The Peroxisome Proliferator-Activated Receptor (PPAR)-γ Antagonist 2-Chloro-5-Nitro-N-Phenylbenzamide (GW9662) Triggers Perilipin 2 Expression via PPARδ and Induces Lipogenesis and Triglyceride Accumulation in Human THP-1 Macrophages.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor family, playing pivotal roles in regulating glucose and lipid metabolism as well as inflammation. While characterizing potential PPARγ ligand activity of natural compounds in macrophages, we investigated their influence on the expression of adipophilin [perilipin 2 (PLIN2)], a well-known PPARγ target. To confirm that a compound regulates PLIN2 expression via PPARγ, we performed experiments using the widely used PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Surprisingly, instead of blocking upregulation of PLIN2 expression in THP-1 macrophages, expression was concentration-dependently induced by GW9662 at concentrations and under conditions commonly used. We found that this unexpected upregulation occurs in many human and murine macrophage cell models and also primary cells. Profiling expression of PPAR target genes showed upregulation of several genes involved in lipid uptake, transport, and storage as well as fatty acid synthesis by GW9662. In line with this and with upregulation of PLIN2 protein, GW9662 elevated lipogenesis and increased triglyceride levels. Finally, we identified PPARδ as a mediator of the substantial unexpected effects of GW9662. Our findings show that: 1) the PPARγ antagonist GW9662 unexpectedly activates PPARδ-mediated signaling in macrophages, 2) GW9662 significantly affects lipid metabolism in macrophages, 3) careful validation of experimental conditions and results is required for experiments involving GW9662, and 4) published studies in a context comparable to this work may have reported erroneous results if PPARγ independence was demonstrated using GW9662 only. In light of our findings, certain existing studies might require reinterpretation regarding the role of PPARγ SIGNIFICANCE STATEMENT: Peroxisome proliferator-activated receptors (PPARs) are targets for the treatment of various diseases, as they are key regulators of inflammation as well as lipid and glucose metabolism. Hence, reliable tools to characterize the molecular effects of PPARs are indispensable. We describe profound and unexpected off-target effects of the PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662) involving PPARδ and in turn affecting macrophage lipid metabolism. Our results question certain existing studies using GW9662 and make better experimental design of future studies necessary.

Macrophage-targeted, enzyme-triggered fluorescence switch-on system for detection of embolism-vulnerable atherosclerotic plaques.

The development of atherosclerotic plaques is a critical step that can result in an arterial embolism. Therefore, detection of these vulnerable plaques is of clinical significance for the diagnosis of atherosclerosis. However, there are few imaging systems able to detect such plaques easily. In this study, we designed a new platform for near-infrared fluorescence (NIRF) imaging of macrophages in atherosclerotic plaques, one using both a liposomal DDS and an activatable fluorescent probe, and evaluated the utility of this imaging for the diagnosis of atherosclerosis. We first synthesized a fluorescent switch-on probe, Peptide-ICG2, which is optically silent under normal conditions but activated in the presence of the lysosomal enzyme, cathepsin B. To achieve macrophage-specific fluorescence activation, we encapsulated Peptide-ICG2 into phosphatidylserine-containing liposome (P-ICG2-PS-Lip), since the accumulation of phosphatidylserine receptor-bearing macrophages is characteristic of embolism-vulnerable plaques. The experiments using macrophage-like RAW264 cells in culture showed that P-ICG2-PS-Lip was selectively taken up into the cells and that significant fluorescence of the probe was observed. For NIRF imaging of the atherosclerotic plaques, P-ICG2-PS-Lip was intravenously injected into ApoE-knockout atherosclerotic model mice or WHHL rabbits, and the fluorescence at the aortae was imaged. The results indicated that ICG fluorescence could be successfully observed at the plaques on the artery walls. The results of the present study thus suggest that NIRF imaging using P-ICG2-PS-Lip would be useful for detecting embolism-vulnerable atherosclerotic plaques.

Transfecting Macrophages.

Transfection is defined as the transfer of foreign nucleic acids into cells. In general, transfection may achieve either overexpression of a gene by the transfer of plasmid DNA or suppression of gene expression by RNA interference after transfer of small interfering RNA. Both approaches allow for the detailed investigation of the function of a particular gene product or mechanisms of gene regulation. Macrophages are considered as hard-to-transfect cells, as they have evolved to recognize foreign nucleic acids and to initiate an immune response to these molecules. The presented electroporation protocol provides an effective tool to efficiently transfect human THP-1 macrophages with siRNA or plasmid DNA while avoiding macrophage activation.

