Use of fiber optic technology to measure the effects of anesthesia on luciferase reaction kinetics

In vivo bioluminescent imaging (BLI) is a sensitive and reliable technique for studying gene expression, although experiments must be controlled tightly to obtain reproducible and quantitative measurements. The luciferase reaction depends on the availability of the reaction substrate, oxygen, and ATP, the distribution of which can vary markedly in different tissues. Here we used in vivo fiber optic technology, combined with stereotaxis-assisted surgery, to assess luciferase reaction kinetics in response to 2 anesthetic regimens, isoflurane and ketamine–xylazine. Transgenic rats that expressed luciferase under the control of the human prolactin promoter were used as a model organism. Anesthesia had a marked effect on luciferase reaction kinetics. The rise time to peak emission differed by 20 min between isoflurane and ketamine–xylazine. Optical imaging using a charge-coupled–device camera confirmed this delay. These results demonstrate that different anesthetics can have substantial effects on luciferase reaction kinetics and suggest that the timing of image acquisition after substrate injection should be optimized in regard to experimental conditions and the tissues of interest

Effect of SARS-CoV-2 infection in pregnancy on CD147, ACE2 and HLA-G expression

Introduction: Recent studies reported a differential expression of both ACE2 and CD147 in pregnant women associated to SARS-CoV-2 placental infection. The aim of this study is to further investigate the placental SARS-CoV-2 infection and the potential effect on protein expression (ACE2, CD147, HLA-G and CD56). Methods: The study was on three subgroups: i) 18 subjects positive for SARS-CoV-2 swab at delivery; ii) 9 subjects that had a positive SARS-CoV-2 swab during pregnancy but resulted negative at delivery; iii) 11 control subjects with physiological pregnancy and with no previous or concomitant SARS-CoV-2 swab positivity. None of the subjects were vaccinated for SARS-CoV-2 infection. The placenta samples were analyzed for SARS-CoV-2 NP (Nucleocapsid protein) positivity and the expression of ACE2, CD147, HLA-G and CD56. Results: We observed a higher percentage of SARS-CoV-2 NP positive placenta samples in the group of SARS-CoV-2 PCR positive at delivery in comparison with SARS-CoV-2 PCR negative at delivery. The localization of SARS-CoV-2 NP positivity in placenta samples was mainly in syncytiotrophoblast (ST) of SARS-CoV-2 PCR positive at delivery group and in extra-villous trophoblast (EVT) of SARS-CoV-2 PCR negative at delivery group. CD147, HLA-G positivity was higher in ST of SARS-CoV-2 PCR positive at delivery group, while CD56-expressing immune cells were decreased in comparison with control subjects. Discussion: We confirmed the ability of SARS-CoV-2 to infect placenta tissues. The simultaneous SARS-CoV-2 swab positivity at delivery and the positivity of the placenta tissue for SARS-CoV-2 NP seems to create an environment that modifies the expression of specific molecules, as CD147 and HLA-G. These data suggest a possible impact of SARS-CoV-2 infection during pregnancy, that might be worthy to be monitored also in vaccinated subjects

Transcription factor Pit-1 affects transcriptional timing in the dual-promoter human prolactin gene

Gene transcription occurs in short bursts interspersed with silent periods, and these kinetics can be altered by promoter structure. The effect of alternate promoter architecture on transcription bursting is not known. We studied the human prolactin (hPRL) gene that contains two promoters, a pituitary-specific promoter that requires the transcription factor Pit-1, and displays dramatic transcriptional bursting activity, and an alternate upstream promoter that is active in non-pituitary tissues. We studied large hPRL genomic fragments with luciferase reporters, and used bacterial artificial chromosome (BAC) recombineering to manipulate critical promoter regions. Stochastic switch mathematical modelling of single-cell time-lapse luminescence image data revealed that the Pit-1-dependent promoter showed longer, higher-amplitude transcriptional bursts. Knockdown studies confirmed that the presence of Pit-1 stabilised and prolonged periods of active transcription. Pit-1 therefore plays an active role in establishing the timing of transcription cycles, in addition to its cell-specific functions

Phenotypic dissection of the mouse Ren-1(d) knockout by complementation with human renin

Normal renin synthesis and secretion is important for the maintenance of juxtaglomerular apparatus architecture. Mice lacking a functional Ren-1d gene are devoid of renal juxtaglomerular cell granules and exhibit an altered macula densa morphology. Due to the species-specificity of renin activity, transgenic mice are ideal models for experimentally investigating and manipulating expression patterns of the human renin gene in a native cellular environment without confounding Renin-angiotensin-system interactions. A 55 kb transgene encompassing the human renin locus was crossed onto the mouse Ren-1d-null background, restoring granulation in juxtaglomerular cells.  Correct processing of human renin in dense core granules was confirmed by immunogold labelling. After stimulation of the renin-angiotensin system, juxtaglomerular cells contained rhomboid protogranules with paracrystalline contents, dilated rough endoplasmic reticulum and electron-lucent granular structures. However, complementation of Ren-1d-/- mice with human renin was unable to rescue the abnormality seen in macula densa structure. The juxtaglomerular apparatus was still able to respond to tubuloglomerular feedback in isolated perfused juxtaglomerular apparatus preparations, although minor differences in glomerular tuft contractility and macula densa cell calcium handling were observed. This study reveals that the human renin protein is able to complement the mouse Ren-1d-/- non-granulated defect and suggests that granulopoiesis requires a structural motif that is conserved between the mouse Ren-1d and human renin proteins. It also suggests that the altered macula densa phenotype is related to the activity of the renin-1d enzyme in a local juxtaglomerular renin-angiotensin system

Role of Estrogen Response Element in the Human Prolactin Gene:Transcriptional Response and Timing

The use of bacterial artificial chromosome (BAC) reporter constructs in molecular physiology enables the inclusion of large sections of flanking DNA, likely to contain regulatory elements and enhancers regions that contribute to the transcriptional output of a gene. Using BAC recombineering, we have manipulated a 160-kb human prolactin luciferase (hPRL-Luc) BAC construct and mutated the previously defined proximal estrogen response element (ERE) located −1189 bp relative to the transcription start site, to assess its involvement in the estrogen responsiveness of the entire hPRL locus. We found that GH3 cell lines stably expressing Luc under control of the ERE-mutated hPRL promoter (ERE-Mut) displayed a dramatically reduced transcriptional response to 17β-estradiol (E2) treatment compared with cells expressing Luc from the wild-type (WT) ERE hPRL-Luc promoter (ERE-WT). The −1189 ERE controls not only the response to E2 treatment but also the acute transcriptional response to TNFα, which was abolished in ERE-Mut cells. ERE-WT cells displayed a biphasic transcriptional response after TNFα treatment, the acute phase of which was blocked after treatment with the estrogen receptor antagonist 4-hydroxy-tamoxifen. Unexpectedly, we show the oscillatory characteristics of hPRL promoter activity in individual living cells were unaffected by disruption of this crucial response element, real-time bioluminescence imaging showed that transcription cycles were maintained, with similar cycle lengths, in ERE-WT and ERE-Mut cells. These data suggest the −1189 ERE is the dominant response element involved in the hPRL transcriptional response to both E2 and TNFα and, crucially, that cycles of hPRL promoter activity are independent of estrogen receptor binding

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements