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Forster resonance energy transfer (FRET) microscopy is an important tool suitable for studying molecular interactions in living cells. Optical section structured illumination microscopy (OS-SIM), like confocal microscopy, has about 200 nm spatial resolution. In this report, we performed quantitative 3-cube FRET imaging in OS-SIM mode and widefield microscopy (WF) mode, respectively, for living cells expressing FRET constructs consisting of Cerulean (C, donor) and Venus (V, acceptor). OS-SIM images exhibited higher resolution than WF images. Four spectral crosstalk coefficients measured under OS-SIM mode are consistent with those measured under WF mode. Similarly, the system calibration factors G and k measured under OS-SIM mode were consistent with those measured under WF mode. The measured FRET efficiency (E) values of C32V and C17V as well as C5V constructs, standard FRET plasmids, in living Hela cells were EC32VOSF=0.32±0.02,EC17VOSF=0.38±0.02 , and EC5VOSF=0.45±0.03 , and the measured acceptor-to-donor concentration ratios ( Rc ) were RC32VOSF=1.07±0.03 , RC17VOSF=1.09±0.03 , and RC5VOSF=1.02±0.04 , consistent with the reported values. Collectively, our data demonstrates that OS-SIM can be integrated into FRET microscopy to build an OS-SIM-FRET with confocal microscopy-like resolution.

Chromosomes, the carriers of genes, were first observed in plant cells in 1842. Visual inspection of chromosomes via cytogenetics laid the foundation for understanding the structure and content of chromosomes. Eventually, Watson and Crick's discovery of DNA, the building block of the chromosomes paved the way for genomics and the DNA sequencing revolution. As we lie on the cusp of scaling genomics to personalized analysis and to the broad diversity of species, the new generation of scientific discoveries relies heavily on computational analysis of complex datasets.This thesis highlights computational methods that we developed for interpreting various data modalities to elucidate the large-scale structure of the genome. We describe two tools, Cerulean and AmpliconArchitect(AA), which aim to interpret sequencing data in different contexts to find an unknown genomic structure. The crux of these tools is the representation of the genomic structure as a graph which encodes connectivity of genomic segments, followed by delineation of the graph into ordered genomic segments. Cerulean performs hybrid de-novo assembly of a novel genome by combining accurate, short sequencing reads with erroneous, long reads which can span longer distances along the genome. AA focuses on a specific feature of cancer genomes called focal amplifications, or regions with a high increase in copy number. These often contain cancer-causing oncogenes and undergo complex rearrangements. AA simultaneously uses short sequencing reads from the cancer genome and information from the human reference genome to predict the structure of the focal amplification.We applied AA to comprehensively characterize the nature of focal amplifications across human cancer. We combined sequence analysis of AA with extensive computational analysis of cytogenetic images of cancer cells. Surprisingly, we found that focal amplification occurs through the formation of circular extrachromosomal DNA(ecDNA) structures breaking off from the human chromosomes in as many as 40% of all cancer cases. Through theoretical modeling we showed that formation of ecDNA drastically accelerates tumor growth and evolution, facilitating rapid development of resistance to targeted drugs. This defines a new paradigm in our understanding of cancer and cancer treatment.

Amaç: Şiddetli akut pankreatit (SAP) yüksek morbidite ve mortalite riski taşır. SAP'nin proenflamatuar yanıt fazı kontrol edilemezse, çoklu organ yetmezliği (ÇOY) ile sonuçlanabilir. Nükleer faktör-kappa B (NF-κB) aktivasyonu ÇOY'un gelişiminde önemli bir rol oynar. Bu çalışmada, bir anti-enflamatuar ve immünsüpresif ajan olan triptolidin SAP'lı sıçanlarda iyileştirici etkilerinin araştırılması amaçlanmıştır.Gereç ve Yöntemler: Toplam 20 Wistar-Albino sıçanı SAP ve triptolide tedavi (TT) grubu olmak üzere iki gruba ayrıldı. SAP her iki grupta da intraperitoneal cerulein (50 mg/kg) enjeksiyonu ile indüklendi. TT grubuna, SAP indüksiyonundan 24 saat sonra tek bir doz (0.2 mg/kg) triptolid uygulandı. Serum ALT, AST, GGT, Lipaz, Glikoz, ALP ve amilaz düzeyleri ve pankreatik doku örnekleri incelendi.Bulgular: Serum glukoz ve amilaz düzeyleri TT grubunda anlamlı olarak daha düşük bulundu (sırasıyla p=0.011 ve p=0.035). Diğer biyokimyasal parametreler açısından gruplar arasında anlamlı bir fark yoktu. Pankreas ödemi, asiner hücre dejenerasyonu, yağ nekrozu, intrapankreatik&perivasküler inflamasyon ve peripankreatik yağ dokusunda inflamasyon her iki grupta da sık görülen histopatolojik bulgulardı. Gruplar arasında histopatolojik değişiklikler açısından anlamlı bir fark yoktu.Sonuç: Cerulein kaynaklı pankreatit, deneysel SAP için başarılı bir yöntemdir. Tek doz triptolid tedavisinin iyileştirici etkileri SAP'nin erken evresinde belirgin değildir. Pankreatik doku histopatolojik ve biyokimyasal parametreler açısından incelendiğinde, triptolidin enflamatuar ve oksidatif stres üzerindeki terapötik etkileri yeterli düzeyde değildi., Aim: Severe acute pancreatitis (SAP) carries high morbidity and mortality risk. If the proinflammatory response phase of SAP cannot be controlled, it may result in multiorgan failure (MOF). Nuclear factor-kappa B (NF-κB) activation plays an important role in the development of MOF. In this study, it was aimed to investigate the healing effects of triptolide, an anti-inflammatory and immunosuppressive agent in rats with SAP.Material and Methods: A total of 20 Wistar-Albino rats were divided into two groups as the SAP and triptolide treatment (TT) groups. SAP was induced by intraperitoneal injection of cerulean (50 mg/kg) in both groups. TT group was administered a single dose (0.2 mg/kg) triptolide 24 hour after the induction of SAP. Serum ALT, AST, GGT, Lipase, Glucose, ALP and amylase levels and pancreatic tissue samples were examined.Results: Serum glucose and amylase levels were found to be significantly lower in the TT group (p=0.011 and p=0.035, respectively). There was no significant difference between the groups in terms of other biochemical parameters. Pancreatic edema, acinar cell degeneration, fat necrosis, intrapancreatic&perivascular inflammation, inflammation in the peripancreatic fat tissue were common histopathological findings in both groups. There was no significant difference between the groups in terms of histopathologic changes.Conclusion: Cerulein-induced pancreatitis is a successful method for experimental SAP. The healing effects of single-dose triptolide treatment are not evident in the early phase of SAP. The therapeutic effects of triptolide on inflammatory and oxidative stress were not significantly approved by histopathological and biochemical parameters by the pancreatic tissue.