A procedure for efficient non-viral siRNA transfection of primary human monocytes using nucleofection.

Monocytes are an important constituent of the innate immune system. Therefore, manipulating gene expression of primary human monocytes is a crucial mean to study and characterize the functions of targeted proteins in monocytes. Gene silencing by transfection of cells with small interfering RNA (siRNA) leading to the degradation of the corresponding mRNA and thus to reduced target protein levels is an important tool to investigate gene and protein function of interest. However, non-viral transfection of primary monocytes is challenging because siRNA uptake by these suspended cells is tricky, and the individual cells vary among different donors and do not proliferate. Here, we describe a procedure for efficient non-viral transfection of primary human monocytes isolated from peripheral blood, which maintains cell viability and cell functions, such as responsiveness to stimuli like LPS and IL-10. Nucleofection was used as an electroporation technique that enables efficient introduction of siRNA and silencing of target genes. Using a modification of our previously published protocol for the fast-proliferating THP-1 monocytic cell line, we transfected primary human monocytes with siRNA targeting 5-lipoxygenase (5-LO). In fact, we successfully downregulated 5-LO mRNA resulting in reduced protein levels and enzymatic activity.

Optimization of the transfection of human THP-1 macrophages by application of Nunc UpCell technology.

We have established an electroporation protocol for transfection of premature adherent human THP-1 macrophages using Lonza Nucleofector technology. For efficient electroporation, detachment of adherent cells is necessary. We tested the Nunc UpCell product line of Thermo Fisher Scientific, which achieves detachment by a change of ambient temperature, as an alternative to enzymatic detachment. Here we present data verifying proper cell morphology and vitality and high transfection efficiency for macrophages cultured on UpCell plates. Appropriate macrophage behavior was confirmed by measuring markers of macrophage differentiation and polarization by reverse transcription quantitative polymerase chain reaction (RT-qPCR). In conclusion, Nunc UpCell materials are a viable alternative to enzymatic detachment.

Highly efficient transfection of human THP-1 macrophages by nucleofection.

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

Identifying the African wintering grounds of hybrid flycatchers using a multi-isotope (δ2H, δ13C, δ15N) assignment approach.

Migratory routes and wintering grounds can have important fitness consequences, which can lead to divergent selection on populations or taxa differing in their migratory itinerary. Collared (Ficedula albicollis) and pied (F. hypoleuca) flycatchers breeding in Europe and wintering in different sub-Saharan regions have distinct migratory routes on the eastern and western sides of the Sahara desert, respectively. In an earlier paper, we showed that hybrids of the two species did not incur reduced winter survival, which would be expected if their migration strategy had been a mix of the parent species' strategies potentially resulting in an intermediate route crossing the Sahara desert to different wintering grounds. Previously, we compared isotope ratios and found no significant difference in stable-nitrogen isotope ratios (δ15N) in winter-grown feathers between the parental species and hybrids, but stable-carbon isotope ratios (δ13C) in hybrids significantly clustered only with those of pied flycatchers. We followed up on these findings and additionally analyzed the same feathers for stable-hydrogen isotope ratios (δ2H) and conducted spatially explicit multi-isotope assignment analyses. The assignment results overlapped with presumed wintering ranges of the two species, highlighting the efficacy of the method. In contrast to earlier findings, hybrids clustered with both parental species, though most strongly with pied flycatcher.

Reduced PMA enhances the responsiveness of transfected THP-1 macrophages to polarizing stimuli.

