Activation of the Leukotriene B4 Receptor 2-Reactive Oxygen Species (BLT2-ROS) Cascade following Detachment Confers Anoikis Resistance in Prostate Cancer Cells

The majority of prostate cancer-related deaths are associated with advanced and metastatic malignancies. Although anoikis resistance has been recognized as one of the hallmarks of metastatic prostate malignancies, the molecular events that cause anoikis resistance are poorly understood. In this study, we found that the detachment of PC-3 prostate cancer cells caused a time-dependent increase in the expression level of the leukotriene B₄ receptor-2 (BLT2) and that BLT2 played a critical role in establishing anoikis resistance in these cells. Blocking BLT2 with the pharmacological inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY255283","term_id":"1257961172"}}LY255283 or with RNAi knockdown clearly abolished anoikis resistance and resulted in severe apoptotic death. Additionally, we demonstrated that the activation of NADPH oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) were downstream of BLT2 signaling and led to the activation of NF-κB, thus establishing anoikis resistance during cell detachment. Furthermore, we observed that the ectopic expression of BLT2 in normal prostate PWR-1E cells rendered the cells resistant to anoikis and apparently diminished apoptotic cell death following detachment. Taken together, our results suggest that BLT2-NOX-ROS-NF-κB cascade induction during detachment confers a novel mechanism of anoikis resistance in prostate cancer cells and potentially contributes to prostate cancer progression.     

Photoacoustic Imaging of Breast Microcalcifications: A Preliminary Study with 8-Gauge Core-Biopsied Breast Specimens

Background

We presented the photoacoustic imaging (PAI) tool and to evaluate whether microcalcifications in breast tissue can be detected on photoacoustic (PA) images.

Methods

We collected 21 cores containing microcalcifications (n = 11, microcalcification group) and none (n = 10, control group) in stereotactic or ultrasound (US) guided 8-gauge vacuum-assisted biopsies. Photoacoustic (PA) images were acquired through ex vivo experiments by transmitting laser pulses with two different wavelengths (700 nm and 800 nm). The presence of microcalcifications in PA images were blindly assessed by two radiologists and compared with specimen mammography. A ratio of the signal amplitude occurring at 700 nm to that occurring at 800 nm was calculated for each PA focus and was called the PAI ratio.

Results

Based on the change of PA signal amplitude between 700 nm and 800 nm, 10 out of 11 specimens containing microcalcifications and 8 out of 10 specimens without calcifications were correctly identified on blind review; the sensitivity, specificity, accuracy, positive predictive and negative predictive values of our blind review were 90.91%, 80.0%, 85.71%, 83.33% and 88.89%. The PAI ratio in the microcalcification group was significantly higher than that in the control group (the median PAI ratio, 2.46 versus 1.11, respectively, P = .001). On subgroup analysis in the microcalcification group, neither malignant diagnosis nor the number or size of calcification-foci was proven to contribute to PAI ratios.

Conclusion

Breast microcalcifications generated distinguishable PA signals unlike breast tissue without calcifications. So, PAI, a non-ionizing and non-invasive hybrid imaging technique, can be an alternative in overcoming the limitations of conventional US imaging.     

Conformation-dependent antibody response to Pseudomonas aeruginosa outer membrane proteins induced by immunization in humans

Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines. In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization. Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different. The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses. In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis. Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma. These data suggest that antibody response to P. aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides.     

Lipids and lipid metabolism in the brown alga, Fucus serratus

The lipids of the brown alga Fucus serratus were isolated, identified and quantified. The major acyl lipids were the three glycosylglycerides, diacylgalactosylglycerol, diacyldigalactosylglycerol and diacylsulphoquinovosylglycerol. These represent over 70% of the total acyl lipids. The fatty acid compositions of the major lipids were examined and most showed rather distinctive fatty acid contents. For example, diacylgalactosylglycerol was enriched in n-3 polyunsaturated fatty acids while phosphatidylcholine and phosphatidylethanolamine had very high levels of arachidonate. Phosphatidylglycerol contained the unusual trans-Δ3-hexadecenoic acid. The labelling of lipids and fatty acids from [¹⁴C]acetate was examined and the distribution of label between individual components as a function of the incubation period and in algae collected at different times of the year is reported. Algae collected in the winter incorporated much more radioactivity into non-esterified fatty acids when compared to algae collected in the summer. All algae could label myristate, palmitate, stearate and oleate at high rates. Longer incubation times allowed the labelling of polyunsaturated fatty acids such as linoleic acid.     

Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca2+ signatures associated with polarized growth,development, and pathogenesis

Calcium is a universal messenger that translates diverse environmental stimuli and developmental cues into specific cellular and developmental responses. While individual fungal species have evolved complex and often unique biochemical and structural mechanisms to exploit specific ecological niches and to adjust growth and development in response to external stimuli, one universal feature to all is that Ca²⁺-mediated signaling is involved. The lack of a robust method for imaging spatial and temporal dynamics of subcellular Ca²⁺ (i.e., “Ca²⁺ signature”), readily available in the plant and animal systems, has severely limited studies on how this signaling pathway controls fungal growth, development, and pathogenesis. Here, we report the first successful expression of a FRET (Förster Resonance Energy Transfer)-based Ca²⁺ biosensor in fungi. Time-lapse imaging of Magnaporthe oryzae, Fusarium oxysporum, and Fusarium graminearum expressing this sensor showed that instead of a continuous gradient, the cytoplasmic Ca²⁺ ([Ca²⁺]_c) change occurred in a pulsatile manner with no discernable gradient between pulses, and each species exhibited a distinct Ca²⁺ signature. Furthermore, occurrence of pulsatile Ca²⁺ signatures was age and development dependent, and major [Ca²⁺]_c transients were observed during hyphal branching, septum formation, differentiation into specialized plant infection structures, cell–cell contact and in planta growth. In combination with the sequenced genomes and ease of targeted gene manipulation of these and many other fungal species, the data, materials and methods developed here will help understand the mechanism underpinning Ca²⁺-mediated control of cellular and developmental changes, its role in polarized growth forms and the evolution of Ca²⁺ signaling across eukaryotic kingdoms.     

Estrogen and n-3 polyunsaturated fatty acid supplementation have a synergistic hypotriglyceridemic effect in ovariectomized rats

The n-3 polyunsaturated fatty acids (PUFAs), EPA and DHA, as well as estrogen have been shown to decrease circulating levels of triglyceride (TG), but their underlying mode of action is unclear. The purpose of this study was to determine the effects of n-3 PUFA consumption and estrogen injection on TG metabolism. Rats (n = 48) were fed a modified AIN-93G diet with 0, 1, or 2 % EPA + DHA relative to the total energy intake during 12 weeks. At 8 weeks, rats were ovariectomized (OVX), and after a 1-week recovery, rats were injected with either 17β-estradiol-3-benzoate (E₂) or corn oil for the last 3 weeks. The n-3 PUFA consumption and E₂ injection independently decreased the hepatic expressions of sterol regulatory element-binding protein 1, acetyl-CoA carboxylase 1, fatty acid synthase (FAS), and diacylglycerol acyltransferase 2 (DGAT2) (P < 0.05). There were interactions between n-3 PUFA consumption and E₂ injection on hepatic expression of FAS and DGAT2. In addition, n-3 PUFA consumption and E₂ injection up-regulated the expression of AMP-activated protein kinase (AMPK), phosphorylated AMPK, peroxisomal proliferator-activated receptor α, and carnitine palmitoyltransferase 1 in liver and skeletal muscle. E₂ injection increased the expression of estrogen receptor α and β in skeletal muscle and liver, but n-3 PUFA consumption increased the expression of both receptors only in skeletal muscle. The present study suggests that the hypotriglyceridemic effects of n-3 PUFA consumption and E₂ injection could be due to the down-regulation of hepatic TG synthesis and up-regulation of TG oxidation in liver and skeletal muscle in OVX rats.     

In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination

Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2–5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1–5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection.     

Uncovering principles that control septin-septin interactions

Septins comprise a conserved family of GTPases important in cytokinesis. These proteins polymerize into filaments from rod-shaped heteromeric septin complexes. Septins interact with one another at two interfaces (NC and G) that alternate within the complex. Here, we show that small mutations at the N terminus greatly enhance the formation of SEPT2 homopolymers. Taking advantage of this mutation to examine polymer formation using SEPT2 alone, we show that both NC and G interfaces are required for filament formation. However, co-expression of wild type SEPT2 with SEPT2 containing mutations at either NC or G interfaces revealed that only the NC mutant suppressed filament formation. NC mutants are able to interact with one another at putative G interfaces, whereas G mutants fail to interact at NC interfaces. In addition, all promiscuous septin pairwise interactions occur at the G interface. These findings suggest that G interface interactions must occur before NC interactions during polymer formation.