Cerulean Warblers (Setophaga cerulea) are among the fastest declining Nearctic-Neotropical migrant wood-warblers (Parulidae) in North America. Despite ongoing conservation efforts, little is known about their non-breeding distribution. In June 2016-2018, we deployed geolocators (n = 30) on adult male Cerulean Warblers in Indiana, USA, to track annual movements of individuals. Recovered geolocators (n = 4) showed that Cerulean Warblers occurred broadly throughout northern South America. Autumn migration lasted 44-71 days (n = 4), whereas spring migration lasted 37-41 days (n = 3). The average migration distance was 5268 km. During autumn migration, Cerulean Warblers made 1-4 stopovers (i.e., ≥2 days; n = 4) and 1-2 stopovers during spring migration (n = 3). When crossing the Gulf of Mexico during autumn migration, two birds stopped over after crossing, but not beforehand. Two others navigated through the Caribbean rather than crossing the Gulf of Mexico. During spring migration, one individual stopped after crossing, one individual stopped before crossing, and one individual stopped before and after crossing the Gulf of Mexico. No birds migrated through the Caribbean Islands during spring migration. These results represent novel information describing annual movements of individual Cerulean Warblers and will inform conservation efforts for this declining species.

Although the overall structure of many classical transient receptor potential proteins (TRPC), including human and murine TRPC6, were recently resolved by cryoelectron microscopy analysis, structural changes during channel activation by 1-oleoyl-1-acetyl-sn-glycerol (OAG), the membrane-permeable analog of diacylglycerol, were not defined. Moreover, data on carboxyl- and amino-terminal interactions were not provided, as the amino-terminal regions of murine and human TRPC6 were not resolved. Therefore, we employed a Förster resonance energy transfer (FRET) approach using a small fluorescein arsenical hairpin (FlAsH) targeted to a short tetracysteine sequence at the unresolved amino-terminus and cerulean, a cyan fluorescent protein, as a tag at the carboxyl-terminus of the murine TRPC6 protein. After OAG as well as GSK-1702934A activation, FRET efficiency was simultaneously and significantly reduced, indicating a decreased interaction between the amino to carboxyl termini in the functional tagged murine TRPC6 tetramer (TRPC6 WT) heterologously expressed in human embryonic kidney 293T cells. There was a significant reduction in the FRET signal obtained from analysis of murine TRPC6 FRET constructs with homologous amino-terminal mutations (M131T, G108S) that had been identified in human patients with inherited focal segmental glomerulosclerosis, a condition that can lead to end-stage renal disease. A novel, designed loss-of-function TRPC6 mutation (N109A) in the amino-terminus in close proximity to the carboxyl-terminus produced similar FRET ratios. SIGNIFICANCE STATEMENT: Our data show for the first time that FlAsH-tagging of ion channels is a promising tool to study conformational changes after channel opening and may significantly advance the analysis of ion channel activation as well as their mutants involved in channelopathies.

In this work samples of historical pigments of green hue were brushed on a canvas and studied by Visible Reflectance, X-Ray Fluorescence and Fourier Transform Infrared Spectroscopy. One hundred samples were investigated, all with green hue, these prepared from pigments themselves green, such as chromium oxide (Cr2O3) or from a mixture of pigments that result in green, for example, chrome yellow (PbCrO4) and Prussian blue (Fe4[Fe(CN)6]3). Because every sample investigated through the spectroscopic techniques were of green hue, the characterization of the pigments present in the mixtures through the visual inspection of spectra has become a complex task in some cases, also, due the large number of recorded spectra. In this work, classification models were developed using the multivariate statistical method Partial Least Squares Discriminant Analysis (PLS-DA) to automate the characterization of the pigments present in the mixtures. The models were developed to classify chromium oxide (Cr2O3), chrome yellow (PbCrO4), cerulean blue (CoO.nSnO2) and yellow ochre (Fe2O3·H2O + clay + silica). The models were developed from the fusion of data from the three spectroscopic techniques. However, before data fusion, pre-treatments of the spectral data were tested for their influence on the PLS-DA models. The models developed with data from the three techniques made it possible to classify the pigments of interest in the samples with up to 100% effectiveness. The results also indicate that fusion of the data from the three techniques allows to obtain fingerprints of the pigments of interest, which is not always possible using data from only one or two of the techniques applied in this work.

