FTO promotes SREBP1c maturation and enhances CIDEC transcription during lipid accumulation in HepG2 cells.

The fat mass and obesity-associated (FTO) gene is tightly related to body weight and fat mass, and plays a pivotal role in regulating lipid accumulation in hepatocytes. However, the mechanisms underlying its function are poorly understood. Sterol regulatory element binding protein-1c (SREBP1c) is a transcription factor that regulates lipogenesis. Cell death-inducing DFFA (DNA fragmentation factor-α)-like effector c (CIDEC) plays a crucial role in lipid droplets (LDs) size controlling and lipid accumulation. In this report, we first observed that FTO overexpression in HepG2 cells resulted in an increase of lipogenesis and up-regulation of SREBP1c and CIDEC, two key regulatory factors in lipogenesis. In contrast, FTO knockdown in HepG2 cells resulted in a decrease of lipogenesis and down-regulation of SREBP1c and CIDEC expression. Moreover, SREBP1c knockdown resulted in a decrease of lipogenesis in HepG2 cells with FTO overexpression. In addition, FTO demethylation defect mutant presented less transcription of the key genes, and less nuclear translocation and maturation of SREBP1c. Further investigation demonstrated that overexpression of SREBP1c in HepG2 cells also promoted high CIDEC expression. Luciferase reporter assays showed that SREBP1c significantly stimulated CIDEC gene promoter activity. Finally, CIDEC knockdown reduced SREBP1c-induced lipogenesis. In conclusion, our studies suggest that FTO increased the lipid accumulation in hepatocytes by increasing nuclear translocation of SREBP1c and SREBP1c maturation, thus improving the transcriptional activity of LD-associated protein CIDEC. Our studies may provide new mechanistic insight into nonalcoholic fatty liver disease (NAFLD) mediated by FTO.

The Diagnosis of Urinary Tract Infections Using a Novel At-home Testing Protocol to Enhance Telemedicine: A Retrospective Analysis.

OBJECTIVE: To retrospectively analyze a novel courier-based home urine collection strategy for patients with symptoms of urinary tract infections (UTIs). This model was developed to provide patient care using telehealth during the coronavirus 2019 pandemic. METHODS: We analyzed data from 2206 patients with symptomatic UTIs to investigate the efficacy of a home urine collection protocol. The primary outcome was the impact of home versus office collection. RESULTS: We analyzed the results of 1112 patient samples collected in-office and 1084 patient samples collected at home. There was no difference in the rate of bacterial identification between females in the office and home collection groups. However, males in the office collection group had a higher rate of bacterial identification (p = .002). The turnaround time was significantly faster in the home collection group than the office collection group (4.08 hours shorter, p < 0.0014). Antibiotic use prior to sample collection was significantly higher in the home collection group for both males (p = .0004) and females (p = .004). Changes in antibiotics were significantly higher in the home collection group than in the office collection group for both males (p = .0009) and females (p = .0006). CONCLUSION: Our home collection protocol is a viable method to provide prompt and reliable outpatient care to urology patients suffering from UTIs. Furthermore, this approach resulted in adequate management and quicker turnaround times. Our findings demonstrate the clinical viability of a decentralized healthcare model to treat UTIs.

A blood biomarker test for brain amyloid impacts the clinical evaluation of cognitive impairment.

OBJECTIVE: The objective of this study was to examine clinicians' patient selection and result interpretation of a clinically validated mass spectrometry test measuring amyloid beta and ApoE blood biomarkers combined with patient age (PrecivityAD® blood test) in symptomatic patients evaluated for Alzheimer's disease (AD) or other causes of cognitive decline. METHODS: The Quality Improvement and Clinical Utility PrecivityAD Clinician Survey (QUIP I, ClinicalTrials.gov Identifier: NCT05477056) was a prospective, single-arm cohort study among 366 patients evaluated by neurologists and other cognitive specialists. Participants underwent blood biomarker testing and received an amyloid probability score (APS), indicating the likelihood of a positive result on an amyloid positron emission tomography (PET) scan. The primary study outcomes were appropriateness of patient selection as well as result interpretation associated with PrecivityAD blood testing. RESULTS: A 95% (347/366) concordance rate was noted between clinicians' patient selection and the test's intended use criteria. In the final analysis including these 347 patients (median age 75 years, 56% women), prespecified test result categories incorporated 133 (38%) low APS, 162 (47%) high APS, and 52 (15%) intermediate APS patients. Clinicians' pretest and posttest AD diagnosis probability changed from 58% to 23% in low APS patients and 71% to 89% in high APS patients (p < 0.0001). Anti-AD drug therapy decreased by 46% in low APS patients (p < 0.0001) and increased by 57% in high APS patients (p < 0.0001). INTERPRETATION: These findings demonstrate the clinical utility of the PrecivityAD blood test in clinical care and may have added relevance as new AD therapies are introduced.

Independent study demonstrates amyloid probability score accurately indicates amyloid pathology.

BACKGROUND: The amyloid probability score (APS) is the model read-out of the analytically validated mass spectrometry-based PrecivityAD® blood test that incorporates the plasma Aß42/40 ratio, ApoE proteotype, and age to identify the likelihood of brain amyloid plaques among cognitively impaired individuals being evaluated for Alzheimer's disease. PURPOSE: This study aimed to provide additional independent evidence that the pre-established APS algorithm, along with its cutoff values, discriminates between amyloid positive and negative individuals. METHODS: The diagnostic performance of the PrecivityAD test was analyzed in a cohort of 200 nonrandomly selected Australian Imaging, Biomarker & Lifestyle Flagship Study of Aging (AIBL) study participants, who were either cognitively impaired or healthy controls, and for whom a blood sample and amyloid PET imaging were available. RESULTS: In a subset of the dataset aligned with the Intended Use population (patients aged 60 and older with CDR ≥0.5), the pre-established APS algorithm predicted amyloid PET with a sensitivity of 84.9% (CI: 72.9-92.1%) and specificity of 96% (CI: 80.5-99.3%), exclusive of 13 individuals for whom the test was inconclusive. INTERPRETATION: The study shows individuals with a high APS are more likely than those with a low APS to have abnormal amounts of amyloid plaques and be on an amyloid accumulation trajectory, a dynamic and evolving process characteristic of progressive AD pathology. Exploratory data suggest APS retains its diagnostic performance in healthy individuals, supporting further screening studies in the cognitively unimpaired.

Site-Specific Polyplex on CCR7 Down-Regulation and T Cell Elevation for Lymphatic Metastasis Blocking on Breast Cancer.

Tumor metastasis contributes to high cancer mortality. Tumor cells in lymph nodes (LNs) are difficult to eliminate but underlie uncontrollable systemic metastasis. The CC chemokine receptor 7 (CCR7) is overexpressed in tumor cells and interacts with CC chemokine ligand 21 (CCL21) secreted from LNs, potentiating their lymphatic migration. Here, a site-specific polyplex is developed to block the CCR7-CCL21 signal and kill tumor cells toward LNs, greatly limiting their lymphatic infiltration. A CCR7-targeting small interfering RNA (siCCR7) is condensed by mPEG-poly-(lysine) with chlorin e6 (Ce6) modification (PPLC) to form PPLC/siCCR7. The knockdown of CCR7 by siCCR7 in tumor cells significantly reduced their response on CCL21 and LN tropism. Additionally, photodynamic therapy-mediated immune activation precisely targets and kills tumor cells released from the primary foci before they reaches the LNs, reducing the number of tumor cells entering the LNs. Consequently, the PPLC/siCCR7 polyplexes inhibited up to 92% of lung metastasis in 4T1 tumor bearing mice and reduced tumor cell migration to LNs by up to 80%. This site-specific strategy optimized anti-metastasis efficacy and promotes the clinical translational development of anti-metastatic therapy.

AMG487 inhibits PRRSV replication and ameliorates lung injury in pig lung xenografts by down-regulating the expression of ANXA2.

