FSHB gene are required for gonadotrope expression of ovine FSHB. These enhancers are associated with regulation by activins and gonadotropin releasing hormone (GnRH). Additional distal promoter sequence between −4741/−750 bp is also required for expression. New transgenic studies presented here focus on this distal region and narrow it to 1116 bp between −1866/−750 bp. In addition, adenoviral constructs were produced to identify these critical distal sequences using purified primary mouse gonadotropes as an in vitro model system. The adenoviral constructs contained −2871 bp, −750 bp or −232 bp of the ovine FSHB promoter. They all showed gonadotrope-specific regulation since they were induced only in purified primary gonadotropes by activin A (50 ng/ml) and inhibited by GnRH (100 nM) in the presence of activin (except −232FSHBLuc). However, basal expression of all three viral constructs (in the presence of follistatin to block cellular induction by activin) was relatively high in pituitary non-gonadotropes as well as gonadotropes. Thus, gonadotrope-specific regulation associated with the proximal promoter was observed as expected, but the model was blind to distal promoter elements between −2871/−750 necessary for gonadotrope-specific expression of ovine FSHB in vivo. The new adenoviral-based in vitro technique did detect, however, a novel GnRH response element between −750 bp and −232 bp of the ovine FSHB promoter. We conclude that adenoviral-based studies in primary gonadotropes can adequately recognize regulatory elements on the ovine FSHB promoter associated with gonadotrope-specific regulation/expression, but that more physiologically based techniques, such as transgenic studies, will be needed to identify sequences between −1866/−750 bp of the ovine FSHB promoter that are also required for tissue/cell specific expression in vivo.The beta subunit of follicle stimulating hormone (FSHB) is expressed specifically in pituitary gonadotropes in vertebrates. Transgenic mouse studies have shown that enhancers in the proximal promoter between −172/−1 bp of the ovine  FSHB transcription has major control over serum FSH and, therefore, affects reproduction directly.It is well known that follicle stimulating hormone (FSH) is important for reproduction in all vertebrates. It is expressed only in pituitary gonadotropes and is secreted primarily through a constitutive pathway FSHB appears to be induced primarily by activins which are associated with a Smad response element on the ovine FSHB promoter between −172/−158 bp FSHB-promoter-luciferase reporter constructs (oFSHBLuc) in LβT2 cells (transformed embryonic mouse gonadotropes). Follow up studies using the same mutated oFSHBLuc constructs in transgenic mice showed that without the −172/158 site, only 0.1–1% of ovine FSHB expression is observed in gonadotropes in vivo Transcription of FSHB promoter FSHB expression may be indirect FSHBLuc expression was used to demonstrate gonadotrope-specific regulation by GnRH because it is more dramatic and more easily measured than the stimulatory effect of GnRH which is observed best when GnRH is delivered in a pulsatile manner.Gonadotropin releasing hormone (GnRH) is also a powerful regulator of gonadotropins, but to date, a specific site has not been clearly identified for positive or negative regulation by GnRH on the ovine FSHB in vivo. These data come from studies that used −4741 bp of ovine FSHB promoter to express luciferase (−4147oFSHBLuc) in transgenic mice FSHBLuc or −750oFSHBLuc were used as transgenes FSHB expression in vivo. These sequences are currently undefined.Strong evidence exists for other site(s) being critical for gonadotrope-specific expression of ovine FSHB in vivo FSHB necessary for induction by activin in vivo FSHBLuc in LβT2 cells varies between 4- and 12-fold. These constructs are also expressed in non-gonadotropes. Five cell lines, including NIH3T3 fibroblasts, express them only 20–50% less than LβT2 cells FSHBLuc in vivo. In transgenic mice, for example, −4741oFSHBLuc is expressed, at least, 1000–2000x more in pituitary gonadotropes than non-gonadotropes and usually expression in non-gonadotropes is undetectable FSHB proximal promoter, but has failed to identify equally important sequences in the distal promoter (−4741/−750 bp) needed for gonadotrope-specific expression in vivo.In vitro studies with LβT2 cells seemed promising for defining all sequences needed for regulating and expressing ovine FSHB promoter are critical for expression of porcine FSHB FSHB promoter has no similar Prop-1 sites. The gene encoding human FSHB needs only −350 bp of 5′ promoter sequences, but absolutely requires distal 3′ sequences between +2138 bp and +3142 bp for transgenic expression in the mouse pituitary FSHB gene have helped to define specific sequences between −750 bp and −4741 bp needed for gonadotrope-specific expression of ovine FSHB.Recent transgenic studies in the rat suggest that Prop-1 sites between −850 bp and −750 bp of the distal porcine FSHB promoter required for gonadotrope-specific expression. One was computer-driven and discovered highly conserved sequence patterns in the ovine, porcine and human upstream 5′ promoters. These sequences were considered potential elements that might direct gonadotrope-specific expression, so they were deleted from wild type −4741oFSHBLuc and the resulting constructs were used as transgenes to determine if the deleted sequences were important for gonadotrope-specific expression. The second approach used viral infection of primary gonadotropes and pituitary non-gonadotropes with viral oFSHBLuc constructs (oFSHBLuc-V) since these adult primary cells had a good chance of producing regulatory factors that create FSHB expression in gonadotropes or repress it in non-gonadotropes. It was thought that although LβT2 cells are of gonadotrope origin, they are transformed and might not produce one or more of these critical factors. To analyze expression of viral constructs (oFSHBLuc-V), we measured basal expression , maximal expression (activin was added to fully induce expression) and GnRH inhibited expression  were obtained from R&D Systems . Gonadotropin releasing hormone (GnRH), oligonucleotide primers for preparing viral constructs, collagenase Type I and Pancreatin were all purchased from Sigma-Aldrich . Fugene 6 was purchased from Roche Applied Science  and restriction enzymes plus luciferase assay kits were from Promega . Integrated DNA Technologies was the source of oligos for PCR. Dulbecco’s modified eagle medium and fetal bovine serum were bought from Invitrogen . Common reagents such as buffers, growth media and agar were purchased from Fisher Scientific, .FSHB promoter-reporter plasmid (−4741oFSHBLuc) was described previously FSHB promoter plus intron 1 driving expression of a luciferase gene in the GL3 basic vector. This plasmid was used to generate the LO- and LS-oFSHBLuc plasmid constructs that contain either −2361 bp or −1866 bp, respectively. Using site directed mutagenesis, a KpnI restriction site was produced at −2361 or −1866 and plasmids were digested and re-ligated to produce LO- or LS-oFSHBLuc, respectively.All transgenic mice were maintained and studied with the approval and oversight of the Institutional Animal Use Committee at the University of North Carolina, Chapel Hill, NC, or North Carolina State University. All transgenic mice were bred and cared for at the Biological Resource Facility of North Carolina State University. The wild type ovine To generate transgenic mice, the constructs were digested with KpnI and BamHI to release them from the plasmid backbone. The digests were sent to the University of North Carolina for purification and injection into the pronuclei of fertilized B6SJL mouse eggs. Transgenic mice were genotyped as described previously FSHBLuc transgenes were sacrificed at 7–40 weeks of age using CO2 and their pituitaries were collected within 5 min before being dispersed into single cell suspensions as described 5 cells/pituitary. Cells were then plated in 96-well Primaria tissue culture plates  at a density of 30,000 cells/well in 50 ul of medium 199 containing 10% charcoal-treated sheep serum plus 100 U/ml penicillin G and 100 ug/ml streptomycin  and allowed to attach for 2 days at 37°C under an atmosphere of 95% air: 5% CO2 in a humidified chamber before treatment. Cells were treated with hormones at the indicated doses and times as described in the figure legends. Treatments were terminated by lysis in 50 µl of 1× passive lysis buffer , and 15 µl of each cell lysate was assayed for luciferase activity. All of the experiments were performed at least three times and each treatment was assayed in triplicate or quadruplicate.Mice carrying LO- or LS-oFor in vivo experiments, mouse pituitaries or other tissues (0.3–1-mg) were snap-frozen in liquid nitrogen before homogenization in 200 µl of cell lysis reagent . Cell debris was removed by centrifugation at 10,000×g for 20 sec, and then 10 µl of all cell lysates were immediately assayed for luciferase activity using the luciferase assay system . Activity was measured for 20 sec using an automated 1420 Victor-Light micro plate luminometer . Finally, protein was assayed using a Qubit fluorometer . For primary pituitary cultures, cells were lysed using 50 µl passive Lysis solution  and 15 ul were analyzed in duplicate using the luciferase assay system.FSHB promoter controlling luciferase expression were produced as reported previously  FSHBLuc constructs in transgenic mice and should be expressed in gonadotropes, but not in non-gonadotropes. The −748oFSHBLuc construct is essentially the same as −750oFSHBLuc and it is the longest construct we tested in transgenic mice that did not express in any cell type in vivo Three plasmids containing different lengths of the ovine Transformed LβT2 cells were obtained as a kind gift from the laboratory of Dr. Pamela Mellon FSHBLuc-V constructs in inappropriate cell types so non-gonadotropes were also purified through two cycles to deplete them of as many gonadotropes as possible. All cells were cultured as noted above.Gonadotropes were purified from hemizygous H2Kk transgenic mice that were 7–40 weeks old FSHLuc, −750oFSHLuc and −2871oFSHBLuc in a viral context. Two of the constructs (−750oFSHLuc-V and −2871oFSHBLuc-V) were generated using the Adeno-X™ Expression System II. These truncated promoters plus intron 1 connected to the luciferase gene were amplified using Advantage-HF 2 PCR Kit  with the following primers:Three adenoviral constructs were obtained using Adeno-X™ Expression Systems from Clontech . These adenoviral constructs contained −232o5′-acggtaccggacatatgaatgcatcagctagcaaaca -3′(−2871 forward),5′-gaattcccgggcatatggaattacacggcatctttc-3′(−2871 & −750 reverse),5′-acggtaccggacatatgcatggagctcttagtctact-3′ (−750 forward).Then the amplified sequences were inserted into the promoterless pDNR-1r donor vector (System II) using the In-Fusion Advantage PCR Cloning Kit , and target sequences on the donor vector were put into the adenoviral backbone by Cre recombinase (System II). The insertion was confirmed by PCR analysis using the Adeno-X™ PCR screening primer set .FSHBLuc-V) was produced using the Adeno-X™ Expression System I since Clonetech system II was discontinued. The differences between System I and II were: System I was not designed for promoterless use so the CMV promoter had to be removed on the shuttle plasmid. Moreover, System I cannot use the efficient recombinase technique. As with system II, the −232oFSHBLuc construct was amplified using the Advantage-HF 2 PCR Kit (Clontech) with the following primers:The third adenoviral construct (−232o5′-ttgattattgactagtcaaggtaaaggagtgggtgg-3′ (−232 forward) and.5′-ccgtttaaacgctagctcttatcatgtctgctcgaa-3′ (−232 reverse).The pShuttle2 plasmid was cut with SpeI and NheI (Promega) to remove the CMV promoter. The amplified sequence was infused into pShuttle2 using the Fusion Advantage PCR Cloning Kit (Clontech). Finally, the sequence was cut out and ligated into the adenoviral backbone according to the protocol. This insertion of the foreign DNA was confirmed by PCR using the Adeno-X™ PCR screening primer set .9 ifu/ml.When all the three adenoviral constructs were made, they were transfected into a batch of HEK293 cells fresh