IntroductionThis study aims to explore the effect and mechanism of miR�489-3p on the proliferation and apoptosis of pancreatic acinar cells in acute pancreatitis (AP).Material and methodsX-linked inhibitor of apoptosis protein (XIAP) and miR-489-3p expression in serum of AP patients, pancreatic AR42J cells, and rat AP tissues were measured using quantitative reverse transcription poly�merase chain reaction and western blot. The effect of miR-489-3p on pro�liferation and apoptosis was determined by MTT assay and flow cytometry. The relationship between XIAP and miR-489-3p was verified using luciferase assay RNA immunoprecipitation assay. Histological changes in rat pancreatic tissues were observed via haematoxylin and eosin staining.ResultsMeasurement of miR-489-3p and XIAP expressions in AP patients revealed a negative correlation between miR-489-3p and XIAP. Increased miR-489-3p expression in AP patients indicated a poor prognosis. Cerulein was used to induce AP in AR42J cells and standard deviation rats. Exper�iments in cells and AP rat models showed that miR-489-3p can increase cell apoptosis and inhibit cell proliferation by regulating XIAP, as shown by elevated expressions of pro-apoptotic proteins (p53 and Bax) and decreased expression of proliferation indicator (Ki-67) after transfection of miR-489- 3p mimics. Meanwhile, knockdown of miR-489-3p abrogated the inhibitory effects of miR-489-3p on cell proliferation and the promotion on cell apop�tosis. Luciferase assay and RNA immunoprecipitation assay confirmed that XIAP can directly bind miR-489-3p.ConclusionsWe concluded that miR-489-3p modulates cell proliferation and apoptosis in AP by targeting XIAP. Given that high expression of miR�489-3p in AP indicated poor prognosis, it raises the possibility that miR�489-3p might be a novel and valuable therapeutic target and a prognosis indicator for AP.

Site fidelity can have significant impacts on population dynamics and may be influenced by both demographic and environmental characteristics, including age, sex, and habitat quality. We examined breeding-site fidelity of Setophaga cerulea (Cerulean Warbler), a small Nearctic–Neotropical migrant that is listed as endangered in Indiana and in decline throughout its range. Our research was part of a long-term demographic study in the research units of the Hardwood Ecosystem Experiment, southern Indiana. From 2013 to 2018, we banded 128 Cerulean Warblers, 10.2% of which returned the year following initial capture and banding. We did not find significant differences in return rates between adult males (n = 92) and adult females (n = 19; P = 0.69), novice (second-year, n = 20) and experienced (after-second-year, n = 91; P = 1) birds, or successful (n = 40) and unsuccessful breeders (n = 26; P = 0.39). We have yet to detect a bird banded as a hatch-year subsequently return the following season as a second-year (n = 0/17). Our return rates are likely affected by a combination of mortality between breeding seasons, our ability to detect returning birds, and dispersal between breeding seasons. We encourage other researchers to publish rates of Cerulean Warbler site fidelity across its range for comparison of populations and habitats to determine source/sink dynamics.

Background: Synaptic dysfunction prevalent in Alzheimer’s disease (AD) brain is closely associated with increased accumulation of amyloid-β (Aβ) peptides in the brain parenchyma. It is widely believed that Aβ peptides trigger synaptic dysfunction by interfering with the synaptic vesicular fusion and the release of neurotransmitters, primarily facilitated by the SNARE protein complexes formed by VAMP-2, SNAP-25, and syntaxin-1. However, Aβ interactions with SNARE proteins to ultimately disrupt synaptic vesicular fusion are not well understood. Objective: Our objective is to elucidate mechanisms by which Aβ peptides perturb SNARE complexes. Methods: Intensity (qualitative) and lifetime (quantitative) based measurements involving Forster (fluorescence) resonance energy transfer (FRET) followed by fluorescence lifetime imaging microscopy (FLIM) were employed to investigate the effect of Aβ peptides on dynamic interactions between VAMP-2, labeled with cerulean (Cer) at the N-terminus (FRET donor), and SNAP-25 labeled with citrine (Cit) on the N-terminus (FRET acceptor). The FRET and FLIM interactions at the exocytosis locations on the pre-synaptic membrane were recorded under spontaneous and high potassium evoked conditions. Moreover, cellular accumulation of fluorescein labeled Aβ (F-Aβ) peptides and their co-localization with Cer-VAMP2 was investigated by confocal microscopy. Results: The F-Aβ40 and F-Aβ42 are internalized by differentiated N2A cells, where they colocalize with Cer-VAMP2. Both Aβ40 and Aβ42 decrease interactions between the N-termini of Cer-VAMP2 and Cit-SNAP25 in N2A cells, as determined by FRET/FLIM. Conclusion: By perturbing the N-terminal interactions between VAMP-2 and SNAP-25, Aβ40 and Aβ42, can directly interfere with the SNARE complex formation, which is critical for the docking and fusion of synaptic vesicles.

Down syndrome (DS) is frequently associated with cataract, but there remains scant information about DS cataract morphology. Supra-nuclear cataracts in DS have been proposed as indicative of beta-amyloid (Aβ) aggregation and thus potential biomarkers for Alzheimer’s (AD). This study employed anterior segment OCT (AS-OCT) and slit-lamp (SL) photography to image the crystalline lens in DS, compared with adult controls. Lens images were obtained post-dilation. Using MATLAB, AS-OCT images were analysed and lens opacities calculated as pixel intensity and area ratios. SL images were classified using LOCS III. Subjects were n = 28 DS (mean ± SD 24.1 ± 14.3years), and n = 36 controls (54.0 ± 3.4years). For the DS group, AS-OCT imaging revealed the frequent presence of small dot opacities (27 eyes, 50%) in the cortex and nucleus of the lens, covering an area ranging from 0.2–14%. There was no relation with age or visual acuity and these dot opacities (p > 0.5) and they were not present in any control lenses. However, their location and morphology does not coincide with previous reports linking these opacities with Aβ accumulation and AD. Four participants (14%) in the DS group had clinically significant age-related cataracts, but there was no evidence of early onset of age-related cataracts in DS.