Porcine reproductive and respiratory syndrome (PRRS) is a pig disease caused by the PRRS virus (PRRSV) that is characterized with diffuse interstitial pneumonia and lung edema. High expressions of chemokine CXCL10 and its receptor CXCR3 are reported in infected porcine lungs. Since CXCR3 is a key player in host inflammatory response, it might be a therapeutic target to treat lung damage caused by PRRSV infection. The size of pigs has long hampered research into molecular mechanisms of PRRS and validating the potential pharmaceutical targets. In this study, a porcine lung xenograft model with PRRSV infection was generated in immunodeficient mice to evaluate the therapeutic effects of the CXCR3 antagonist AMG487 on PRRSV infection-induced lung injury. The porcine lung tissues developed normally two weeks after xeno-transplantation in the mouse kidney capsule. Infection of PRRSV resulted in its efficient replication in the xenografts and histological damage to the porcine lung tissue structure, with no or little effects on mouse lungs. AMG487 administration dramatically reduced the number of PRRSV genome copies and significantly alleviated the porcine lung injury. Furthermore, treatment of AMG487 in cultured porcine macrophages consistently suppressed PRRSV replication with significant downregulation of Annexin A2 (ANXA2), a cellular protein facilitating viral replication. These findings provide a suitable model for evaluating new antiviral therapies as well as a possible therapeutic option for virus infection-induced lung injury.

Concordance Between Antibiotic Resistance Genes and Susceptibility in Symptomatic Urinary Tract Infections.

PURPOSE: Studies have shown that multiple genes influence antibiotic susceptibility, but the relationship between genotypic and phenotypic antibiotic susceptibility is unclear. We sought to analyze the concordance between the presence of antibiotic resistance (ABR) genes and antibiotic susceptibility results in urine samples collected from patients with symptomatic urinary tract infection (UTI). PATIENTS AND METHODS: Urine samples were collected from patients presenting to 37 geographically disparate urology clinics across the United States from July 2018 to February 2019. Multiplex polymerase chain reaction was used to detect 27 ABR genes. In samples containing at least one culturable organism at a concentration of ≥ 104 cells per mL, pooled antibiotic susceptibility testing (P-AST), which involves simultaneous growing all detected bacteria together in the presence of antibiotic and then measure susceptibility, was performed against 14 antibiotics. The concordance rate between the ABR genes and the P-AST results was generated for the overall group. The concordance rates for each antibiotic between monomicrobial and polymicrobial infection were compared using chi-square test. RESULTS: Results from ABR gene detection and P-AST of urine samples from 1155 patients were included in the concordance analysis. Overall, there was a 60% concordance between the presence or absence of ABR genes and corresponding antimicrobial susceptibility with a range of 49-78% across antibiotic classes. Vancomycin, meropenem, and piperacillin/tazobactam showed significantly lower concordance rates in polymicrobial infections than in monomicrobial infections. CONCLUSION: Given the 40% discordance rate, the detection of ABR genes alone may not provide reliable data to make informed clinical decisions in UTI management. However, when used in conjunction with susceptibility testing, ABR gene data can offer valuable clinical information for antibiotic stewardship.

MNK as a potential pharmacological target for suppressing LPS-induced acute lung injury in mice.

Acute lung injury (ALI) or its more severe form, known as acute respiratory distress syndrome (ARDS), is characterized by an initial exudative phase, expression of proinflammatory mediators, activation of inflammatory leukocytes, and impairment of the lung endothelium and epithelium. Despite numerous, novel therapeutic strategies have been developed regarding the pathophysiology of ALI, current treatment is mainly supportive, as specific therapies have not been established in the past few decades. The MAP kinase-interacting kinases (MNK1 and MNK2) are serine threonine kinases which are activated by mitogen-activated protein kinases (MAPKs), regulate protein synthesis by phosphroylating eukaryotic translation initiation factor 4E (eIF4E). Although studies have shown that MAPKs pathway is involved in anti-inflammatory and preventing tissue injury processes, the role of MNKs in ALI has, until now, remained relatively unexplored. Here, we investigated whether partial inhibition of MAPKs pathway by targeting MNKs was effective in the prevention and treatment of ALI. C57BL6 mice were pretreated with MNK1 and MNK2 inhibitor (CGP57380, 30 mg/kg) for 30 min and then challenged with 5 mg/kg LPS for 6 h. The results showed that pretreatment with CGP57380 not only significantly attenuated LPS-induced lung wet/dry ratio, as well as protein content, total cells and neutrophils in bronchoalveolar lavage fluid (BALF), but also decreased the production of pro-inflammatory mediators such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and keratinocyte-derived chemoattractant (KC). In addition, CGP57380 was observed to significantly suppress LPS-stimulated phosphorylation of eIF4E and MAPKs in the mouse bone marrow-derived macrophages (BMDMs). The involvement of MNK2 in lung injury was further evident by MNK2 knockout mice. MNK2 deficiency resulted in the attenuated lung histopathological changes, as also reflected by reductions in neutrophil counts, and the less LPS-induced the production of IL-6, TNF-α and KC in mouse BALF. Taken together, these findings demonstrated for the first time that MNK inhibition could effectively reduce the LPS-induced ALI in mice, suggesting a novel and potential application for MNK-based therapy to treat this serious disease.