from ATCC which were cultured the same as LβT2 cells (see above). The viruses were amplified and harvested according to the Clonetech protocol. After 3–4 generations of amplification, the virus titers reached 108 ifu/ml.Virus titers were determined using the Adeno-X™ Rapid Titer Kit . All three viral constructs were adjusted o the same titer of 10Primary cell culture was performed as described above. The infection was with the MOI of ∼100 ifu/cell in a volume of 10ul.2O, 0.1ul of each primer and probe . The primers and probes for 18S, mouse prolactin cDNA, and mouse FSHB cDNA were as follows:Cellular mRNA was extracted using TRI REAGENT . Then mRNA was converted into cDNA using the iScriptTM cDNA Synthesis Kit . For real time PCR, the setting for each well in the 96-well plate was: 1ul cDNA, 15ul Universal PCR Master Mix , 13.7ul dHthe primers and probe for 18S were5′-gaaactgcgaatggctcattaa-3′ (forward),5′-gaatcaccacagttatccaagtagga-3′ (reverse),and 5′−/56FAM/atggttcctttggtcgctcgctcc/3BHQ_1/−3′ (probe);for mouse prolactin they were.5′-tctcaaggtcctgaggtgccaaat-3′ (forward),5′-caattgcacccaagcatgcactga-3′ (reverse) and5′−/56-FAM/acaactgctaaacccacattcagtcca/3BHQ_1/−3′ (probe);FSHB they werefor mouse 5′-agagaaggaagagtgccgtttctg -3′ (forward),5′- acatactttctgggtattgggccg-3′ (reverse),and 5′−/56FAM/atcaataccacttggtgtgcgggcta/3BHQ_1/−3′ (probe);for the H2Kk they were5′-agacaaggcagctgtctacggaaa-3′ (forward),5′-gcagattgctctccagcaacagaa-3′ (reverse) and.5′−/56 FAM/agcatccacagttaccaagtgccc/3BHQ_1/−3′ (probe).The PCR cycle setting was 50C for 2 minutes, 95C for 10 minutes, then 95C for 15 seconds for 40 cycles followed by 60 C for 1 minute.All experiments were performed either in triplicate or quadruplicate and repeated 3 times or more. The data in FSHB promoter directs robust expression of the luciferase reporter gene specifically to the pituitary, but −750 bp or −215 bp of the same promoter cannot generate any expression FSHB promoters found unique promoter regions that show ≥75% homology in all three species in 1.1 kb of the distal promoter  and 1866 bp (LS). The LO promoter lacks 3 of the upstream high homology sections whereas LS lacks all 4 distal high homology sections. Three founder lines were produced for each LO and LS construct. All founder lines were verified to contain the correct construct using PCR and all founders were fertile with a normal transmission frequency. Pituitaries and other tissues from the homozygous offspring of all founder lines were analyzed for luciferase activity .To show that the shortest transgene (LS) was regulated by activin and GnRH the same as −4741oFSHBLuc, −748oFSHBLuc and −2932oFSHBLucFSHBLuc-V, −750oFSHBLuc-V and −2871oFSHBLuc-V where the “V” designates a viral construct. Although the adenoviral and plasmid constructs were slightly different in lengths due to the use of different cloning sites, both short constructs contained complete enhancer sequences associated with activin induction (and GnRH inhibition) FSHBLuc) is the longest construct we have used in transgenic mice that did not express FSHBLuc) contained all the sequences needed for robust transgenic expression in mice.Adenovirus constructs were used to infect primary pituitary cells since plasmid transfection was ineffective. One criterion for judging if adenovirus constructs behaved like plasmid constructs was to compare their expression and regulation in LβT2 cells 8ifu/ml). Each construct was used to express luciferase in 40,000 LβT2 cells and then the cells were treated with 50 ng/ml activin for 24 hours. All plasmid and viral constructs produced linear dose-responses with luciferase expression being similar for both plasmids and viruses  or adenovirus  were linear  and 4A iBoth adenoviral and plasmid constructs behaved alike in LβT2 cells as shown in It should be noted that the data in Mouse pituitaries from transgenic mice expressing H2Kk on the surface of their pituitary gonadotropes were dispersed and purified by magnetic immunomicrobead technology as reported earlier Gonadotrope and non-gonaotrope populations were assayed by real-time rtPCR for murine prolactin and FSHB mRNAs plus 18s RNA . Lactotropes comprise 30–40% of mouse pituitary cells and FSHB comes solely from gonadotropes which comprise ∼5% mouse pituitary cells FSHBLuc-V and −750oFSHBLuc-V are expressed much better than the longer −2871oFSHBLuc-V construct. These same results have been observed in LβT2 cells The data in FSHBLuc-V and −2871oFSHBLuc-V constructs, but surprisingly it did not inhibit expression of −232oFSHBLuc-V. This phenomenon was not observed in LβT2 cells and indicates a significant difference between regulation by GnRH in primary and LβT2 transformed gonadotropes.As expected GnRH inhibited the −750oFSHBLuc-V constructs in non-gonadotropes. There should not have been any expression in these cells if proper cell-specific targeting had occurred. A statistical comparison of the data in FSHBLuc-V constructs is not significantly different in gonadotropes compared to non-gonadotropes. Finally, it should be noted that neither activin nor GnRH (100 nM) had any significant effect on expression of any of the oFSHBLuc-V constructs in non-gonadotropes as expected.FSHB promoter linked to the luciferase gene and all but two expressed high levels of luciferase (1–10 million RLU/mg protein) in the pituitary th (∼5%) of pituitary cells are gonadotropes and they are the only pituitary cells producing luciferase FSHBLuc in gonadotropes versus non-gonadotropes is calculated to be 20×100 = 2,000∶1.Prior to this study, 12 founder mice were produced that contained 4741 bp of ovine FSHBLuc and 3 transgenic founders containing −750oFSHBLuc were also produced and none expressed luciferase in any tissue FSHB in vivo exist between −750 bp and −4741 bp of the ovine FSHB promoter. One of the −4741oFSHBLuc transgenes that did not express in vivo had a deletion from −2755 bp to −3275 bp suggesting that important elements within this sequence might be especially important for gonadotrope expression. FSHB promoter contains three sequences between −2755 bp and −3275 bp that have high homology to similar sequences on the human and porcine FSHB promoters. The 5′ sequences of the human promoter that are homologous to the ovine sequences are so far upstream of the transcription start site (−5 to −6.1 kb) they have never been included or tested for importance in any human FSHBLuc transgene. The concept that these conserved sequences might help control FSHB expression in many vertebrates needed to be tested. Therefore, we produced transgenics that carried ovine FSHBLuc constructs missing the conserved homologous regions, −2361oFSHBLuc (LO) and −1866oFSHBLuc (LS). As noted in results, we created 8 different constructs longer than LO and all expressed at least as well as LO and LS (see FSHBLuc) by follistatin, activin and activin plus GnRH as well as LS  was expressed even better than the LO transgene (−2361oFSHBLuc) and equally well as −4741oFSHBLuc previously reported FSHB expression. Therefore, the critical 5′ sequences lie between −1866 bp and −750 bp for gonadotrope-specific expression of ovine FSHB. Since the porcine promoter sequence is different from the ovine sequence in this region and the human FSHB gene requires 3′ sequences for expression, it may be that unique requirements exist for the expression of many vertebrate FSHB genes. This later scenario seems highly unlikely, however, for a gene so important for the preservation of all vertebrates.We show here that the LS transgene  to study gonadotrope specific expression of ovine FSHB: −232oFSHBLuc-V, −750oFSHBLuc-V, and −2871oFSHBLuc-V.Since the homology approach did not locate 5′ distal promoter sequences needed for gonadotrope-specific expression of ovine FSHBLuc-V, contained sequences previously known to be important for gonadotrope-specific regulated expression FSHBluc and −2871FSHBLuc in primary gonadotropes (FSHBLuc) in primary pituitary cultures  size The data in dotropes . These cFSHB gene are necessary for FSHB expression in vivo. Unfortunately, neither primary gonadotropes nor LβT2 cells seem suitable for identifying these important sequences. It has been shown, however, that primary gonadotropes can be used to further investigate oFSHB promoter sequences between −750/−232 bp that negatively regulate activin-induced expression in vivo. Finally, it is worth noting that transgenic studies could efficiently and rapidly identify the critical sequences needed for gonadotrope-specific expression given the information presented herein.IN CONCLUSION, it has been shown here that 5′ distal promoter sequences between −1866/−750 bp of the ovine


A robust and copper-free clicked quantum dot–DNA sensor for sensitive and ratiometric detection of specific DNA and protein targets at the pM level is reported. via highly efficient copper-free “click chemistry” (CFCC) between a dihydro-lipoic acid–polyethylene glycol–azide (DHLA–PEG–N3) capped QD and a cyclooctyne modified DNA. This represents an excellent balance between the requirements of high sensitivity, robustness and specificity for the QD-FRET (Förster resonance energy transfer) based sensor as confirmed by a detailed FRET analysis on the QD–DNA conjugate, yielding a relatively short donor–acceptor distance of ∼5.8 nm. We show that this CFCC clicked QD–DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA–PEG–N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor. Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD–DNA conjugate.We report herein the successful preparation of a compact and functional CdSe–ZnS core–shell quantum dot (QD)–DNA conjugate  Although the FRET efficiency can be enhanced by increasing the ratio of acceptors on each QD, such designs are inefficient at low target to QD ratios.2 Therefore for biosensing, it is important to balance the requirements of sensitivity and robustness because they are often incompatible. In this regard, QDs capped with PEGylated small-molecule ligands appeared to be highly attractive; they are relatively compact yet display good stability and, more importantly, effective resistance to non-specific adsorption of biomolecules.2The unique, size-dependent, highly stable and bright fluorescence of quantum dots (QDs) make them powerful tools in broad ranges of bio-related applications.i) catalysed azide–acetylene cycloaddition, best known as the “click chemistry” (CuCC),5 is highly powerful and versatile; it offers exquisite functional group selectivity and high yield. It has been used successfully in preparing a wide range of functional nanoparticle bioconjugates  for sensing and biomedical applications.5 However, the CuCC is unsuitable for the QD, because the Cu(i) catalyst used in the CuCC can efficiently and irreversibly quench the QD fluorescence.6 The Cu-free “click chemistry” (CFCC) between strained cyclooctynes and azides happens rapidly and efficiently, and moreover, it does not require any Cu catalyst.7 Therefore, the CFCC appears to be ideal for efficient QD-bioconjugation without compromising the QD fluorescence.7 Indeed, the CFCC has been successfully used to make functional QD–protein/small-molecule conjugates recently for live virus labelling/imaging and intra-cellular trafficking studies.8 Despite such developments, the QDs used in these studies were all capped with polymer based ligands (and hence of relatively big sizes) because the QD sizes here are less critical for such applications.8 To our knowledge, the CFCC has yet to be used to develop QD-FRET based biosensors where the compact size of the QD-bioconjugate is known to be of critical importance. Herein, we report the successful preparation of the first compact and functional QD–DNA conjugate via the CFCC between a dihydrolipoic acid–polyethylene glycol–azide (DHLA–PEG–N3) capped CdSe–ZnS core–shell QD and a cyclooctyne modified DNA, giving a good balance between the requirements of high sensitivity, specificity and robustness. This is supported by a FRET analysis showing a relatively short QD-dye distance of ∼5.8 nm for the QD–DNA FRET system. Moreover, the CFCC clicked QD–DNA conjugate is found not only to retain the native fluorescence quantum yield (QY) of the parent QD, but also well-suited for robust biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability even in complex media, e.g. 10% human serum, on a conventional fluorimeter. It can also directly detect, at the pM level, a specific protein via the encoded DNA aptamer sequence.Besides surface capping, a robust and efficient QD-bioconjugation chemistry that can offer high bioactivity without compromising the QD fluorescence is also important. In this regard, the Cu head group, a polyethylene glycol moiety of a molecular weight of 600  and a terminal azide group (for efficient DNA conjugation via the CFCC), DHLA–PEG600–N3, was prepared  and the structural compactness (for high sensitivity).2 Then a hydrophobic CdSe–ZnS core–shell QD  was made water-soluble by ligand exchange with the DHLA–PEG600–N3 in a mixed solvent of CHCl3–ethanol using our previously established procedures,3 yielding the QD–DHLA–PEG600–N3 which was readily soluble in polar solvents. Its fluorescence QY was found to decrease to ∼6.0% (and hence a decrease of ca. 70%), which is in good agreement with most other reports in the literature where most hydrophobic CdSe–ZnS core–shell QDs typically showed a QY decrease of 50–80% following the ligand exchange and transfer to aqueous media.4 A single-stranded (ss) target DNA encoded with a 29 mer anti-thrombin (TB) aptamer sequence with strong affinity for TB  ester activated cyclooctyne to yield TBA–cyclooctyne, which was then reacted with the QD–DHLA–PEG600–N3 in a mixed solvent of ethanol–water at a molar ratio of 30 : 1. This led to QD–TBA covalent conjugation via the efficient CFCC approach. Approximately 20 strands of TBAs were found to be conjugated to each QD, denoted as QD–TBA20 hereafter, this gave a DNA conjugation efficiency of ∼67%. The detailed experimental procedures for the ligand synthesis and QD–DNA conjugation are given in the ESI.20 was determined as ∼5.9% using rhodamine 6G in ethanol as the calibration standard (QY 95%),3 which is effectively the same as that of the QD–DHLA–PEG600–N3 (ca. 6.0%).20 was found to be highly soluble and stable in aqueous media. It showed no change of physical appearance or fluorescence after being stored in a fridge at 4 °C for over two months. More importantly, the QD–TBA20 was found to have effectively retained the native QY of the parent QD–DHLA–PEG600-N3. In contrast, conjugation of the H2N–TBA to a water-soluble, glutathione capped QD (the same batch of QD) by using 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC)–NHS mediated covalent coupling resulted in significantly reduced QY. The fluorescence intensity of the former was ∼6.4 times as strong as the latter  of different molar ratios was carried out to ensure a relatively small donor–acceptor distance (r) for high sensitivity. This is because the FRET efficiency (E) decreases dramatically with the increasing r value following the Förster dipole–dipole interaction formula:R0 is the Förster radius of the single donor (QD)–single acceptor (Atto647N) FRET pair here, for which E = 50%. R0 can be estimated from the spectral overlap (I) and the QY of the QD donor via the following equation:nD is the refractive index of the medium (estimated as 1.4 here), NA is Avogadro's number (6.02 × 1023), kp2 is the orientation factor (2/3 here assuming randomly orientated fluorophores). The integral of the spectral overlap, I, is defined as:Prior to using the CFCC clicked QD–DNA conjugate for sensing, a FRET analysis on the CFCC clicked QD–TBAI is the integral of the donor–acceptor spectral overlap over all wavelengths λ, FD(λ) is the normalized donor emission and εA is the absorption extinction coefficient of the acceptor.I, can be calculated from Fig. S2B,15 M–1 cm–1 nm4. These combined with the QY of the QD (5.9%) and the parameters above yielded a R0 value of 4.25 nm for the QD–Atto647N FRET pair (at 1 : 1 molar ratio).The normalised absorption and emission spectra of the QD and Atto647N and the corresponding spectral overlap function are shown in the ESI, Fig. S2.n identical acceptor systems, E is given by the following equation:E can be estimated directly from the acceptor fluorescence enhancement via the following equation:IA and ID are the integrated acceptor and donor fluorescence, respectively. Here a ratiometric FRET analysis is used which can be more reliable than those only based on donor quenching because it can be essentially insensitive to instrument noise and signal fluctuations, making the analysed result potentially more accurate. Moreover, IA here only comes from the QD-sensitised FRET because the acceptor is not directly excited under our experimental conditions (see the next section below) and any unbound species will be too far away to participate in the FRET process, and hence do not interfer with target detection, allowing highly convenient and separation-free measurements.For a single-donor (QD here) simultaneously FRET with 20 sample after hybridization with different molar equivalents of DNA29  with a relatively short donor–acceptor distance r of 5.82 ± 0.01 nm. This result confirms that the CFCC clicked QD–DNA conjugate FRET system is indeed compact, and moreover, all Atto647N labels on the DNA29 strands are bound to the QD at an identical spatial separation .20 based FRET sensor in detecting complementary DNA probes was further evaluated by using DNA29. Hybridization of DNA29 with the QD–TBA20 should bring the Atto647N dye (acting as the FRET acceptor) in the close proximity to the QD, leading to the QD sensitized Atto647N FRET signal upon excitation of the QD. An advantage of the FRET based signal readout over other approaches (e.g. donor quenching or life time change) is its ratiometric signal, which can be effectively insensitive to signal fluctuation and instrument noise, allowing for more reliable and accurate detection.2 Moreover, since FRET only happens over short distances (ca. < 10 nm), any free, unbound species will be too far to participate in FRET with the QD donor and hence undetected, allowing for convenient probe detection to be carried out in a separation-free format.3n Interestingly, the QD–TBA20 (CQD = 2 nM) fluorescence in PBS  was found to be enhanced significantly after treatment with a cysteine–histidine6 short peptide and/or bovine serum albumin  and a more rapid phase at higher concentrations , such free DNA29 strands are unable to participate in the FRET process and hence undetected. Note here that 40 nM corresponds to the total [TBA] in the 2 nM QD–TBA20 conjugate, suggesting that all TBAs conjugated to the QD are functional and available for hybridization. The detection limit (DL) for DNA29, based on 3 times the standard deviation/slope of linear calibration over the lower concentration range (3σ/slope), is estimated as ∼91 pM,3 making it one of the most sensitive QD-FRET based sensors for direct DNA quantification without probe pre-amplification using conventional fluorescence spectroscopy ,12 surface plasmon resonance 12 and quartz crystal microbalance 12 detection .13 The excellent linear relationship observed here clearly confirmed that all DNA29 strands were hybridized with the QD at identical spatial separation between the QD donor and the dye acceptor. This result is also in good agreement with the earlier FRET analysis where E can be fitted very well (R2 = 0.991) by the single-donor FRET with multiple identical acceptor models.2 The two-stage linear dependence observed here may indicate two different phases of DNA hybridization: the slower increase over the 0–5 nM range is likely due to incomplete hybridization of DNA29 with the QD–TBA20, arising presumably from the low, sub-Kd levels of [DNA29], whereas the faster increase over the 10–40 nM range may be attributed to more effective, complete binding of the introduced DNA29 to the QD–TBA20 under such conditions. Given that Kd values of 17, 19 and 41 nM have been reported for 25,14 20,14 and 12  were determined as 2.563 and 0.00314, respectively, yielding an outstanding signal discrimination ratio of 816 between the full- and non-complementary DNA probes  were found to be 0.112, 0.091, 0.062, 0.036 and 0.011 nM–1 for DNA29, DNA18, DNA15, DNA12 and DNA12-SM, respectively. Therefore the slope of the IDye/IQD increase rate for the DNA29 is ∼3 times that of DNA12, while that for DNA12 is a further ∼3.3 times that of DNA12-SM, the same length (12 mer) probe containing just a single base mismatch with TBA, equivalent to single nucleotide polymorphism (SNP). The CFCC clicked QD–DNA sensor can therefore offer a SNP discrimination ratio of ∼3.3 for the 12 mer DNA probes. Similar levels of probe length dependence and SNP discrimination ratio (ca. > 3 between DNA12 and DNA12-SM) were also obtained from the IDye/IQD ratios at 40 nM probe concentration  that forms more stable duplex with TBA can effectively displace a shorter labelled DNA  pre-hybridized with the QD–TBA20, leading to a decreased FRET as the unlabelled probe readout signal c and a recently optimised electrochemical DNA sensor (ca. 8-fold).12 An advantage of our approach here is its ratiometric signal, which is insensitive to instrument noise and signal fluctuation, allowing more reliable target detection. In addition, the DNA displacement assay was found to work equally efficiently in complex media, such as PBS with a large excess of BSA (10 μM) and in 10% human serum, suggesting that it may have good potential for clinical applications.n-linear , where 5n-linear , inset, 20 can be readily extended for label-free protein sensing via the anti-thrombin DNA aptamer sequence encoded within the TBA sequence, where the formation of thrombin (TB)–TBA complex can effectively displace the pre-hybridised reporter DNA12-SM, leading to the FRET decrease  was not as high as that obtained in DNA29-NF detection (∼29), suggesting that the TB binding here is less efficient in displacing the DNA12-SM reporter strands from the QD–TBA20 conjugate as compared to DNA29-NF. Given that the binding affinity between the 29 mer anti-TB aptamer and TB (Kd ∼ 0.5 nM)11 is as strong as that of the TBA/DNA29 duplex (most likely to be in the low nM range as described above) here, the relatively low efficiency in displacing the reporter strands observed for TB here is therefore attributed to the significantly greater size of the TB–aptamer complex as compared to the TBA/DNA29 duplex, leading to steric hindrance and reduced accessibility for TB binding on the QD–DNA conjugate, especially under high [TB] conditions. Similar to the DNA29-NL based displacement assay above, a non-linear response curve between the I605/I665 signal and [TB] was also observed  between the full- and non-complementary DNA probes, which is the highest for the QD-FRET based sensors. Moreover, it can discriminate between the perfect-match and the SNP targets in 10% serum. The sensor has also been exploited for sensitive label-free detection, at the pM level, of thrombin