The persistence of the latent HIV-1 reservoir is a major obstacle to cure HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). We investigated HIV-1 promoter activity after integration into specific sites in BACH2. The HIV-1-based vector LTatCL[M] contains two fluorophores: 1.) Cerulean, which reports the activity of the HIV-1 promoter, and 2.) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean−/mCherry+) HIV-1 promoters were characterized. Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, active HIV-1 promoters were gradually silenced as reflected by decrease in Cerulean expression over a period of 162 days in culture. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. Our results show that the HIV-1 promoter is silenced when integrated into BACH2 without impairing BACH2 mRNA and protein expression. This might contribute to HIV-1 persistence, enabling infected T-cells to complete differentiation into a memory phenotype, persist, and clonally expand over time.

In the last decade, genome editing has been the center of attention as a novel tool for mechanistic investigations and for potential clinical applications. Various genome editing tools like meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (Cas), have been developed in recent years. For the optimal use as well as continued developments of these genome editing tools, the evaluation of their efficiencies and accuracies is vital. Here, we present a protocol for a reporter based on frameshift fluorescence protein which we recently developed to evaluate the efficiency and accuracy of genome editing tools. In this method, a ~20 bp target sequence containing frame-shifting is inserted after the start codon of a cerulean fluorescence protein (CFP) to inactivate its fluorescence, and only a new insertion/deletion event in the target sequence will reactivate the CFP fluorescence. To increase the traceability, an internal ribosome entry site and a red fluorescence protein, mCherryFP, are placed downstream of the reporter. The percentage of CFP-positive cells resulted from in/del mediated fluorescence restoration can be quantified by fluorescence measuring devices as the readout for genome editing frequency. As a demonstration, we present the usage for CRISPR-Cas9 technique here with flow cytometer as the readout for fluorescence changes.

A set of transgenic zebrafish lines, called PhOTO (Photoconvertible Optical Tracking Of…), were previously introduced and have wide applicability for accurate cell tracking during highly dynamic events, including embryo development, tissue regeneration, and cancer/disease progression. Cell shapes, interactions, divisions, and dynamics can be monitored by employing the photoconvertible fluorescent protein Dendra2 and the blue fluorescent protein Cerulean as compartment-specific labels of the nucleus and the plasma membrane. The combination of PhOTO zebrafish with confined primed conversion, a recently introduced axially confined photoconversion method, enables nontoxic, instantaneous, and precise cell tracking during any stage of the life cycle of zebrafish. The implementation of advanced imaging platforms and smart bioimaging software to perform high-fidelity lineage tracing of primed converted PhOTO zebrafish will greatly benefit the goal of acquiring a refined mechanistic view of development and disease.

A recent paper published in The Condor: Ornithological Applications by Raybuck et al. (2017) described the results of an analysis of potential effects of light-level geolocators on reproductive parameters and apparent annual survival of Cerulean Warblers (Setophaga cerulea). In a comparison of geolocator-tagged birds and color-banded control birds, the authors reported no discernible differences in several standard reproductive parameters, but a significant difference in apparent annual survival. Here, we show that the reported overall geolocator effect on annual survival may have obfuscated a large effect in one year, when one geolocator model and harness method were used, compared with no effect on annual survival in the second year of their study, when a different geolocator model and harness method were used. These more nuanced results suggest that Raybuck and colleagues may have identified variation in effects between geolocator marking methods for Cerulean Warblers, rather than a general ge...

Territorial male birds of many north temperate species sing prolifically before sunrise in a behaviour known as ���dawn song,��� after which males sing at lower rates, use different song types, or cease singing altogether. From May to July 2017 in southern Indiana, we recorded 100 dawn song performances from 12 paired male Cerulean Warblers (Setophaga cerulea) and we quantified the relative influences of social (breeding stage of the focal pair) and abiotic factors (Julian date and weather) on six characteristics of dawn song. We found that dawn song was influenced by both biotic and abiotic factors, but weather had the most model support. Males were more likely to sing dawn song early in the season, and most males nesting late in the season ceased dawn song altogether. Dawn song start times (in relation to sunrise) varied little over the season, but males ended dawn song earlier, and sang at lower peak rates, as the season progressed. Additionally, males initiated and ended dawn song later on windy and rainy mornings. Multiple factors apparently influence dawn singing behaviour in paired male Cerulean Warblers, yet weather may have a greater effect on dawn song than do Julian date or social behaviour.

Transglutaminases (TGs) comprise a protein family in which the members catalyze the formation of isopeptide bonds between glutamine and lysine residues in various proteins. Expression studies on its three major members, FXIII, TG1, and TG2, have been performed in a relatively large number of mammalian tissues in comparison with those on the other isozymes. We previously identified a highly reactive substrate peptide, including glutamine, for each isozyme from a phage display library and developed a method for detecting isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. Here, we describe genetically encoded Förster resonance energy transfer (FRET)-based probes composed of each fluorescence protein (Cerulean and EVenus) fused with substrate peptides. The probe pairs, designated as Trac-MTG (His-CerΔ11-LQ/EV-K-His) containing linker and substrate peptide sequence for microbial TG (MTG), increased the EVenus:Cerulean fluorescence intensity ratio by more than 1.5-fold. Furthermore, we demonstrated that Trac-TG1 (His-CerΔ11-K5) and Trac-TG2 (His-CerΔ11-T26) containing substrate peptide sequence for mammalian TGs successfully detected the isozyme-specific activity of TG1 and TG2, respectively. In this study, we developed a rapid and convenient experimental system for measuring the isozyme-specific activity of TGs. The application of these probes for analyses in cells and tissues will be helpful for elucidating the physiological and pathological functions of TGs.