International guidelines for the flow cytometric evaluation of peripheral blood for suspected Sézary syndrome or mycosis fungoides: Assay development/optimization, validation, and ongoing quality monitors.

Introducing a sensitive and specific peripheral blood flow cytometric assay for Sézary syndrome and mycosis fungoides (SS/MF) requires careful selection of assay design characteristics, and translation into a laboratory developed assay through development/optimization, validation, and continual quality monitoring. As outlined in a previous article in this series, the recommended design characteristics of this assay include at a minimum, evaluation of CD7, CD3, CD4, CD8, CD26, and CD45, analyzed simultaneously, requiring at least a 6 color flow cytometry system, with both quantitative and qualitative components. This article provides guidance from an international group of cytometry specialists in implementing an assay to those design specifications, outlining specific considerations, and best practices. Key points presented in detail are: (a) Pre-analytic components (reagents, specimen processing, and acquisition) must be optimized to: (i) identify and characterize an abnormal population of T-cells (qualitative component) and (ii) quantitate the abnormal population (semi/quasi-quantitative component). (b)Analytic components (instrument set-up/acquisition/analysis strategy and interpretation) must be optimized for the identification of SS/MF populations, which can vary widely in phenotype. Comparison with expert laboratories is strongly encouraged in order to establish competency. (c) Assay performance must be validated and documented through a validation plan and report, which covers both qualitative and semi/quasi-quantitative assay components (example template provided). (d) Ongoing assay-specific quality monitoring should be performed to ensure consistency.

The tissue specific regulation of miR22 expression in the lung and brain by ribosomal protein L29.

Endogenous miR22 is associated with a diverse range of biological processes through post-translational modification of gene expression and its deregulation results in various diseases including cancer. Its expression is usually tissue or cell-specific, however, the reasons behind this tissue or cell specificity are not clearly outlined till-date. Therefore, our keen interest was to investigate the mechanisms of tissue or cell-specific expression of miR22. In the current study, miR22 expression showed a tissues-specific difference in the poly(I:C) induced inflammatory mouse lung and brain tissues. The cell-specific different expression of miR22 was also observed in inflammatory glial cells and endothelial cells. The pattern of RPL29 expression was also similar to miR22 in these tissues and cells under the same treatment. Interestingly, the knockdown of RPL29 exerted an inhibitory effect on miR22 and its known transcription factors including Fos-B and c-Fos. Fos-B and c-Fos were also differentially expressed in the two cell lines transfected with poly(I:C). The knockdown of c-Fos also exerted its negative effects on miR22 expression in both cells. These findings suggest that RPL29 might have regulatory roles on tissue or cell-specific expression of miR22 through the transcription activities of c-Fos and also possibly through Fos-B.

High-content imaging characterization of cell cycle therapeutics through in vitro and in vivo subpopulation analysis.

Although the cycling of eukaryotic cells has long been a primary focus for cancer therapeutics, recent advances in imaging and data analysis allow even further definition of cellular events as they occur in individual cells and cellular subpopulations in response to treatment. High-content imaging (HCI) has been an effective tool to elucidate cellular responses to a variety of agents; however, these data were most frequently observed as averages of the entire captured population, unnecessarily decreasing the resolution of each assay. Here, we dissect the eukaryotic cell cycle into individual cellular subpopulations using HCI in conjunction with unsupervised K-means clustering. We generate distinct phenotypic fingerprints for each major cell cycle and mitotic compartment and use those fingerprints to screen a library of 310 commercially available chemotherapeutic agents. We determine that the cell cycle arrest phenotypes caused by these agents are similar to, although distinct from, those found in untreated cells and that these distinctions frequently suggest the mechanism of action. We then show via subpopulation analysis that these arrest phenotypes are similar in both mouse models and in culture. HCI analysis of cell cycle using data obtained from individual cells under a broad range of research conditions and grouped into cellular subpopulations represents a powerful method to discern both cellular events and treatment effects. In particular, this technique allows for a more accurate means of assessing compound selectivity and leads to more meaningful comparisons between so-called targeted therapeutics.