Deeper investigations at earlier stages of development revealed that msw is involved in the specification and differentiation of precursors of the Kupffer's vesicle, a structure homologous to the mammalian node. To test the hypothesis that msw, by controlling Kupffer's vesicle morphogenesis, controls lateralized behaviors related to diencephalic asymmetries, we analyzed left- and right-parapineal offspring in a “viewing test”. As a result, left- and right-parapineal individuals showed opposite and complementary eye preference when scrutinizing a model predator, and a different degree of lateralization when scrutinizing a virtual companion. As maternal effect genes are expected to evolve more rapidly when compared to zygotic ones, our results highlight the driving force of maternal effect alleles in the evolution of vertebrates behaviors.In the present work we report evidence compatible with a maternal effect allele affecting left-right development and functional lateralization in vertebrates. Our study demonstrates that the increased frequency of reversed brain asymmetries in a zebrafish line isolated through a behavioral assay is due to selection of  Solea spp) to the almost perfect external appearance of many animals. From the evolutionary point of view it can be supposed that levels of bilateral symmetries can be limited or promoted by natural selection due to the social and individual trade offs associated with survival and reproduction nodal and lefty families and, subsequently, to that of the transcription factor pitx2 in the left lateral plate mesoderm (LPM) of chick, mice, frog and zebrafish situs inversus in humans nodal and Pitx orthologues expression in two species of snails with opposite body handedness and direction of shell coiling. Authors found that nodal and Pitx are both expressed in the embryo on the right side in dextral species and on the left side in sinistral species. These results suggest that the asymmetrical expression of nodal and Pitx may represent an ancestral feature conserved in the evolution of Bilateria During the evolution of animal body plans, symmetry has been broken at least three times, possibly for purposes linked to feeding and escape behaviors. It can be easily recognized that the last broken symmetry is the left-right  whose level ranges from extreme situations  line, found that adult zebrafish with normal heart position preferred to bite targets on their right, while fish with reversed heart position did the opposite. Moreover, fry with either right or left heart were found to have an opposite pattern of eye use in the mirror test, although they did not differ in two other lateralized measures. Recently, Dadda et al. At least two more studies provided evidences of a correlation between neuroanatomical, visceral and behavioral asymmetries. Barth et al. mother-of-snow-white (msw), affects morphogenesis of Kupffer's vesicle and controls the proportion of LR asymmetries in the progeny. A second purpose of this work was to evaluate the role of the msw maternal allele in the development of neuroanatomical asymmetries which seem to be associated with behavioral asymmetries. To establish whether a concordance exists between parapineal position and eye use we compared adults with opposite parapineal position in a modification of the “viewing test” used by Facchin et al. In this study we have continued the genetic analysis of the alleles isolated by Facchin et al. We found that in zebrafish a polymorphic maternal effect variant called Facchin et al. could isolate in five generations two strains of zebrafish: GTLE and TLRE that used preferentially the left or the right eye, respectively, when inspecting a mirror. Histological analysis of larvae of the two strains revealed that TLRE fish had a very high percentage of larvae with parapineal on the right side of the body leftover expression. The expression of this habenular marker has been reported to be stronger in the left dorsal habenula than in the right in about 95–97% of the embryos. This is a consequence of the neural connection of the left-sided parapineal organ with the ipsilateral habenular nucleus leftover is stronger in the right habenula (6) = 0.391, p = 0.709): embryos with reversed brain asymmetries (R-PPO) were in mean 3.6%±2.6 (n = 441). Conversely, mating pairs between four different females from TLRE line and males from GTLE produced offspring in which a mean of 23.9%±5.6 of embryos showed reversed epithalamic asymmetries (n = 699). The same frequencies of R-PPO offspring were reported when TLRE females were crossed to males of wild type strains . No significant difference has been found between the crosses of TLRE females either with GTLE or WT males  = 0.575, p = 0.586) ((4) = −0.014, p = 0.990)  . Moreove990) see .mother of snow-white (msw) after the fact that the maternal allele that controls the offspring phenotype has been selected using the mirror test.These results led to the most suitable hypothesis that the behavioral selection promoted the isolation of a spontaneous polymorphism in a maternal effect gene that we called 0), in which GFP is expressed also in the pineal complex thus allowing the in vivo scrutiny of the position of the parapineal organ 1 generation were screened for the position of the parapineal organ at 3 dpf and fish with left  and right  parapineal organ were raised separately. Then, F1 males and females with right or left parapineal were mated to obtain the F2 generation zf15 line  = 184.812, p<0.001). For simplicity embryos derived from msw?/? females will be referred to as M-msw?/? embryos.Twenty-four lefty1, which is expressed in the dorsal diencephalon before development of epithalamic structures msw−/− embryos had normal left-sided lefty1 expression (60.5%) compared to M-msw+/+ and M-msw+/− .We next examined the transcription of the asymmetric marker -msw+/− . The thrsouthpaw (spaw), the earliest nodal-related gene with a LR asymmetrical expression described in zebrafish  and predicted to promote the expression nodal-related genes, cyclops (cyc/ndr2) and lefty1 in the left dorsal diencephalon during later somitogenesis stages southpaw in the LPM in 15-18-somite stage embryos although similar to those of lefty1 expression in the left dorsal diencephalon  while M-msw+/+embryos do not significantly differed from wild type .Based on these results, we decided to focus on a previous step in zebrafish bilateral development, the expression of the gene ebrafish . spaw iscephalon , show a spaw expression has been shown to occur after the onset of the cilia-driven leftward fluid flow generated by the zebrafish Kupffer's vesicle (KV) spaw expression in M-msw−/− embryos suggested that the characterization of this maternal effect allele may extend to earlier developmental stages. Elegant studies demonstrated how disrupted leftward flow affects the normal left sided expression of Nodal in the left LPM msw−/− embryos. Furthermore it has been shown that events disrupting normal Kupffer's vesicle morphogenesis also lead to laterality defects msw females. In line with a previous study on the morphological characterization of the zebrafish Kupffer's vesicle The activation of msw−/− embryos , in addition, a percentage of 12.5% of M-msw−/− embryos completely lacked Kupffer's vesicle (msw+ embryos lacking KV. The AP average diameter is 48.6±14.3 µm in M-msw−/− embryos (n = 39); 57±13.0 µm in M-msw+/−embryos (n = 206) and 65.5±7.7 µm in M-msw+/+ embryos (n = 25). The difference between the three maternal genotypic classes of embryos is statistically significant  = 34.08, p<0.001). Post hoc analyses (LSD method) revealed no significant difference between WT control (n = 110) and M-msw+/+ embryos (p = 0.291).Results showed a significant reduction in the size of KV of M- vesicle . We coulmsw−/− embryos (n = 39); 58.6±12.4 µm in M-msw+/−embryos (n = 206) and 66.7±9.2 µm in M-msw+/+ embryos (n = 25). Again, the difference between the three maternal genotypic classes of embryos is statistically significant  = 34.65, p<0.001). Post hoc analyses (LSD method) revealed no significant difference between WT control (n = 104) and M-msw+/+ embryos (p = 0.287).The LR average diameter is 50.5±13.6 µm in M-msw−/−, M-msw+/− and M-msw+/+ embryos is 1903.3±1061.8 µm2 (n = 38), 2565.3±1051.5 µm2 (n = 205) and 3156.8±695.5 µm2 (n = 25). Differences in the size of KV between the three maternal genotypic classes of embryos  = 36.10, p<0.001), but post hoc analyses (LSD method) revealed no significant difference between WT control (n = 104) and M-msw+/+ embryos (p = 0.074). A correlation analysis has also been performed to verify if there was a correspondence between the percentage of embryos with reversed parapineal produced by females of the three classified genotypic classes and the size of KV of their offspring. We analyzed KV and brain asymmetries of embryos and larvae respectively derived from two msw+/+ females, three msw+/− females and two msw−/− females. As reported in the graph  the directional flow generated by cilia-beating into the KV in M-msw−/− embryos, thus leading to a randomization in the expression of nodal-related genes and nodal-dependent downstream genes. Beads microinjection procedure msw−/− females. We successfully injected 15 M-msw−/− embryos, all showing a counterclockwise movement of fluorescent beads inside the lumen of KV. Similarly 25 control embryos have been successfully injected all showing a counterclockwise movement of fluorescent beads inside the lumen of KV , a group of cells expressing sox17 in the most posterior region of the embryos. This group of cells does not undergo involution or epiboly, rather, they reorganize in shape and differentiate in a mature KV containing counterclockwise beating cilia sox17 expression at that developmental point in WT and M-msw−/− embryos (msw−/− embryos (n = 55) compared to WT (n = 58)  = 9.096; p<0.001; see somatostatin in the pancreas (data not shown).In order to understand the origin of KV defects in M- embryos . Resultsmsw−/− embryos were separated according to their parapineal organ position and grown to adulthood.We then decided to test the hypothesis that a natural occurring polymorphism in a maternal effect allele involved in LR development and lateralization is maintained in the population due to its possible adaptive benefits for predator escape behavior. For this purpose, M-(23) = 2.596, p = 0.016) while R-PPO use preferentially the right eye (t(23) = 2.742, p = 0.012); the difference between L-PPO and R-PPO is significant  = 13.499, p = 0.002). When looking at their mirror image L-PPO use preferentially the right eye (t(23) = 2.168, p = 0.041) while R-PPO showed a marginally non-significant tendency to use the left eye (t(23) = 1.756, p = 0.092); the difference between L-PPO and R-PPO is significant  = 7.691, p = 0.008).L-PPO and R-PPO revealed different patterns of eye use for the both the dummy predator and the mirror image inspection . L-PPO l(23) = 1.070, p = 0.296; R-PPO t(23) = 1.420, p = 0.169) and did not differ between themselves  = 3.146, p = 0.183).Both L-PPO and R-PPO showed no lateralization in viewing a neutral stimulus  = 14.860, p<0.001) and no effect of sex  = 2.606, p = 0.166) or order of presentation  = 0.714, p = 0.496). No other factors or interactions were significant.To control for possible effects of sex and order of presentation, we performed a general ANOVA with position of the parapineal organ (L-PPO or R-PPO), sex and order of presentation as between-subjects factors and stimulus type  as within-subject factor. We found a significant interaction between parapineal position and stimulus type (F(46) = 2.044, p = 0.047) but not in the other two tests (mirror t(46) = 0.200, p = 0.842; neutral stimulus t(46) = 0.749, p = 0.458). A general ANOVA showed that, on the whole, zebrafish were more lateralized when looking at the predator  = 33.131; p<0.001); L-PPO and R-PPO did not differ in absolute lateralization  = 2.204; p = 0. 