretina and retinal pigment epithelium (RPE). ApoA1 was assessed by immunofluorescence (confocal laser microscopy) and Western blot analysis. The presence of early diabetic retinal damage was evaluated by measuring the rate of apoptosis and glial activation. RESULTS: ApoA1 mRNA levels and ApoA1 immunofluorescence obtained in RPEs and in neuroretinas from diabetic donors were significantly higher than those obtained from nondiabetic donors. ApoA1 was expressed in all retina layers and it was more abundant in RPE than in the neuroretina in both diabetic and nondiabetic donors. In addition, ApoA1 immunofluorescence was significantly higher in all the layers of the neuroretina from diabetic patients. Densitometric analysis of immunoblots showed higher ApoA1 in the retinas from diabetic donors in comparison with nondiabetic donors, but the differences were at significant levels only for the RPE. CONCLUSIONS: ApoA1 overexpression is an early event in the retina of diabetic patients and can be involved in the physiopathology of diabetic retinopathy. In addition, RPE is the main source of ApoA1 within the retina. These findings my be relevant to aiming new treatment strategies toward reducing the development of diabetic retinopathy.