Enhanced cyan fluorescent protein (ECFP) was derived fromAequorea victoriagreen fluorescent protein (avGFP), notably with S65T/Y66W mutations. Its chromophore consists of a tripeptide comprised of Thr65, Trp66 and Gly67 (TWG) residues, while that ofavGFP consists of a Ser65, Tyr66 and Gly67 (SYG) tripeptide. Cerulean and SCFP3A were derived from ECFP-S72A/H148D (a double mutation) with additional Y145A and S175G mutations, respectively, while Cerulean-S175G has both mutations (Y145A and S175G). The crystal structures of these ECFP variants at neutral pH were reported to adopt two distinct major conformations calledECFPandCerulean. In this study, Cerulean-S175G was revealed to adopt only theCeruleanconformation, while Cerulean has been reported to adopt both theECFPand theCeruleanconformations in its crystal structures. Sharing the same S175G mutation with SCFP3A, Cerulean-S175G showed a slightly increased quantum yield, like SCFP3A, but did not adopt theECFPconformation adopted by SCFP3A. Detailed comparison of Cerulean-S175G and other ECFP variants revealed that the notable conformational changes in ECFP variants can be understood mainly in terms of the interaction between the Trp66 residue of the chromophore and residues 145–148 of β-strand 7.

Within species of birds, variation in plumage may allow potential mates or competitive rivals to quickly assess the quality of an individual. Little is known about the role of white tail feather patches (“tail white”) in male Cerulean Warblers (Setophaga cerulea) and whether variation in patch size could serve as a signal. We hypothesized that the size of tail white patches in males acts as an honest signal of quality, with larger white patches indicating high quality males. We measured and compared the area of tail white to four estimates of quality (age, structural size, body mass, and blood parasite load) in 71 male Cerulean Warblers at the Queen’s University Biological Station in eastern Ontario. We found that males 2 years old or older had significantly larger tail white patches than 1 year old males, and that structurally larger males (estimated by wing length) had significantly larger tail white patches than smaller males. Our best-performing statistical model suggested that heavier indivi...

BackgroundThe persistence of the latent HIV-1 reservoir is a major obstacle to curing HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the geneBTB domain and CNC homology 2 (BACH2).MethodsWe investigated HIV-1 promoter activity after integration into specific sites inBACH2in Jurkat T-cells. The HIV-1-based vector LTatCL[M] contains two fluorophores: (1) Cerulean, which reports the activity of the HIV-1 promoter and (2) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 ofBACH2of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation ofBACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean–/mCherry+) HIV-1 promoters were characterised.ResultsUpon targeted integration of the 5.3 kb vector LTatCL[M] intoBACH2, the HIV-1 promoter was gradually silenced as reflected by the decrease in Cerulean expression over a period of 162 days. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation.BACH2mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M].ConclusionSuccessful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity.