Higher Expression Levels of Aquaporin Family of Proteins in the Kidneys of Arid-Desert Living Lepus yarkandensis.

Lepus yarkandensis specifically lives in arid climate with rare precipitation of Tarim Basin in western China. Aquaporins (AQPs) are a family of channel proteins that facilitate water transportation across cell membranes. Kidney AQPs play vital roles in renal tubule water permeability and maintenance of body water homeostasis. This study aimed to investigate whether kidney AQPs exhibit higher expression in arid-desert living animals. Immunohistochemistry results revealed localization of AQP1 to the capillary endothelial cells in glomerulus and epithelial cells in proximal tubule and descending thin limbs, AQP2 to the apical plasma membrane of principal cells in the cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and IMCD cells in the initial inner medullary collecting duct (IMCD1) and middle IMCD (IMCD2), and AQP3 and AQP4 to the basolateral plasma membrane of principal cells and IMCD cells in CCD, OMCD, IMCD1, and IMCD2 in L. yarkandensis kidneys. Quantitative real-time PCR analysis showed higher mRNA levels of AQP1, AQP2, AQP3, and AQP4 in L. yarkandensis kidneys compared with Oryctolagus cuniculus. Similar results were obtained by western blotting. Our results suggested that higher expression levels of AQP1, AQP2, AQP3, and AQP4 in L. yarkandensis kidneys favored for drawing more water from the tubular fluid.

Well-developed ligand-binding assays demonstrate robust performance using singlet analysis.

Aim: Replicate sample testing has long been regarded as a necessity for bioanalytical laboratory testing, especially in the realm of ligand-binding assays (LBAs). In an era in which results were derived from crude test tube-based assays, the replication of results was warranted. Those assays were often imprecise and required multiple replicates to arrive at results that approached accuracy. However, given technological advancements and excellent accuracy and precision of many modern LBAs, the practice of replicate testing should be re-evaluated. Although most regulatory guidelines allow for singlet testing when sufficient robustness and precision are demonstrated during validation, duplicate testing is still common practice. Recently however, several articles have been published that support singlet analysis for LBAs performed on a platform with automated liquid handling. Results: Data from five pharmacokinetic assay validations and five clinical and preclinical studies originally run in duplicate were re-evaluated in singlet and found to be nearly identical to the original duplicate results. Conclusion: We confirm that well-developed LBAs produce comparable data whether evaluated in singlet or duplicate. Additionally, automation is not requisite for singlet testing.

Improving The Oral Absorption Of Nintedanib By A Self-Microemulsion Drug Delivery System: Preparation And In Vitro/In Vivo Evaluation.

OBJECTIVE: Nintedanib (NDNB) is a triple receptor tyrosine kinase inhibitor with poor solubility in neutral conditions and low bioavailability. A self-microemulsifying drug delivery system (SMEDDS) of NDNB was developed to improve drug solubility in physical conditions and absorption in vivo. METHODS: The NDNB-SMEDDS formulation was optimized via pseudo-ternary phase diagrams. The physicochemical properties of NDNB-SMEDDS, viz., morphological observation, droplet size, stability, compatibility and in vitro release were investigated. The permeability of NDNB-SMEDDS was detected using both a Caco-2 cell monolayer in vitro and an intestinal perfusion study in vivo. Furthermore, the pharmacokinetic characteristics of NDNB-SMEDDS were evaluated. RESULTS: The optimal formulation was composed of MCT as an oil phase, RH 40 as a surfactant and ethylene glycol as a co-surfactant. The average droplet size of the microemulsion was about 23 nm with good stability within 30 days. The formulation did not exhibit any obvious cytotoxic effect on Caco-2 cells. Permeability of nintedanib in a Caco-2 cell monolayer was enhanced by 2.8-fold upon incorporation in SMEDDS compared with the drug solution. The intestinal perfusion study demonstrated that the P app of NDNB-SMEDDS increased by 3.0-fold in the entire intestine and 3.2-fold in the colon in comparison with the drug solution. The pharmacokinetics study showed that the AUC of the NDNB-SMEDDS increased significantly. CONCLUSION: This study showed that the self-microemulsion formulations could improve the absorption of nintedanib, and can thus serve as a promising carrier for the oral delivery of nintedanib.