Macrophages are versatile cells of the immune system which react to various external stimuli through different polarization patterns which adjust the cells to the required function whether it is removal of pathogens or necrotic cells, tissue repair or propagation of inflammation. As much of macrophage behavior is determined by their polarization, appropriate models to study macrophage polarization are required. Previously we have published a protocol for transfection of THP-1 macrophages, which in brief pre-differentiates THP-1 monocytes for 48h using 100ng/ml PMA, followed by detachment of the cells and eletroporation using Lonza nucleofector technology and finally includes further 48h of differentiation with 100ng/ml PMA. When we applied this protocol to study interleukin (IL) 10 dependent polarization, the cells were inert to the IL10 stimulus, as indicated by a failure to induce IL10 target genes such as SOCS3. Further investigation revealed that the cells were classically activated by the differentiation agent phorbol 12-myristate 13-acetate (PMA) as shown by induction of chemokine receptor CCR7. Subsequent reduction of PMA concentration during THP-1 macrophage differentiation significantly improved their response to IL10 as SOCS3 increased more than 40-fold. This increased responsiveness of the THP-1 macrophages was also confirmed for polarization with LPS and IFNγ. Up-regulation of classical activation markers CCL3, CCR7 and TNFα was enhanced from 18-, 21- and 70-fold, respectively, to 48-, 222- and 951-fold, respectively. Reduction of PMA concentration did not negatively affect macrophage differentiation or transfection efficiency. Expression of macrophage differentiation markers CD11b and CD68 as well as cell morphology remained unchanged. In addition transfection efficiency and rates of apoptosis and necrosis remained unaffected. Thus our revised protocol combines high transfection efficiency and cell vitality with a strong response to polarizing stimuli and therefore constitutes a suitable model for studying macrophage polarization in vitro.

Evidence for an alternative genomic structure, mRNA and protein sequence of human ABCA13.

ABC transporters form one of the major families of transport proteins. In humans, the ABC family comprises seven subfamilies named A to G, of which the A subfamily contains twelve members. Among these are several well-characterized transporters, including ABCA1, which is involved in cellular cholesterol transport and HDL formation, and ABCA4, which is a transporter for vitamin A derivatives in photoreceptor cells. The function of another subfamily member termed ABCA13 is unknown. The human ABCA13 gene has been reported to span 450kb of genomic DNA at chromosomal locus 7p12.3 and to encode a 5058 amino acid protein that includes two unusually large exons close to the N-terminus. We now show that the gene as well as the corresponding mRNA and protein may be considerably shorter than previously thought. We used PCR and RACE to identify a genomic sequence spanning about 350kb and encoding a protein of 2323 amino acids. This corresponds to the C-terminal half of the previously reported ABCA13 protein but lacks the residues reportedly encoded by the two very big N-terminal exons. Using immunoprecipitation and Western blot analyses we identified a protein of about 260kDa in size likely representing the shorter protein proposed here. Computer analyses showed that our proposed sequence contains all the structural elements of an ABCA protein and agrees well with the mouse ABCA13 protein sequence. Additionally, we identified a putative promoter region containing well-conserved TATA and CAAT boxes just upstream of our transcription start site. Overall, our data provide good evidence for an alternative human ABCA13 transcript and protein.

Seasonality determines patterns of growth and age structure over a geographic gradient in an ectothermic vertebrate.

Environmental variation connected with seasonality is likely to affect the evolution of life-history strategies in ectotherms, but there is no consensus as to how important life-history traits like body size are influenced by environmental variation along seasonal gradients. We compared adult body size, skeletal growth, mean age, age at first reproduction and longevity among 11 common frog (Rana temporaria) populations sampled along a 1,600-km-long latitudinal gradient across Scandinavia. Mean age, age at first reproduction and longevity increased linearly with decreasing growth season length. Lifetime activity (i.e. the estimated number of active days during life-time) was highest at mid-latitudes and females had on average more active days throughout their lives than males. Variation in body size was due to differences in lifetime activity among populations--individuals (especially females) were largest where they had the longest cumulative activity period--as well as to differences between populations in skeletal growth rate as determined by skeletochronological analyses. Especially, males grew faster at intermediate latitudes. While life-history trait variation was strongly associated with latitude, the direction and shape of these relationships were sex- and trait-specific. These context-dependent relationships may be the result of life-history trade-offs enforced by differences in future reproductive opportunities and time constraints among the populations. Thus, seasonality appears to be an important environmental factor shaping life-history trait variation in common frogs.

Improved protocol for efficient nonviral transfection of premature THP-1 macrophages.