145) but there was a significant interaction stimulus x parapineal position  = 3,388; p = 0.038).A low laterality index, as observed with mirror image in R-PPO fish or with the neutral stimulus in both groups, may derive either from the subject being aligned in direction but poorly lateralized or from the group being composed of an equal proportion of left and right lateralized individuals. To unravel this point, we calculated for L-PPO and R-PPO an absolute index of laterality , which provides a measure of the degree of lateralization independently from its direction. L-PPO and R-PPO zebrafish differed in the degree of lateralization when viewing the predator  of shell coiling in snails (Limnaea sp.)nodal and Pitx orthologues have been isolated in two species of snails with opposite direction of shell coiling. The authors found that the side of the embryo that expresses nodal and Pitx is related to body chirality: both genes are expressed on the right side of the embryo species with dextral shell coiling (Lottia gigantea) and on the left side in the species with sinistral coiling Based on a preliminary Mendelian analysis of this trait, we performed selective crosses to validate the hypothesis that the transmission of the allele reflected those of a recessive maternal effect. Indeed, homozygous recessive females could be identified with the screening of their progeny for the position of the parapineal organ at the 3 dpf stage. Analysis revealed that females could be classified into three groups according to the percentage of reversed brain asymmetries in their offspring and, in addition, the three phenotypic groups of females fit the expected mendelian proportions. In facts, the incross between offspring generated by the outcross of putative nodal activates its own expression and also the expression of its inhibitors of the Lefty/Antivin family squint (ndr1), cyclops (ndr2) southpaw (ndr3). In zebrafish the spaw gene is the earlier Nodal gene with asymmetric transcription along the left lateral plate mesoderm and has been predicted to activate the expression of cyclops (cyc/ndr2) and lefty1 in the left dorsal diencephalon at later somitogenesis stages msw−/− embryos we found altered, bilateral or right-sided, expression of lefty1 and spaw thus reflecting the subsequent organization of neuroanatomical asymmetries. Therefore, embryos expressing spaw and lefty1 on the left side can be predicted to have normal asymmetries, i.e. those with spaw and lefty1 expression on the right side (reversed asymmetries), while embryos with bilateral expression of spaw and lefty1 can be predicted to develop normal or reversed asymmetries with equal frequencies as already discussed for heart looping by other authors msw allele is likely to be involved in the differentiation and specification of precursors of the Kupffer's vesicle. In fact, as evidenced by sox17 analysis, at the 50% epiboly/shield stage M-msw−/− embryos have a smaller KV due to the reduced number of its precursors: the dorsal forerunner cells (DFCs). Events leading to the formation of a reduced or even to an absent KV are known to result in disrupted expression of asymmetric L-R signals as well as subsequent randomization of organ laterality msw allele is involved in the mechanism of DFCs differentiation and KV formation without affecting ciliogenesis and cilia motility. Briefly, the maternal effect allele msw is reported to control LR development of epithalamic structures by regulating KV morphogenesis.Our data are consistent with the hypothesis of a maternal effect allele controlling the generation of LR asymmetries at the gene expression level. Accordingly, we decided to perform a backward stepwise analysis of key developmental stages of L-R development. Previous work has demonstrated that during L-R development Lymnaea stagnalis the direction of body chirality, which is controlled by a maternal effect gene Our findings point out the evolutionary importance of maternal genetic control on the behavioral phenotype of the progeny. In fact we hypothesize that in zebrafish, as in other lower vertebrates with external fertilization and lack of parental/maternal care, the main source for genetic control of phenotypic plasticity and evolutionary response to selection could be represented by the maternal genetic factors provided in the egg msw−/− fish in a “viewing test”. The results of the viewing tests in fish of the F3 generation are largely consistent with the pattern of lateralization already described in a previous study using different laterality tests on fish of the msw pedigree (generation F1) To support the hypothesis that the msw allele controls lateralized behaviors related to diencephalic asymmetries, we analyzed left-and right-parapineal M-Numerous studies have dealt with the genetics of lateralization, focusing their attention in mammals and other vertebrates Recently Klar Evidence also comes from studies of non-human species. In chimpanzees, handedness is usually measured by means of a tube task where the subject is requested to obtain peanut butter from a tube using one hand. The percentage of right-handed offspring from right-handed mothers ranged from 46 to 86% as a consequence of developmental instability experienced by the offspring indicating that other mechanisms, rather than simple mendelian genetic factors, affect lateralization In conclusion, as maternal effect genes are expected to evolve more rapidly when compared to zygotic ones msw and wt Zebrafish strains 2 females were housed separately to allow their identification. For behavioral tests msw−/− females were mated to males from the transgenic line tg(foxD3:GFP)zf15 Wild type stocks used in this work and for selection are from Giotto Leo  or from Tupfel Longfin (TL) strain. lovsox17ntllefty1spawWhole-mount in situ hybridization was performed as described previously This procedure has been performed according to protocols described in previous works of other authors Embryos at the 5–10 somite stage were mounted and microinjected as previously described The area of DFCs cell mass after in situ hybridization has been measured as depicted in Viewing tests have been successfully used in previous studies to estimate asymmetries in eye use in fish Twenty-four adult zebrafish with left parapineal  and 24 adult zebrafish with right parapineal  were used for this experiment.Echinodorus bleheri. To prevent any side bias the original picture was duplicated and then rotated horizontally in order to obtain a symmetric stimulus. The second stimulus, the hazardous one, was a dummy predator, previously adopted for similar purposes in other species of small fish The apparatus used was similar to that described elsewhere Artemia salina nauplii and each apparatus was lit for 12 h a day (using a single 58W fluorescent lamp), in order to make the test fish familiar with the novel environment (no target was present in this phase).Six identical apparatus were provided. Fish were housed singly into the apparatus for a 5-day period. During this period subjects were fed twice a day with The experiment has been completed in three sessions spaced 3 h apart. Two fish (1 R-PPO and 1 L.PPO) were first tested with the neutral stimulus, two fish with the dummy predator and the remaining two fish with the mirror as target. The behavior of the fish was recorded from its entering the swimway for 5 min. The original video recordings were subsequently edited (Adobe Premiere® Pro2.0) in order to obtain 5 fps clips. For each frame, fish positions were scored using a computer program (written in Delphi 4 – Borland®) that calculated the degree of alignment (angle in degrees formed by prolongation of the major body axis of the fish with respect to the glass partition) between the subject and the glass partition. Data were discarded when the fish was perpendicular to the partition (binocular stimulation) or when it formed an angle larger than 90° with respect to the partition. From the analysis of the recordings we derived an index of eye preference as:[(frequency of right-eye use):(frequency of right-eye use + frequency of left-eye use)].Figure S1leftover expression in embryos from WT females. The graph represents the percentage of right lov expression in crosses of WT females with either GTLE or WT males. Mean and SD are espressed.(TIF)Click here for additional data file.Figure S2Calculation of DFC's area. 8bit images as in (TIF)Click here for additional data file.Movie S1Normal leftward flow inside the KV in Mmsw+/+embryos. Dorsal view inside the KV of a live 10-somite stage M+/+msw embryo. Fluorescent beads injected into the KV show a net counterclockwise movement.(MOV)Click here for additional data file.Movie S2Leftward flow inside the KV in wtembryos. Dorsal view inside the KV of a live 10-somite stage wt embryo. Fluorescent beads injected into the KV show a net counterclockwise movement, evidencing no difference compared to M+/+msw embryos.(MOV)Click here for additional data file.


Loxodonta africana) was annotated using available genome data. The elephant IgH locus on scaffold 57 spans over 2,974 kb, and consists of at least 112 VH gene segments, 87 DH gene segments , six JH gene segments, a single μ, a δ remnant, and eight γ genes . The Igκ locus, found on three scaffolds , contains a total of 153 Vκ gene segments, three Jκ segments, and a single Cκ gene. Two different transcriptional orientations were determined for these Vκ gene segments. In contrast, the Igλ locus on scaffold 68 includes 15 Vλ gene segments, all with the same transcriptional polarity as the downstream Jλ-Cλ cluster. These data suggest that the elephant immunoglobulin gene repertoire is highly diverse and complex. Our results provide insights into the immunoglobulin genes in a placental mammal that is evolutionarily distant from humans, mice, and domestic animals.The genomic organization of the IgH (Immunoglobulin heavy chain), Igκ (Immunoglobulin kappa chain), and Igλ (Immunoglobulin lambda chain) loci in the African elephant ( Elephas maximus) and the African elephant (Loxodonta africana). The three lineages of this family: Loxodonta, Elephas, and Mammuthus are thought to have originated 4–6 million years ago. Whereas some species of the former two lineages are still alive today, the last representative of the Mammuthus lineage, the woolly mammoth (Mammuthus primigenius), became extinct very recently (about 3.7 thousand years ago) Trichechus  and Procavia  The elephant is the biggest terrestrial placental mammal alive today. It belongs to the order Proboscidea and the family elephantidae, which contains only two existing species: the Asian elephant  and two identical light (L) chains. In some exceptional cases, such as shark IgNAR and selected subclasses of camelid IgGs, only heavy chains are used H, DH, and JH gene segments are followed by consecutive μ, δ, γ, ε, and α gene segments λ gene segments is followed by multiple sets of clustered Jλ gene segments, each linked to a single Cλ gene. Differentially, the cluster of Vκ gene segments is followed by a cluster of Jκ gene segments, and then by a single Cκ gene In the mammals studied so far, the locus of unique immunoglobulin heavy chain genes and loci of λ and κ light chain genes are commonly organized in a “translocon” pattern IgH and IgL loci have been characterized in different mammalian species Loxodonta africana), provided by the Broad Institute via whole genome shotgun, can be obtained from the Ensembl database (http://www.ensembl.org). LoxAfr3, an assembly of the genome of African Elephant, has been sequenced to 7× coverage . The elephant immunoglobulin gene sequences were retrieved from the UCSC genome browser (http://genome.ucsc.edu/).The genome sequence of the African Elephant (http://embossgui.sourceforge.net/demo/fuzznuc.html) was used to find adjacent recombination signal sequences (RSSs) for identification of variable, diversity, and joining gene segments. Five or more mismatched bases were allowed to cover all genes. The locations of the annotated elephant gene sequences on the elephant genome are shown in Human immunoglobulin gene sequences were used as queries to search the elephant genome scaffolds that contained immunoglobulin genes. A conventional TBLASTN approach was used to identify constant region genes of the elephant immunoglobulins. FUZZNUC, an online software  was used for comparing two sequences to identify a possible alignment of characters between the sequences.Phylogenetic studies were carried out using MrBayes3.1 and viewed with the TreeView package. All the trees were obtained with 1 million generations for the chains, a sample frequency of a 100, and a burn in of 2,500 . The site by site rate variation was set to a gamma distribution (rates = gamma) for all the Bayesian trees and a General Time-Reversible (GTR) (nst = 6) model of substitution was chosen. The sequences from other species used in phylogenetic analyses are presented in H and VL gene segments can be grouped into families based on their nucleotide sequence similarity H and VL segments having similarity greater than 70% into the same family.In mammals, germline VLoxodonta africana), sequenced to 7× coverage. The high genome coverage of this assembly confers a high reliability on the gene analysis. BLAST searching localized the elephant IgH locus to genomic scaffold 57. It spans approximately 2,974 kb from the most 5′ VH segment (VH2-112p) to the most 3′ γ gene  and Tryptophan (W) Most mammals also express a δ gene, which is always situated immediately downstream of the μ, and the distance between μ and δ usually does not exceed 7 kb. A BLAST search against the elephant whole genome using both DNA and amino acid sequences of the δ genes of other mammalian species showed no intact δ gene. However, approximately 10 kb downstream of the elephant µ (no sequence gaps for 90 kb downstream), we identified a short fragment encoding a polypeptide  homologoIn addition to the eight γ genes (γ1 to γ8) in scaffold 57 , an addiGGGCT and GAGCT). The elephant Sμ region shows substantial nucleotide similarity with those of human, mouse, and pig , as in humans and mice  and pig . The sixH segments were identified in the elephant IgH locus. 