AMPK promotes the survival of colorectal cancer stem cells.

BACKGROUND: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females worldwide in 2012. In the past 20 years, strong evidence suggests that cancer stem cells are the main culprit of cancer metastasis, chemotherapy resistance, and relapse. METHODS: To further understand the unique biological properties of cancer stem cells and uncover novel molecular targets to eradicate them, we first established a panel of patient-derived xenograft (PDX) tumor models using tumors surgically removed from human colorectal cancer patients. We then isolated CRC cancer stem cells based on their ALDH activity using fluorescent-activated cell sorting (FACS) and characterized their metabolic properties. RESULTS: Interestingly, we found that the CRC cancer stem cells (ie, CRC cells with higher ALDH activity, or ALDH+) express higher level of antioxidant genes and have lower level of reactive oxygen species (ROS) than non-CRC cancer stem cells (ie, CRC cells with lower ALDH activity, or ALDH-). The CRC cancer stem cells also possess more mitochondria mass and show higher mitochondrial activity. More intriguingly, we observed higher AMP-activated protein kinase (AMPK) activities in these CRC cancer stem cells. Inhibition of the AMPK activity using 2 AMPK inhibitors, Compound C and Iodotubercidin, preferentially induces cell death in CRC cancer stem cells. CONCLUSION: We propose that AMPK inhibitors may help to eradicate the CRC cancer stem cells and prevent the relapse of CRCs.

Modification of Kolmogorov-Smirnov test for DNA content data analysis through distribution alignment.

The Kolmogorov-Smirnov (K-S) test is a statistical method often used for comparing two distributions. In high-throughput screening (HTS) studies, such distributions usually arise from the phenotype of independent cell populations. However, the K-S test has been criticized for being overly sensitive in applications, and it often detects a statistically significant difference that is not biologically meaningful. One major reason is that there is a common phenomenon in HTS studies that systematic drifting exists among the distributions due to reasons such as instrument variation, plate edge effect, accidental difference in sample handling, etc. In particular, in high-content cellular imaging experiments, the location shift could be dramatic since some compounds themselves are fluorescent. This oversensitivity of the K-S test is particularly overpowered in cellular assays where the sample sizes are very big (usually several thousands). In this paper, a modified K-S test is proposed to deal with the nonspecific location-shift problem in HTS studies. Specifically, we propose that the distributions are "normalized" by density curve alignment before the K-S test is conducted. In applications to simulation data and real experimental data, the results show that the proposed method has improved specificity.

Analysis of pathway activity in primary tumors and NCI60 cell lines using gene expression profiling data.

To determine cancer pathway activities in nine types of primary tumors and NCI60 cell lines, we applied an in silica approach by examining gene signatures reflective of consequent pathway activation using gene expression data. Supervised learning approaches predicted that the Ras pathway is active in approximately 70% of lung adenocarcinomas but inactive in most squamous cell carcinomas, pulmonary carcinoids, and small cell lung carcinomas. In contrast, the TGF-beta, TNF-alpha, Src, Myc, E2F3, and beta-catenin pathways are inactive in lung adenocarcinomas. We predicted an active Ras, Myc, Src, and/or E2F3 pathway in significant percentages of breast cancer, colorectal carcinoma, and gliomas. Our results also suggest that Ras may be the most prevailing oncogenic pathway. Additionally, many NCI60 cell lines exhibited a gene signature indicative of an active Ras, Myc, and/or Src, but not E2F3, beta-catenin, TNF-alpha, or TGF-beta pathway. To our knowledge, this is the first comprehensive survey of cancer pathway activities in nine major tumor types and the most widely used NCI60 cell lines. The "gene expression pathway signatures" we have defined could facilitate the understanding of molecular mechanisms in cancer development and provide guidance to the selection of appropriate cell lines for cancer research and pharmaceutical compound screening.