INTRODUCTION: The human monocytic leukemia cell line THP-1 is a widely used model for investigating monocyte and macrophage biology. Successful transfection of THP-1 monocytes with subsequent phorbol 12-myristate 13-acetate (PMA)-induced differentiation into macrophages is not a trivial matter, because according to previous transfection protocols, cell viability is lost almost completely within 24 h of PMA treatment following transfection. This protocol constitutes an optimized version of a previously published protocol by our group. It describes a procedure for transfecting premature THP-1 macrophages, which subsequently can be further differentiated into mature macrophages by PMA without a loss of cell viability. Transfection of THP-1 cells with plasmids or small interfering RNA (siRNA) is achieved by electroporation using the Lonza Nucleofector technology (Basel, Switzerland). This technique allows for the efficient nonviral delivery of plasmids, DNA, RNA, or siRNA into primary cells or cell lines even if the cells are not or are only slowly proliferating. Such cells are usually rather difficult to transfect by nonviral approaches. This means that only viral approaches would be left, which are expensive and labor-intensive and require laboratories complying with the respective safety regulations. The protocol described here is an efficient and convenient alternative.

Selection of reliable reference genes during THP-1 monocyte differentiation into macrophages.

BACKGROUND: Reliable reference genes are a vital prerequisite for any functional study employing quantitative real-time RT-PCR (RT-qPCR) for analyzing gene expression. Yet a proper selection and assessment of the chosen reference genes is only rarely included into a study. To date, no reference genes have been validated for differentiation of THP-1 monocytes. Here we report on the selection of validated reference genes during differentiation of THP-1 monocytes into macrophages induced by phorbol 12-myristate 13-acetate (PMA). RESULTS: The mRNA expression of 21 preselected potential reference genes was measured by RT-qPCR at several time-points over six days of PMA-induced THP-1 monocyte-to-macrophage differentiation. A ranking according to expression stability was calculated. Calculations were performed using Microsoft Excel-based applets GeNorm, NormFinder and BestKeeper. Our results indicated ACTB (ß-actin) (Cq ± SD, 14.1 ± 0.3) and RPL37A (ribosomal protein L37a) (14.5 ± 0.3) as the most stable genes. While other frequently used reference genes such as GAPDH (glycereraldehyde-3-phosphate dehydrogenase) (20.8 ± 0.8) or G6PD (glucose-6-phophate dehydrogenase) (16.1 ± 1.0) were found to be not as reliable and were therefore unsuited for use as reference genes. These findings were validated by investigating mRNA expression of macrophage scavenger receptor CD36, known to be regulated during monocyte-to-macrophage differentiation. Using ACTB and RPL37A as reference genes a profound and significant regulation of CD36 could be demonstrated, while use of G6PD resulted in a much less pronounced apparent regulation of CD36. CONCLUSION: Consequently, it is recommended to normalize any real-time PCR-based expression data obtained during THP-1 monocyte differentiation using ACTB and RPL37A.

Passerine extrapair mating dynamics: a bayesian modeling approach comparing four species.

In many socially monogamous animals, females engage in extrapair copulation (EPC), causing some broods to contain both within-pair and extrapair young (EPY). The proportion of all young that are EPY varies across populations and species. Because an EPC that does not result in EPY leaves no forensic trace, this variation in the proportion of EPY reflects both variation in the tendency to engage in EPC and variation in the extrapair fertilization (EPF) process across populations and species. We analyzed data on the distribution of EPY in broods of four passerines (blue tit, great tit, collared flycatcher, and pied flycatcher), with 18,564 genotyped nestlings from 2,346 broods in two to nine populations per species. Our Bayesian modeling approach estimated the underlying probability function of EPC (assumed to be a Poisson function) and conditional binomial EPF probability. We used an information theoretical approach to show that the expected distribution of EPC per female varies across populations but that EPF probabilities vary on the above-species level (tits vs. flycatchers). Hence, for these four passerines, our model suggests that the probability of an EPC mainly is determined by ecological (population-specific) conditions, whereas EPF probabilities reflect processes that are fixed above the species level.

Sex chromosome-linked species recognition and evolution of reproductive isolation in flycatchers.