51 of them appear to be potentially functional, because they have leader exons, normal open reading frames (ORF), downstream RSSs, and V gene domain (framework regions (FRs) and complementarity determining regions (CDRs)). The remaining 61 segments contain either in-frame stop codons or frameshifts, and are thus designated as pseudogenes. In addition, there are 17 partial segments of about 200 bp in length, which are regarded as truncated VH sequences. There are gaps above 10 kb in the elephant genome among the VH gene segments  were confirmed to be homologous with the corresponding human VH gene families. The elephant VH4 family contains the most members (72 VH segments), which could be further divided into three groups . In humans and mice, the germline DH segments can be classified into families based on the extent of sequence similarity H segments could be divided into seven families. Members within the same family share at least 70% nucleotide identity (data not shown), while some members in a family have completely identical sequences   correspond to the six human Vκ gene families. In addition, scaffold 86 includes 24 Vκ segments showing the same transcriptional orientation as the Jκ and Cκ, and 18 Vκ segments showing a reverse transcriptional direction. Three Jκ segments and one Cκ gene on scaffold 86 are displayed in κ segments located on scaffolds 202 and 50 also possess two different transcriptional directions.Immunoglobulin κ chain genes of elephant were identified on three scaffolds: 202, 50, and 86. A schematic diagram is shown in Vκ1∼Vκ8) , which cλ gene segments belonged to six families , human (seven families), and horse (seven families). The few gene families or single gene family group includes dog (three families), rabbits (one family), cattle (one family), camel (one family), and swine (one family) H gene families, should be put into the first group. The mammalian VH families can be further classified into three clans: I, II, and III, which have co-existed in the genome for more than 400 Myr H families also conform to three clans: families 1, 5, and 7 form clan I, families 2, 4, and 6 form clan II, and family 3 forms clan III. The largest group of elephant VH genes is the VH4 family of clan II. It has been demonstrated that the unique VH family identified in cattle belonged to clan II H genes are also categorized into clan II H genes.Our analysis also suggested a high degree of complexity in the elephant IgVH locus. At least 112 VH locus, where at least 87 germline D segments could be mapped to a 450-kb DNA region; the largest number in mammals examined so far. The presence of more DH segments may greatly increase the Ig diversity generated through DNA rearrangement. The size of the elephant DH coding regions ranges from 10 to 37 bp, similar to that of human (11 to 37 bp) H segments were translated in three reading frames abundant in polar/hydrophobic amino acids, which is different to dog Close attention should also be paid to the elephant Dκ germline genes are more abundant than Vλ . Different mammalian species possess different ratios of Vκ and Vλ. In humans, roughly 60% of the variable light chain repertoire is κ . The germline Vκ genes of mice are dominant by as much as 95% or more For the light chain genes, elephant VH (61/112), Vκ (100/153), and Vλ (13/15) loci. In some species, the base-pair changes could be inferred using an existing pseudogene or germline gene as a template, and therefore pseudogenes in the V loci constitute a potential donor pool for gene conversion to generate immunoglobulin diversity Interestingly, a great number of pseudogenes exist in the elephant VThe study of structure and organization of the immunoglobulin gene loci is vital to the understanding of the nature of antibody molecules. This study provides information for comparative studies of mammalian Ig genes, as well as data for further studies of the elephant immunoglobulin genes.Figure S1Alignment of IgM amino acid sequences from several vertebrate species. Elephant IgM was compared with a panel of vertebrate IgM sequences. Dots indicate similar residues as in elephant μ, whereas dashes indicate gaps introduced for optimal alignment. The cysteine residues C and W important for intra-domain disulfide bonds are shown on the first line of the alignment.(TIF)Click here for additional data file.Figure S2Alignment of the elephant IgD remnant with the IgD CH3 domains of several mammalian species. Amino acid residues that are identical to the top counterpart in every panel are shown as dots; Gaps and missing data are indicated by hyphens. Stop codons are indicated by stars.(TIF)Click here for additional data file.Figure S3Phylogenetic tree of the immunoglobulin gamma heavy chains of some mammalian species. The phylogenetic tree was constructed from the amino acid sequences of the CH3 exons of the immunoglobulin gamma heavy chains of various mammalian species. The credibility value for each node is shown.(TIF)Click here for additional data file.Figure S4H segments.87 Elephant germline D The nonamers (9-mer) and heptamers (7-mer) are displayed. Heptamer components that are different from the consensus (5′: CACTGTG and 3′: CACAGTG) are shadowed. The deduced amino acids of all three reading frames of the coding region of D segments are shown. Except DH, which has a 10 bp spacer, all the DH segments were attached by 12 bp spacer.(TIF)Click here for additional data file.Figure S5The alignment of amino acid sequences of J and C genes from elephant IgL chains. A, alignment of the deduced amino acid sequences of the three elephant Jκ gene segments. B, alignment of the amino acid sequences of the Cκ proteins from several mammalian species. C, alignment of the deduced amino acid sequences of the two elephant Jλ gene segments. D, alignment of the deduced amino acid sequences of three elephant Cλ genes and several mammalian species Cλ genes. Amino acid residues that are identical to the top counterpart in every panel are shown as dots; Gaps and missing data are indicated by hyphens.(TIF)Click here for additional data file.Table S1The elephant immunoglobulin heavy chain and light chain DNA segments located in scaffolds 57, 202, 50, 86, and 68.(RAR)Click here for additional data file.Table S2GenBank accession numbers or references of the gene sequences from other species used in this paper.(RAR)Click here for additional data file.Table S3κ gene families from scaffolds 202, 50, and 86.The eight elephant V(TIF)Click here for additional data file.


The real sample temperatures during the nuclear resonant vibrational spectroscopy on biological samples have been assessed and significantly reduced (116 → 52 K) by improving the sample-loading procedures. 4N)[FeCl4] to verify these trends; and (iii) proposing a new sample-loading procedure to achieve significantly lower real sample temperatures and to balance among the intertangled experimental issues in biological NRVS measurements.There are several practical and intertangled issues which make the experiments of nuclear resonant vibrational spectroscopy (NRVS) on biological samples difficult to perform. The sample temperature is one of the most important issues. In NRVS the real sample temperatures can be very different from the readings on the temperature sensors. In this study the following have been performed: (i) citing and analyzing various existing NRVS data to assess the real sample temperatures during the NRVS measurements and to understand their trends with the samples’ loading conditions; (ii) designing several NRVS measurements with (Et It scans an extremely monochromatic (∼1 meV) X-ray beam through the e.g. photochemically or electrochemically produced states), making the sample temperature the leading issue in almost every biological X-ray experiment. However, it is not a simple matter to lower the sample temperature in a NRVS experiment measuring biological samples. Issues of the NRVS signal levels, the samples’ air sensitivity and the samples’ real temperatures are all intertangled. These issues are described in detail as follows:Biological samples  have several practical issues which often make their NRVS experiments difficult. A higher sample temperature will lead to faster radiation damage and less stable nascent chemical states  to be as short as possible, e.g. ∼1 mm  Most biological molecules have very low metal concentration and hence a very weak spectral signal. For a NRVS experiment, this issue is even worse because the extremely monochromatic beam has an intensity of the order of 102), 77 K, and are kept at this temperature. During the sample loading to the cryostat base, the sample would be exposed to air at a temperature higher than 77 K. To minimize this potential problem, experimentalists need to (a) shorten the sample-loading time, defined as the period between the samples leaving the LN2 and the samples being attached firmly to the base (and the cryostat being cooled down); (b) maintain the cryostat base at a temperature as low as possible.(ii) Many biological molecules are air-sensitive. Protein samples are often prepared and quenched in liquid nitrogen (LNa1) versus Fig. 1(a2)].In a cold-finger-type cryostat there are two current practices for mounting biological NRVS samples onto the cryostat base. The first method is to use cryogenic adhesive to attach the sample onto the base. In this method there will be no extra materials in front of the NRVS samples, so the sample–detector distance can be as short as possible. Also, the time to mount samples is very short (∼15 s). However, the cryostat base has to be warmed up above the melting point of the cryogenic adhesive. If the loading temperature is too low, the adhesive will become too sticky, and the sample–base contact could be a point-to-point contact rather than a surface-to-surface contact  have been performed to verify these trends; (iii) the sample-loading procedure has been changed to improve the real sample temperatures (T2). This study has illustrated how the new sample-loading procedure significantly lowers T2 and meanwhile balances all the intertangled experimental issues.In this study, (i) various existing NRVS data  have been analyzed to assess the real sample temperatures during the NRVS measurements and to understand their trends with the samples’ loading conditions; (ii) several standard NRVS measurements with . Delayed nuclear fluorescence and Fe Kα fluorescence were recorded using a 2 × 2 APD detector array. The maximum resonant peak varied from sample to sample between 50 and 3000 counts s−1. In addition, a few NRVS spectra measured at ESRF (BL18) were also cited and discussed.et al., 2005et al., 2005PHOENIX software package , normalized to I0, averaged and converted to the 57Fe partial vibrational density of states (PVDOS or DOS for abbreviation). The spectral conversion was optimized when the observed Stokes/anti-Stokes imbalance matched the imbalance calculated using the entered temperature as a parameter. Therefore, NRVS-measured temperature for the X-ray-irradiated sample portion was obtained during NRVS spectral analysis.Spectral analysis was performed following the published procedure  using a liquid-helium-flow cold-finger cryostat. The base temperature was raised to a few K above the cryogenic adhesive’s melting point during the sample loading. Lucite boxed samples were directly attached onto the recessed trench on the top of the cryostat base with cryogenic adhesive Fig. 1a.etc., were loaded from LN2 onto the cryostat base, which was kept at 180 K at the time of the sample loading. This was to ensure an as-low-as-possible sample temperature during the loading processes. Procedure A2: ordinary biological samples, such as iron–sulfur proteins, were loaded from LN2 onto the cryostat base, which was kept at 190 K instead. Procedure A3: chemical complexes were loaded from RT onto the base kept at 200 K. Procedure A4: the first calibration sample (Et4N)[FeCl4] was loaded before cooling down the cryostat, i.e. both the sample and the cryostat base were at RT.Our old sample-loading procedures used HIVAC-G cryogenic grease  as adhesive, denoted LT grease hereafter. The