Staphylococcus aureus (S. aureus) is a gram-positive bacterium with a “split personality”. Staphylococci are part of the normal skin flora, but can also cause skin and wound infections as well as fatal septicaemia. The mechanism of transition from apathogenic colonisation to invasive infections is modulated by several gene regulators. An important gene regulator is the Accessory Gene Regulator (Agr) system, which is responsible for the modulation of hundreds of genes. The agr locus consists of two transcriptional units expressed from two divergent promoters P2 and P3. The Agr system is a typical two-component system and is regulated by a quorum sensing mechanism, whereby staphylococci produce an extracellular autoinducing peptide (AIP). AIP binds to its receptor, the histidine kinase AgrA and causes the autoinduction of the Agr operon itself through the agr P2 promoter, as well as the activation of various target genes through the agr P3 promoter. Gene expression is activated by a regulatory RNAIII molecule (originating from the agr P3 promoter) and, among other regulatory functions, leads to the expression of the capA-P biosynthesis cluster, which leads to the synthesis of capsular polysaccharide (CP). The capA-P operon is controlled by the capP promoter upstream of capA. CPs protect staphylococci from opsono-phagocytosis, but also mask surface molecules and therefore reduce the ability of staphylococci to adhere to tissue surfaces. Analysis using fluorescence-activated cell sorting and immunofluorescence staining have revealed that not all staphylococci within a single culture produce CP in the post-exponential phase, but rather present phenotypically heterogeneous. The aim of this study was to establish reporter strains to simultaneously analyse the activity of both the agr-P3 and the cap-P promoter on a single cell level. For this, promoters with fluorescence proteins were cloned into integration vectors and integrated into the S. aureus chromosome. We used integration vectors that either used the S. aureus pathogenicity island I (SaPI-I) as an attachment site, or integrated with a phage (attachment site geh). Plasmids with various combinations of fluorescence genes Venus (ven) or Cerulean (cer) and different selection markers were tested. The generated strains were verified using PCR to confirm correct integration, and phenotypically analysed using confocal fluorescence microscopy. We were able to show that all reporter strains had successfully integrated the vector plasmid at the correct attachment site. In one case, partial deletion of the integration plasmid was observed. In another case, we were able to detect multiple integration of the vector. Fluorescence microscopy revealed that 1. Agr-activity is largely homogeneous and is highest in the post-exponential phase. 2. cap-A activity is also at highest levels in the post-exponential phase. 3. cap-P activity is highly heterogeneous within a single culture. 4. Promoter fusion constructs with Venus displayed a stronger fluorescence signal than fusion constructs with Cerulean. 5. The analysis of the double reporter strain gave inconclusive results and needs to be analysed further. In the second part of this work, we aimed to establish a 3D collagen model, in which reporter strains could be observed over an extended period. Guggenberger et al. have shown that embedding staphylococci in collagen with fibrinogen led to the formation of a pseudocapsule from fibrinogen deposits, as well as a surrounding microcolony-associated meshwork (Guggenberger C, Wolz C, Morrissey JA, Heesemann J (2012) Two Distinct Coagulase-Dependent Barriers Protect Staphylococcus aureus from Neutrophils in a Three Dimensional in vitro Infection Model. PLoS Pathogens). These structures are similar to those found in an abscess, therefore we aimed to simulate an in vitro abscess with our model. Pseudocapsule and a microcolony-associated meshwork formation was observed partially. Analysis of promoter activity within the collagen/fibrinogen-structure revealed preliminary results: 1. The intensity of fluorescence signal was weaker for both promoter fusion genes compared to growth in liquid medium. 2. cap-Promoter activity seems significantly higher than agr activity. 3. Bacteria in the periphery of a microcolony displayed higher levels of fluorescence. These results represent first steps towards the establishment of a system to analyse promoter activity within an in vitro abscess model., Staphylococcus aureus (S. aureus) ist ein grampositives Bakterium mit „gespaltener Persönlichkeit“. Staphylokokken kommen als Teil der natürlichen Hautflora vor, können aber auch als pathogenes Bakterium Haut- und Wundinfektionen bis hin zu tödlich verlaufenden Blutstrominfektionen verursachen. Der Übergang von apathogener Kolonisation zu invasiver Infektion wird durch eine Vielzahl von Genregulatoren moduliert. Ein wichtiger Genregulator bildet das Accessory Gene Regulator (Agr) System, das für die Modulation von über hundert Genen verantwortlich ist. Der agr locus besteht aus zwei Transkriptionseinheiten, die von zwei divergenten Promotoren, agr P2 und agr P3 kontrolliert werden. Das Agr System ist ein klassisches Zwei-Komponentensystem und wird über einen Quorum Sensing Mechanismus gesteuert, wobei das autoinduzierende Protein autoinducing peptide (AIP) von Staphylokokken in den Extrazellulärraum sezerniert wird. Durch Rückbindung an seinen Rezeptor, der Histidinkinase AgrA, kommt es zur Selbstaktivierung des Agr Operons über den agr P2 Promotor, sowie zur Aktivierung von verschiedenen Zielgenen über den agr P3 Promotor: Die aktivierte Genexpression der regulatorischen RNAIII (ausgehend von dem Promoter agr P3) führt u.a. zur Expression des capA-P Biosynthesegencluster welches für Enzyme zur Synthese Kapselpolysaccharide (CP) führt. Das capA-P Operon wird durch einen Promoter upstream von capA (cap-P) gesteuert. CPs schützen Staphylokokken vor der Opsonophagozytose durch Immunzellen, führen aber gleichzeitig zur Maskierung von Oberflächenmolekülen und mindern somit die Fähigkeit zur Adhärenz an Gewebeoberflächen. Analysen mittels fluoreszenzaktivierter Durchflusszytometrie, sowie Immunfluoreszenzmethoden haben gezeigt, dass nicht alle Staphylokokken in der postexponentiellen Phase innerhalb einer Kultur CP produzieren, sondern sich phänotypisch heterogen präsentieren. Das Ziel der Studie war, Reporterstämme herzustellen, mit denen die Aktivitäten der Promotoren agr-P3 und cap-P auf der Ebene einzelner Zellen simultan analysiert werden können. Hierzu wurden Promotern mit Fluoreszenzproteingenen in Integrationsvektoren kloniert und chromosomal in das Staphylokokkenchromosom integriert. Dabei wurden Plasmide verwendet die entweder in die „attachment site“ für die Pathogenitätsinsel I (SaPI) oder die mittels eines Phagen (attachment site in geh) integrieren. Es konnten Plasmide mit verschiedenen Kombinationen von den Fluoreszenzgenen Venus (ven) oder Verulean (cer) und verschiedenen Selektionsmarkern getestet werden. Dadurch sollte es möglich sein, Doppel-Integranten mit agr-P3-cer, agr-P3 ven, capA-cer, capA-ven herzustellen. Die generierten Stämme wurden mittels PCR auf korrekte Integration überprüft und die Fluoreszenz mikroskopisch detektiert. dienen. Es konnte gezeigt werden, dass alle Reporterstämme das Vektorplasmid an den zu erwartenden Stellen chromosomal integriert hatten. In einem Fall kam es zur partiellen Deletion des Integrationsplasmids und in mindestens einem Fall konnte gezeigt werden, dass es zur Mehrfachintegration des Vektors kam. Die Fluoreszenzuntersuchungen zeigten, dass 1. die agr-P3 Aktivität größtenteils homogen ist und maximal in der post-exponentiellen Wachstumsphase ist. 2. die cap-P Aktivität ist ebenfalls maximal in der post-exponentiellen Wachstumsphase. 3. cap-P Aktivität ist sehr heterogen innerhalb einer Kultur. 4. Promoterfusionen mit ven zeigten ein stärkeres Signal als Fusionen mit cer. 5. Die Analyse der Doppel-Integrante lieferte inkonsistente Ergebnisse und bedarf der weiteren Überprüfung. Im zweiten Teil dieser Arbeit sollte einer 3D-Kollagenmodells, in dem die Reporterstämme über einen längeren Zeitraum beobachtet werden können, etabliert werden. Guggenberger et al. konnten zeigen, dass Staphylokokken, die in Kollagen mit Fibrinogen eingebettet werden, eine Pseudokapsel aus Fibrinablagerungen und eine Mikrokolonie-assoziiertes Netzwerk ausbildeten (Guggenberger C, Wolz C, Morrissey JA, Heesemann J (2012) Two Distinct Coagulase-Dependent Barriers Protect Staphylococcus aureus from Neutrophils in a Three Dimensional in vitro Infection Model. PLoS Pathogens). Diese Strukturen sind einem Abszess sehr ähnlich und der Versuch kann daher ein in vitro Abszess-Modell darstellen. Es gelang abszessähnliche Strukturen von S. aureus innerhalb einer Kollagen-Matrix zu generieren. Allerdings konnte die Ausbildung der Pseudokapsel oder des Mikrokolonie-assoziierten Netzwerks nur partiell beobachtet werden. Die Analyse der Promotor-Aktivität innerhalb der Kollagen/Fibrin-Struktur zeige folgende, vorläufige Ergebnisse: 1. die Fluoreszenzintensität war für beide Promotor-Fusionen deutlich geringer als bei Wachstum der Stämme in flüssigem Kulturmedium. 2. Die cap-P Promoteraktivität scheint deutlich höher als die agr-P Aktivität zu sein. 3. Bakterien an der Peripherie der Struktur zeigten höhere Fluoreszenz. Die Ergebnisse legen einen Grundstein zur weiteren Etablierung eines Systems zur Analyse von Promoter-Aktivitäten innerhalb eines in vitro Abszess-Models.