Testosterone regulates 3T3-L1 pre-adipocyte differentiation and epididymal fat accumulation in mice through modulating macrophage polarization.

Low testosterone levels are strongly related to obesity in males. The balance between the classically M1 and alternatively M2 polarized macrophages also plays a critical role in obesity. It is not clear whether testosterone regulates macrophage polarization and then affects adipocyte differentiation. In this report, we demonstrate that testosterone strengthens interleukin (IL) -4-induced M2 polarization and inhibits lipopolysaccharide (LPS)-induced M1 polarization, but has no direct effect on adipocyte differentiation. Cellular signaling studies indicate that testosterone regulates macrophage polarization through the inhibitory regulative G-protein (Gαi) mainly, rather than via androgen receptors, and phosphorylation of Akt. Moreover, testosterone inhibits pre-adipocyte differentiation induced by M1 macrophage medium. Lowering of serum testosterone in mice by injecting a luteinizing hormone receptor (LHR) peptide increases epididymal white adipose tissue. Testosterone supplementation reverses this effect. Therefore, our findings indicate that testosterone inhibits pre-adipocyte differentiation by switching macrophages to M2 polarization through the Gαi and Akt signaling pathways.

Comparative analysis and integrative classification of NCI60 cell lines and primary tumors using gene expression profiling data.

BACKGROUND: NCI60 cell lines are derived from cancers of 9 tissue origins and have been invaluable in vitro models for cancer research and anti-cancer drug screen. Although extensive studies have been carried out to assess the molecular features of NCI60 cell lines related to cancer and their sensitivities to more than 100,000 chemical compounds, it remains unclear if and how well these cell lines represent or model their tumor tissues of origin. Identification and confirmation of correct origins of NCI60 cell lines are critical to their usage as model systems and to translate in vitro studies into clinical potentials. Here we report a direct comparison between NCI60 cell lines and primary tumors by analyzing global gene expression profiles. RESULTS: Comparative analysis suggested that 51 of 59 cell lines we analyzed represent their presumed tumors of origin. Taking advantage of available clinical information of primary tumor samples used to generate gene expression profiling data, we further classified those cell lines with the correct origins into different subtypes of cancer or different stages in cancer development. For example, 6 of 7 non-small cell lung cancer cell lines were classified as lung adenocarcinomas and all of them were classified into late stages in tumor progression. CONCLUSION: Taken together, we developed and applied a novel approach for systematic comparative analysis and integrative classification of NCI60 cell lines and primary tumors. Our results could provide guidance to the selection of appropriate cell lines for cancer research and pharmaceutical compound screenings. Moreover, this gene expression profile based approach can be generally applied to evaluate experimental model systems such as cell lines and animal models for human diseases.

The loss in power when the test of differential expression is performed under a wrong scale.

One of the most common and important goals of microarray studies is to identify genes that are differentially expressed between cells of different conditions. T-test and ANOVA models on the expression data are common practices to gauge the significance of the observed difference in expression levels. Transformation of the microarray data is often applied in order to satisfy the model assumptions being entertained. However, the distributional properties of the expression are gene specific, and it is impractical to find a single transformation that is universally optimal for all the genes. This difficulty results in the situation that some genes have to violate the assumptions of the model (e.g., homogeneity in variance, normality). It is thus the interest of this paper to evaluate the impact on the inference of differential expression when the test is performed under an inappropriate scale. Particularly, we quantitatively assess the loss of power when the test is performed under a wrong scale. Normal distribution and log-normal distribution of the expression data are considered. The loss in power is investigated in two scenarios: a transformation is misused, or a transformation fails to be applied. Log transformation and power transformation are particularly considered due to the fact that Box-Cox types of transformation are commonly used in practice. The impact of using a wrong scale is investigated analytically and based on simulations. The loss in power is assessed both as a function of the degree to which the assumptions are violated and as a function of the effect size. Simulations are conducted to quantitatively assess the power loss when tests are performed under a wrong scale. A public experimental microarray dataset is used to illustrate the impact of transformation on the results of testing differential expression. The results show that the loss of power is a function of CV and fold-change (effect size). The loss in power depends on the true model and on how severely the assumptions are violated.