Interbreeding between species (hybridization) typically produces unfit offspring. Reduced hybridization should therefore be favored by natural selection. However, this is difficult to accomplish because hybridization also sets the stage for genetic recombination to dissociate species-specific traits from the preferences for them. Here we show that this association is maintained by physical linkage (on the same chromosome) in two hybridizing Ficedula flycatchers. By analyzing the mating patterns of female hybrids and cross-fostered offspring, we demonstrate that species recognition is inherited on the Z chromosome, which is also the known location of species-specific male plumage traits and genes causing low hybrid fitness. Limited recombination on the Z chromosome maintains associations of Z-linked genes despite hybridization, suggesting that the sex chromosomes may be a hotspot for adaptive speciation.

Does migration of hybrids contribute to post-zygotic isolation in flycatchers?

In the face of hybridization, species integrity can only be maintained through post-zygotic isolating barriers (PIBs). PIBs need not only be intrinsic (i.e. hybrid inviability and sterility caused by developmental incompatibilities), but also can be extrinsic due to the hybrid's intermediate phenotype falling between the parental niches. For example, in migratory species, hybrid fitness might be reduced as a result of intermediate migration pathways and reaching suboptimal wintering grounds. Here, we test this idea by comparing the juvenile to adult survival probabilities as well as the wintering grounds of pied flycatchers (Ficedula hypoleuca), collared flycatchers (Ficedula albicollis) and their hybrids using stable isotope ratios of carbon (delta13C) and nitrogen (delta15N) in feathers developed at the wintering site. Our result supports earlier observations of largely segregated wintering grounds of the two parental species. The isotope signature of hybrids clustered with that of pied flycatchers. We argue that this pattern can explain the high annual survival of hybrid flycatchers. Hence, dominant expression of the traits of one of the parental species in hybrids may substantially reduce the ecological costs of hybridization.

Hepatitis C virus NS3-4A serine protease inhibitors: SAR of P'2 moiety with improved potency.

We have discovered that introduction of appropriate amino acid derivatives at P'2 position improved the binding potency of P3-capped alpha-ketoamide inhibitors of HCV NS3 serine protease. X-ray crystal structure of one of the inhibitors (43) bound to the protease revealed the importance of the P'2 moiety.

Use of methylene blue for detection of specialized intestinal metaplasia in GERD patients presenting for screening upper endoscopy.

The ability of randomly obtained biopsy specimens to identify intestinal metaplasia in the distal esophagus is low. Use of vital staining has been suggested, as stains are taken up by areas histologically identified as specialized intestinal metaplasia (SIM). This study evaluated the role of methylene blue (MB) staining for identification of SIM in GERD patients undergoing a screening endoscopy. Chromoendoscopy of the distal esophagus using 1% MB was performed on 52 GERD patients presenting for their first endoscopy. Biopsies were obtained from areas that were stained darkly, stained lightly, unstained, or macroscopically abnormal. In patients with no MB staining, four-quadrant biopsy of the distal esophagus was performed. Twenty-seven patients (52%) showed staining with MB, while 25 patients did not. Two hundred sixty-six biopsies were obtained. SIM was detected in 11 (21%) subjects (SIM+) but not in the remaining 41 (SIM-). One hundred sixty-five biopsies were unstained (25 SIM+, 140 SIM-) and 101 were stained (12 SIM+, 89 SIM-). The per-biopsy sensitivity and specificity of MB for detection of SIM were 32.4 and 85%, while the per-patient sensitivity and specificity were 63.3 and 51.2%. MB staining for detection of SIM in GERD patients without a macroscopic appearance suggestive of a columnar-lined esophagus is a poor screening tool for SIM.

The utility of prescreening for hepatitis A in military recruits prior to vaccination.

The U.S. Army administers the hepatitis A virus (HAV) vaccination for prophylaxis against HAV infection. There is little comparative data as to whether prescreening for previous HAV infection before immunization is less costly than universal vaccination. We designed a study to determine the prevalence of previous HAV infection in U.S. Army recruits and then perform a cost analysis. The cost analysis compared selective vaccination versus universal vaccination. Basic demographic information, including age, gender, geographic origin, and ethnicity, were collected after which patients were tested for HAV antibodies. A total of 1,332 individuals was prospectively enrolled with 183 individuals (13.74%) having evidence of previous HAV infection. Minority recruits were found to have a higher prevalence than Caucasian recruits (p = 0.0451. The cost analysis demonstrates that vaccination without prescreening was the least costly of two vaccination strategies for this cohort. To achieve current vaccination goals, all U.S. military recruits should be vaccinated without evaluation for previous HAV immunity.