From its vast expanses of cerulean waters to its azure heavens above, our world is dominated by blue, so it is no wonder that the color blue is favored by many across cultures and throughout the ag...

The aggregation of α-synuclein (Syn or S) to form insoluble fibrils is important in the pathogenesis of Parkinson's disease, but key risk factors remain ill-defined. We have developed Fluorescence Resonance Energy Transfer (FRET)-based assays for α-synuclein aggregation, using Green Fluorescent Protein variants Cerulean (C) or Venus (V), fused to each other (CV, VC) or to human synuclein (SC, SV etc). Bacterially expressed proteins were purified to homogeneity, and C-terminal fusions SC and SV largely retained their ability to aggregate in vitro. FRET signals from mixtures of SC and SV were used to monitor aggregation. These fusion genes were linked to the C. elegans unc-54 myosin promoter to generate integrated transgenic strains. Increased FRET signals, indicative of S aggregation, were observed following treatment of unc-54::SC + unc-54::SV double transgenic worms with low concentrations of mercury or chlorpyrifos, or with RNAi against hsp-70 and hip-1. Opposite changes in Yellow Fluorescent Protein (YFP) fluorescence in an unc-54::SV strain (NL5901) are likely to reflect FRET from Yellow Fluorescent Protein to aggregates of Syn fusion protein. This could provide the basis for a high throughput screening assay, which could be used for studying the effects of toxic chemicals and environmental pollutants on the aggregation of proteins such as Syn in vivo.

Construction of nests containing multiple layers or “stories” has been observed in a number of passerine species. These atypical nest structures are frequently a response to brood parasitism, wherein the host female will bury the parasite’s egg under a new layer of nesting material. They may also occur as a result of either intra- or interspecific competition for limited nest sites, with one individual usurping another’s nest and adding new material on top. However, such structures have not previously been documented in the Cerulean Warbler (Setophaga cerulea), a Neotropical migrant species that breeds in the eastern U.S. and Canada. Here, we report our observations of an unusual “double-decker” nest that was constructed in 2014 in Brown County, southern Indiana. A female Cerulean Warbler first built a typical nest, and several days later we discovered a second visibly distinct nest superimposed on top of the original one. Subsequent retrieval and inspection of the two-story nest revealed no buri...

Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photoinsensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.

This report presents a simple method named as sp-ECR to determine the molar extinction coefficient ratio ( γ ( λ ex )) of acceptor-to-donor in living cells at excitation wavelength λ ex , which is closely associated with the acceptor cross-excitation, the hardest issue of FRET quantification. sp-ECR determines γ ( λ ex ) by spectrally unmixing the emission spectrum of a donor–acceptor tandem construct under λ ex excitation without any additional references, such that this method can be performed under optimal imaging condition. We used sp-ECR to measure the γ (458) of Venus/Cerulean in living HepG2 cells on a confocal microscope, and the measured values were consistent with those obtained by lux-FRET method. We also used sp-ECR to measure the γ (458) values of Venus/Cerulean and YFP/CFP as well as YFP/GFP, the commonly used FRET FPs pairs in other two kinds of cancer cell lines on the confocal microscope, and found that the extinction coefficients of FPs depended on cell lines. After predetermining the γ (458) of Venus to ECFP, we used sp-ECR method to monitor the staurosporine (STS)-induced dynamical caspase-3 activation in single live A549 cells expressing SCAT3 by spectrally resolving the absolute FRET efficiency of SCAT3, and found that STS-induced caspase-3 activation in single cells is a very rapid process within 20 min.

International audience; ECFP, the first usable cyan fluorescent protein (CFP), was obtained by adapting the tyrosine-based chromophore environment in green fluorescent protein to that of a tryptophan-based one. This first-generation CFP was superseded by the popular Cerulean, CyPet, and SCFP3A that were engineered by rational and random mutagenesis, yet the latter CFPs still exhibit suboptimal properties of pH sensitivity and reversible photobleaching behavior. These flaws were serendipitously corrected in the third-generation CFP mTurquoise and its successors without an obvious rationale. We show here that the evolution process had unexpectedly remodeled the chromophore environment in second-generation CFPs so they would accommodate a different isomer, whose formation is favored by acidic pH or light irradiation and which emits fluorescence much less efficiently. Our results illustrate how fluorescent protein engineering based solely on fluorescence efficiency optimization may affect other photophysical or physicochemical parameters and provide novel insights into the rational evolution of fluorescent proteins with a tryptophan-based chromophore.