LT grease is still widely used in the NRVS community as it is convenient to use over a wide range of temperatures (including RT). Procedure A1: all the critical or sensitive biological samples, such as CO-bound nitrogenase T2. To lower both temperatures, a lower-melting-point adhesive has to be used. Solvent 1-propanol with a melting point at 147 K  was selected as the new cryogenic adhesive. In our new sample-loading procedures, the cryostat base was kept at <150 K for all the samples. Procedure B1: all the protein samples were loaded from LN2 onto the cryostat base. Procedure B2: all the chemical complexes {including the calibration sample (Et4N)[FeCl4]} were loaded from RT onto the cryostat base.As will be discussed in detail in §32.3.4N][FeCl4] was synthesized following the published procedures  (raw NRVS) and 2(b)−1), a lattice mode near 5 meV (40 cm−1), and some residual NRVS intensity between 5 and 14 meV (40–112 cm−1). The highest-frequency modes have the greatest NRVS amplitude and hence the largest amount of iron motion [FeCl4], many other NRVS spectra were cited and analyzed in this study.The tetrahedral , we found (i) the acceptable temperature range during the process of spectral analysis is about ±5 K (1σ), and (ii) the temperature repeatability for the same sample in the same sample load but with different spectral scans is about ±3 K (1σ). The total error bar for the temperature analysis depends on various issues, and the above two values or their sum should not be treated as the total error bar but rather as a reference for the repeatability under certain experimental condition(s). Nevertheless, it cannot be concluded that the large temperature deviation of σ = 34 K is simply due to a large error bar either.During our analysis on (Et4N)[FeCl4] in each beam time always obtained very low sample temperatures (blue circles below the −1σ line). It has a sample temperature that is lower than other chemical complexes and even lower than the same (Et4N)[FeCl4] sample measured later during the same beam time; (iii) all the temperature data points which were above the +1σ line were from the critical protein samples, such as CO-bound nitrogenase and H2-bound Hmd hydrogenase etc.With more careful analysis, the above temperature data further illustrated the following trends: (i) model chemical complexes (blue circles), in general, had lower sample temperatures than protein samples (red circles); (ii) the first calibration measurements on  NRVS spectrum could contribute to a large error bar in calculating sample temperatures. Although it sounds reasonable, a higher error bar does not mean a higher temperature itself. Besides, this proposal could not explain (i) why all the initial calibration measurements (in each beam time) always had the lowest temperatures  and (ii) why all the critical protein samples had the highest temperatures (higher than other protein samples).The first possible explanation for protein samples having higher temperatures could be that the lower counts sb)versus complexes).The trends with the experimental conditions appear when the real sample temperatures were re-classified according to their sample-loading procedures and re-illustrated as in Fig. 3(a1) versus (a2)], hindering the heat transfer (kc) and resulting in a higher sample temperature (T2). A large error bar (σ = ±26 K) in this loading procedure (A1) also suggests that the thermal contact situation be random.All the critical biological samples were loaded at 180 K (in an effort to save the samples). At 180 K the LT grease became very sticky and froze almost immediately after applying. Although macroscopically the sample stuck to the cryostat base, microscopically it could have a point-to-point contact rather than a surface-to-surface contact with the cryostat base  was always loaded at RT before the cryostat was cooled down and thus had a very nice surface-to-surface contact and should have a very low T2, which is indeed the case. The decreasing statistical error bars also suggest better thermal contacts between the sample and the base when the sample-loading procedure changes: A1 → A2 → A3 → A4.The less-critical protein samples were loaded at 190 K and the model complexes were loaded at 200 K (both referring to the temperatures of the cryostat base). Most complex samples themselves were at RT before loading. At these loading temperatures the LT grease had less viscosity and facilitated a better surface-to-surface contact between the sample and the base. Assuming a fixed cryostat base temperature [FeCl4] sample (∼2000 counts s−1), mimicking the four loading procedures . In one case (A1), the LT grease was applied to the cryostat base at 180 K and the (Et4N)[FeCl4] sample was soaked in LN2 before loading onto the cryostat base. This is to simulate the real situation for loading the critical protein samples. Such mounted (Et4N)[FeCl4] indeed had very high sample temperatures (154 K and 162 K) as shown in Fig. 3(b)4N)[FeCl4] has a T2 of 131 K and 116 K (squares inside the orange circle), lower than those loaded at 180 K but still much higher than those loaded at RT .To further prove the above speculation that the samples’ loading procedures rather than the samples themselves actually affect Although other mechanisms cannot be absolutely ruled out, the adhesive-affected sample–base thermal conductivity was the major reason for the high sample temperatures in our NRVS measurements with the old sample-loading procedures . A lower loading temperature led to a higher sample temperature during the NRVS measurement, and a higher loading temperature led to a lower sample temperature. Unfortunately, most biological samples need to be loaded at a temperature even lower than 180 K. Therefore, it is necessary to search for an alternative adhesive which has a freezing point lower than 175 K.3.4.i.e. 175 K. Then liquid organic solvents were also considered. Although the organic solvents are not adhesive at all at RT, it is an excellent bonding medium at cryogenic temperatures. In our new sample-loading procedures, 1-propanol was selected as the cryogenic adhesive because it has a low freezing point of 147 K and a relatively high boiling point of 370 K, a suitable viscosity constant and no hazards to the experimenters. Our experimental tests showed that 1-propanol became fluid at ∼140 K and the best sample-loading temperature was about 145–150 K. This made the sample-loading temperatures at least 30 K lower than the ones using the LT grease as adhesives, i.e. 180 K.Grease-form adhesives are easy to use at all temperatures. However, no such adhesive was found to have a freezing point lower than the LT grease, The 1-propanol  has much better fluidity, hence providing a much better surface-to-surface contact. Therefore lower sample temperatures were expected with the use of 1-propanol as cryogenic adhesive, which was indeed the case. Using the new loading procedures immediately produced a much lower sample temperature of 50 ± 7 K during the particular beam time in July 2009 alone. Afterwards, we continued to use 1-propanol as adhesive and measured various NRVS samples in several beam times. As illustrated in Fig. 4versus 51.8 ± 9.1 K). This further concluded that the new sample-loading procedures led to a better and more stable surface-to-surface thermal contact in between the sample and the cryostat base, under which all the samples have a similar and low temperature.Although the total error bar is not obtainable in this study, the above statistical error bar of ±10 K is based on seven beam times and 51 various samples, and is repeatable. Also, using 1-propanol as the adhesive, the proteins and chemical complexes have almost the same mean sample temperature and almost the same standard deviation (52.3 ± 10.8 K kT)], which is the reason why X-ray experiments should be performed at as-low-as-possible temperatures. According to Garman & Nave [FeCl4] during two NRVS beam times with a loading temperature of ∼160 K. Those samples were found to have about the same real sample temperatures as those loaded with 1-propanol. Lower T2 was not detected.Table 1T2 was made possible owing to the better fluidity of the solvents versus the LT grease at low temperatures. It is not because the solvents have better thermal conductivity than the LT grease. Thus it is not likely to lower the sample temperature further simply by choosing different solvents. The different solvents are listed and discussed here just in case a different sample-loading temperature is required. For example, dimethylether has a melting point of 134 K.It must be noted that the improvement on IntroductionHow about a comparison between the 1-propanol loaded samples and the mechanically attached samples? At APS (ID03), the NRVS samples were mounted mechanically. The advantage of this procedure is that the cryostat base can be cooled to an as-low-as-possible temperature. However, the temperatures during the NRVS measurements for the similar samples were found at 70–110 K (or ∼90 ± 20 K), lower than those loaded with the LT grease but still much higher than those loaded with 1-propanol as the adhesive. As mentioned in the 4.In this study we have evaluated the real sample temperatures during the NRVS experiments on various biological samples and chemical complexes. We have understood the relation between the samples’ loading conditions and their real sample temperatures. Changing the cryogenic adhesive from the LT grease to 1-propanol has reduced the samples’ loading temperatures by at least 30 K (180 K → 150 K) and meanwhile reduced the real sample temperatures by 64 K (116 K → 52 K). This improvement is real, repeatable and stable.i.e. the as-short-as-possible sample–detector distance, the as-low-as-possible sample-loading temperatures, the as-fast-as-possible sample-loading process, and the as-low-as-possible real sample temperatures during the NRVS measurement.This approach is so far the best balance among all the intertangled issues in a biological NRVS experiment, Synchrotron radiation rings, undulators and NRVS beamlines continue to improve. In the future, NRVS will have a greater role in studying iron-specific chemistry/biochemistry, and meanwhile will face a greater challenge over the issue of sample radiation damage. This study begins a journey to deal with this potential problem. According to Garman & Nave 2009, lowerinClick here for additional data file.10.1107/S0909049512001380/kt5030sup1.pdfSupplementary material file. DOI:


Interferon β (IFNβ) is an antiviral cytokine secreted in response to pathogenic exposure that creates a restrictive intracellular environment through the action of downstream interferon-stimulated genes (ISG). The objective of this study was to examine the expression of IFNβ and ISG in both human uterine epithelial cells (UEC) and the ECC-1 uterine epithelial cell line and determine if expression changes with TLR stimulation and hormone exposure. Stimulation of primary uterine epithelial cells and ECC-1 cells with the TLR3 agonist poly (I∶C) induced the mRNA expression of IFNβ, MxA, OAS2 and PKR. Other TLR agonists including imiquimod and CpG had no effect on either IFNβ or ISG expression. In contrast to ECC-1 cell responses which were slower, maximal IFNβ upregulation in UEC occurred 3 hours post-stimulation and preceded the ISG response which peaked approximately 12 hours after poly (I∶C) exposure. Unexpectedly, estradiol, either alone or prior to treatment with poly (I∶C), had no effect on IFNβ or ISG expression. Blockade of the IFN receptor abrogated the upregulation of MxA, OAS2 and PKR. Furthermore, neutralizing antibodies against IFNβ partially inhibited the upregulation of all three ISG. Estradiol, directly and in the presence of poly (I∶C) had no effect on IFNβ and ISG expression. These results indicate that uterine epithelial cells are important sentinels of the innate immune system and demonstrate that uterine epithelial cells are capable of mounting a rapid IFN-mediated antiviral response that is independent of estradiol and is therefore potentially sustained throughout the menstrual cycle to aid in the defense of the uterus against potential pathogens. Neisseria gonorrhoeae and Chlamydia trachomatis, which cause morbidity and mortality in large numbers of women. The innate immune response in the FRT is a key component of host defense against these incoming pathogens about which much still remains to be elucidated.The female reproductive tract (FRT) is a unique mucosal site that must reconcile two competing functions: host defense versus reproduction. It is the primary site of infection by sexually transmitted diseases (STDs) including Herpes Simplex Virus (HSV), Human Immunodeficiency Virus (HIV), Epithelial cells are essential for mucosal defense providing a physical and structural barrier against pathogen entry into the body. They are the first cells exposed to invading pathogens and thus, their responses could determine whether an infection occurs. Human uterine epithelial cells (UEC) express a full complement of pattern recognition receptors (PRR) including Toll-like receptors (TLR) 1–9, RIG-like receptors (RLR) and NOD-like receptors (NLR) Type I interferons (IFNs) are a family of cytokines that can rapidly induce a protective antiviral response and create a restrictive environment for pathogen survival Every cell in the premenopausal FRT exists in an environment that is constantly exposed to varying levels of the sex hormones estradiol and progesterone. Previously, we have shown that estradiol regulates several aspects of the innate immune system in the FRT. In human UEC estradiol stimulates the production of antimicrobial compounds such as elafin and secretory leukocyte protease inhibitor (SLPI) that are essential in restricting viral and bacterial infections We examined the Type I IFN response of UEC to a panel of viral TLR agonists and demonstrated that UEC are exquisitely sensitive to dsRNA stimulation which transiently (over 24 hours) leads to a rapid intracellular antiviral response. Treatment with other viral TLR ligands did not induce a Type I IFN response. We examined the role of estradiol in modulating the IFN pathway and found that hormonal treatment does not alter the response profile of ISG in UEC. Overall, this study demonstrates the presence of a functional Type I IFN pathway in primary polarized UEC and the ECC-1 cell line that is responsive to the presence of dsRNA, is unaffected by the presence of estradiol and therefore provides a constant level of protection across the menstrual cycle.Human uterine tissue was obtained immediately following surgery from premenopausal women who had undergone hysterectomies at Dartmouth-Hitchcock Medical Center . Tissues used in this study were distal to the sites of pathology and were determined to be unaffected with disease upon inspection by a trained pathologist. All investigations involving human subjects were conducted according to the principles expressed in the Declaration of Helsinki and carried out with the approval from the Committee for the Protection of Human Subjects (CPHS), Dartmouth Hitchcock Medical Center, and with written informed consent obtained from the patients before surgery.Epithelial cells were isolated as previously described g for 10 min, and analyzed for cell number and viability.Epithelial cell sheets were separated from stromal cells by filtration through a 20-µm nylon mesh filter . Epithelial sheets were retained on the 20-µm filter, while stromal cells passed through. Epithelial sheets were recovered by rinsing and backwashing the filter with Complete medium. Epithelial sheets were collected, centrifuged at 500× To establish a cell culture system of polarized human UEC with both apical and basolateral compartments, human UEC were cultured in Falcon cell culture inserts coated with Human Extracellular Matrix  in 24-well culture plates . Apical and basolateral compartments had 300 and 850 µl of complete medium respectively. The medium was changed every 2 days.ECC-1 cell line is a well-differentiated human UEC line that is responsive to sex hormones Peripheral blood mononuclear cells (PBMC) were isolated from a blood cone and cultured in RPMI medium (Gibco) supplemented with 20 mM HEPES, 2 mM L-glutamine, 50 mg/ml primocin and 10% heat-inactivated defined FBS prior to TLR stimulation.Polarized epithelial cells were apically stimulated with various TLR agonists at the following concentrations unless otherwise stated: poly (I∶C)  25 µg/ml; imiquimod  100 µM and CpG oligonucleotide (Invivogen) 1 µM.Recombinant human IFNβ  was used to stimulate polarized UEC or ECC-1 cells for 24 hours. IFNβ neutralization experiments were conducted using a rabbit polyclonal anti-human IFNβ neutralizing antibody (αIFNβ) . Interferon receptor blockade experiments were conducted using a mouse monoclonal anti-human interferon receptor 2 (IFNAR2) blocking antibody (R&D Systems)−3 M, evaporated to dryness and resuspended in Complete media containing charcoal dextran-stripped FBS to a concentration of 1×10−5 M. Further dilutions were made to achieve final working concentrations of estradiol ranging from 5×10−8 M to 5×10−12 M. As a control, an equivalent amount of ethanol without dissolved hormone was initially evaporated. In all cases, hormone was added to both the apical and basolateral compartments.For all hormone experiments, 17β-estradiol  was dissolved in 100% ethanol for an initial concentration of 1×10In all experiments with TLR agonists, IFN blockade and neutralization, or sex hormones, Complete medium containing 10% heat-inactivated FBS was replaced with Complete medium supplemented with 10% heat-inactivated charcoal/dextran-treated Stripped FBS .IFNβ secretion was analyzed by an enzyme-linked immunosorbent assay (R&D Systems). Briefly, cell culture conditioned media was recovered and centrifuged at 500 g for 5 minutes. The resulting supernatant was stored at −80°C until required.As an indicator of tight junction formation of epithelial cell monolayers, TER was periodically assessed using an EVOM electrode and Voltohmmeter . All cell cultures had TER values greater than 1000 Ω/insert when experiments were performed. Cell viability was assessed using the CellTiter Cell Viability Assay  as per the manufacturer's instructions.t) for each reaction and this was used to quantify the amount of starting template in the reaction. The Ct values for each set of duplicate reactions were averaged for all subsequent calculations. A difference in Ct values (ΔCt) was calculated for each gene by taking the mean Ct of each gene of interest and subtracting the mean Ct for the housekeeping gene β-actin for each cDNA sample. Assuming that each reaction functions at 100% PCR efficiency, a difference of one Ct represents a 2-fold difference. Relative expression levels were expressed as a fold-increase in mRNA expression and calculated using the formula 2−ΔΔCt.Total RNA was isolated from cells using TRIzol Reagent according to the manufacturer's recommendations (Invitrogen) and purified on RNeasy columns  with on-column DNase digestion using the RNase-Free DNase set (Qiagen). 400 ng of total RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's recommendations. Relative mRNA expression levels of genes of interest were measured using the 5′ fluorogenic nuclease assay in real-time quantitative PCR using TaqMan chemistry on the ABI 7300 Prism real-time PCR instrument . The IFN-β, OAS2, MxA, PKR, β-actin, progesterone receptor primer/MGB probe sets were obtained from Applied Biosystems assays-on-demand . PCR was conducted using the following cycle parameters: 95°C, 12 min for 1 cycle , for 40 cycles. Analysis was conducted using the sequence detection software supplied with the ABI 7300. The software calculates the threshold cycle  for a range of time-points up to 24 hours. As shown in In parallel studies with polarized human UEC, a similar pattern was observed. As seen in 1, 102 & 103 U/ml), IFNβ neutralizing antibody (αIFNβ) or IFNAR2 blocking antibody (αIFNAR2) for 24 hours prior to analysis of ISG (1 U/ml) and 120- (103 U/ml) fold over unstimulated levels, while MxA went up 6 and 20-fold respectively. In contrast, PKR was the least sensitive to IFNβ, increasing maximally by 6-fold at 103 U/ml.The Type I IFN response has not been studied extensively in the human FRT epithelium despite it being a major site of STD transmission. To establish that polarized uterine cells are responsive to IFNβ, ECC-1 cells were incubated with a combination of recombinant human IFNβ (10s of ISG . IFNβ do3 U/ml) to UEC (Addition of exogenous IFNβ (10) to UEC  also ind) to UEC  over unt) to UEC . To furt−8 M) apically and basolaterally. As seen in −8 M to 5×10−12 M over the same time (3–48 hr) periods. Irrespective of the dose of estradiol used, no change in IFNβ, MxA, and OAS2 or PKR expression was detected (data not shown).Epithelial cells in the upper FRT exist in an environment that is constantly exposed to levels of estradiol which vary with the stage of the menstrual cycle −8 M) for 24 hours followed by 12–24 hours incubation with poly (I∶C) during which estradiol was maintained in the culture media. As shown in Previous studies have shown that estradiol can regulate TLR-induced responses in the FRT These studies demonstrated that human UEC and ECC-1 upregulate IFNβ in response to poly (I∶C), a dsRNA viral mimic. When added to the culture media of polarized epithelial cells, IFNβ induced expression of MxA, OAS2 and PKR – a subset of ISG that protect against viral pathogens. Interestingly, inhibition of IFNβ action, either by blockade of IFNAR2 or by IFNβ neutralization, partially abrogated increases of MxA, OAS2 and PKR in the presence of poly (I∶C). The sex hormone estradiol had no effect on IFNβ or ISG expression, either alone or with poly (I∶C) stimulation. Overall, these studies indicate that ISG are present in uterine epithelial cells and under appropriate conditions, are up-regulated by IFNβ independently of estradiol.in vivo. HIV, for example, is recognized by TLR7 in specific subsets of immune cells. Could the inability of uterine epithelial cells to mount an effective Type I IFN response to TLR7/8 stimulation also imply they are insensitive to the presence of HIV and would this allow for increased viral transmission?Type I IFNs are secreted in response to pathogenic stimulation and rapidly induce an antiviral state in adjacent cells via their ubiquitously expressed cell-surface receptor complex consisting of IFNAR1 and 2 What confers epithelial sensitivity to dsRNA is unresolved but may involve TLR3 and potentially two additional PRRs known to recognize dsRNA: the cytosolic retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) which bind poly (I∶C) Unexpectedly, we found that when secretions from poly (I∶C)-stimulated cells were neutralized with an IFNβ-specific antibody, ISG upregulation in UEC was only partially abrogated (30–40%). Inadequate antibody blockade is unlikely since we used a concentration of antibody that abrogated IFNβ signaling in ECC-1 cells and was previously shown to completely block HSV-2-induced IFNβ signaling in UEC Epithelial cells are poised to respond to viral pathogens by rapidly upregulating IFNβ and subsequently, the ISG. Similar to findings elsewhere in vitroThe speed of the antiviral response probably allows uterine epithelial cells to confer protection against viral pathogens such as HIV and HSV that periodically enter the upper FRT after vaginal sexual intercourse. Type I IFNs are believed to have essential roles in restricting these pathogens Previous studies by us demonstrated that estradiol and progesterone regulate a spectrum of uterine epithelial cell immune functions, including tight junction formation, cytokine and antimicrobial secretion, as well as antigen presentation The present studies demonstrated that pretreatment of epithelial cells with estradiol had no effect on poly (I∶C)-induced IFNβ and ISG expression. These findings are in contrast to our previously published findings that cytokine and chemokine secretion in response to TLR agonists are dampened when epithelial cells are pretreated with estradiol In conclusion, we demonstrated the presence of a robust IFNβ-mediated response to poly (I∶C) which is unaffected by estradiol in both human UEC and ECC-1 cells. The lack of an estradiol effect suggests a degree of protection that is unaffected by hormonal flux across the menstrual cycle thus indicating the importance of maintaining a constant level of Type I IFN-mediated innate immune protection.