Background: The present study evaluates the effect of Spatholobus suberectus stem extract (SS) in the management of pancreatitis alone and in combination with heparin.Material and methods: Pancreatitis was induced pancreatitis by cerulean (50μg/kg, i.p.) five times at an interval of 1 h without any pretreatment of drug. Rats were treated with SS (100 and 200 mg/kg, p. o.) and heparin (150 U/kg, i.p.) alone and in combination for the duration of a week. Later pancreatic weight and blood flow was estimated and different biochemical parameters like concentration of D-dimer and Interleukin 1β (IL-1β) and activity of amylase and lipase were determined in blood of pancreatitis rats. Moreover effect of drug treatment on DNA synthesis and histopathology was also estimated on cerulean induced pancreatitis rats.Result: Results of this study suggest that treatment with SS alone and in combination with heparin significantly increase in prothrombin time and pancreatic blood flow than negative control group. There was significant decrease in concentration of IL-1β and D-dimer and activity of amylase and lipase in SS and heparin treated group than negative control group. Pancreatic DNA synthesis was also found to be reduced in SS and heparin alone and in combination treated group. Histopathology study also reveals that treatment with SS and heparin alone and in combination reduces edema, hemorrhages, leukocyte infiltration in the TS of pancreatic tissues.Conclusion: Present study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway and potentiates the effect of heparin in the management of pancreatitis.Keywords: Spatholobus suberectus, Pancreatitis, Heparin, Cerulean

Far from being passive, apoptotic cells influence their environment. For instance, they promote tissue folding, myoblast fusion and modulate tumor growth. Understanding the role of apoptotic cells necessitates their efficient tracking within living tissues, a task which is currently challenging. In order to easily spot apoptotic cells in developing Drosophila tissues, we generated a series of fly lines expressing different fluorescent sensors of caspase activity. We show that three of these reporters (GFP, Cerulean and Venus derived molecules) are detected specifically in apoptotic cells and throughout the whole process of programmed cell death. These reporters allow the specific visualization of apoptotic cells directly within living tissues, without any post-acquisition processing. They overcome the limitations of other apoptosis detection methods developed so far and notably, they can be combined with any kind of fluorophore.

Forest openings, or canopy gaps, are an important resource for many forest songbirds, such as Cerulean Warblers (Setophaga cerulea). We examined canopy gap selection by this declining species to determine if male Cerulean Warblers selected particular sizes, vegetative heights, or types of gaps. We tested whether these parameters differed among territories, territory core areas, and randomly-placed sample plots. We used enhanced territory mapping techniques (burst sampling) to define habitat use within the territory. Canopy gap densities were higher within core areas of territories than within territories or random plots, indicating that Cerulean Warblers selected habitat within their territories with the highest gap densities. Selection of regenerating gaps with woody vegetation >12 m within the gap, and canopy heights >24 m surrounding the gap, occurred within territory core areas. These findings differed between two sites indicating that gap selection may vary based on forest structure. Differe...

Item does not contain fulltext The introduction of weak, hydrophobic interactions between fluorescent protein domains (FPs) can substantially increase the dynamic range (DR) of Forster resonance energy transfer (FRET)-based sensor systems. Here we report a comprehensive thermodynamic characterization of the stability of a range of self-associating FRET pairs. A new method is introduced that allows direct quantification of the stability of weak FP interactions by monitoring intramolecular complex formation as a function of urea concentration. The commonly used S208F mutation stabilized intramolecular FP complex formation by 2.0 kCal/mol when studied in an enhanced cyan FP (ECFP)-linker-enhanced yellow FP (EYFP) fusion protein, whereas a significantly weaker interaction was observed for the homologous Cerulean/Citrine FRET pair (DeltaG0(o-c) = 0.62 kCal/mol). The latter effect could be attributed to two mutations in Cerulean (Y145A and H148D) that destabilize complex formation with Citrine. Systematic analysis of the contribution of residues 125 and 127 at the dimerization interface in mOrange.linker.mCherry fusion proteins yielded a toolbox of new mOrange-mCherry combinations that allowed tuning of their intramolecular interaction from very weak (DeltaG0(o-c) = .0.39 kCal/mol) to relatively stable (DeltaG0(o-c) = 2.2 kCal/mol). The effects of these mutations were also studied by monitoring homodimerization of mCherry variants using fluorescence anisotropy. These mutations affected intramolecular and intermolecular domain interactions similarly, although FP interactions were found to be stronger in the latter. The knowledge thus obtained allowed successful construction of a red-shifted variant of the bile acid FRET sensor BAS-1 by replacement of the self-associating Cerulean-Citrine pair by mOrange.mCherry variants with a similar intramolecular affinity. Our findings thus allow a better understanding of the subtle but important role of intramolecular domain interactions in current FRET sensors and help guide the construction of new sensors using modular design strategies.

Female song is more widespread than previously assumed, and is adaptive in many species; however, it may also be an aberrant, nonfunctional, behavior in others. We document novel vocalizations, including female song, from 2 paired female Cerulean Warblers (Setophaga cerulea) recorded in southern Indiana, USA, in June 2017. This is the first documentation of female song in this species. When assessed spectrographically, one female's vocalizations were similar to the typical zeet call of this species, but with appreciable differences in duration, composition, and frequency bandwidth. The other female's vocalizations, which we define as a song, had large frequency modulations as well as harmonics. The females' vocalizations resembled neither each other's nor the typical song of an adult male Cerulean Warbler. Unlike most other instances of infrequent female song in temperate-breeding passerines, these vocalizations only occurred during the incubation and nestling stages, rather than during mate acquisition. Based on their context, they appeared to function in intersexual communication with the male mate. We discuss potential explanations, both adaptive and nonfunctional, and urge researchers of this species to pay particular attention to the vocalizations of females in their study populations.

Hybridization events have long interested biologists because of their implications for species concepts and taxonomy. Documenting hybridization events is important because hybridization rates are commonly used as support for accepted taxonomic divisions. In July 2017, we observed a nest where a male Cerulean Warbler (Setophaga cerulea) and a female Blackthroated Blue Warbler (Setophaga caerulescens) were feeding nestlings. The Cerulean Warbler and Blackthroated Blue Warbler communicated with each other using song (male) and calls (female), similar to a nonhybrid pair of either parent species, and the pair was not observed to engage in aggressive behaviors toward each other. Interestingly, the Black-throated Blue Warbler is not known to breed in Indiana, although it is a regular migrant in the spring and fall. We describe the behaviors of the hybrid pair at the nest, provide photographs and audio recordings for documentation, and hypothesize that this instance of hybrid pairing may have occurred due to mate scarcity.