Search

Abstract: High-resolution dual X and γ spectrometry has been optimized to improve the sensitivity in analyzing radioxenon from environmental samples, especially for the metastable isotopes (131Xem and 133Xem). The full-energy peak (FEP) efficiency curve in the 20–700keV range has been established with a global uncertainty better than ±3% using a Monte Carlo simulation. The minimum detectable activity concentrations (MDC) achievable at the laboratory for 131Xem, 133Xem, 133Xe and 135Xe are presented and the influence of 133Xe activity on the metastable radioxenon MDCs is evaluated. [Copyright &y& Elsevier]

Single particle tracking (SPT) is a powerful technique for real-time microscopic visualization of the movement of individual biomolecules within or on the surface of living cells. However, SPT often suffers from the suboptimal performance of the photon-emitting labels used to tag the biomolecules of interest. For example, fluorescent dyes have poor photostability, while quantum dots suffer from blinking that hampers track acquisition and interpretation. Upconverting nanoparticles (UCNPs) have recently emerged as a promising anti-Stokes luminescent label for SPT. In this work, we demonstrated targeted SPT using UCNPs. For this, we synthesized 30 nm diameter doped UCNPs and coated them with amphiphilic polymers decorated with polyethylene glycol chains to make them water-dispersible and minimize their nonspecific interactions with cells. Coated UCNPs highly homogeneous in brightness (as confirmed by a single particle investigation) were functionalized by immunoglobulin E (IgE) using a biotin-streptavidin strategy. Using these IgE-UCNP SPT labels, we tracked high-affinity IgE receptors (FcεRI) on the membrane of living RBL-2H3 mast cells at 37 °C in the presence and absence of antigen and obtained good agreement with the literature. Moreover, we used the FcεRI-IgE receptor-antibody system to directly compare the performance of UCNP-based SPT labels to organic dyes (AlexaFluor647) and quantum dots (QD655). Due to their photostability as well as their backgroundless and continuous luminescence, SPT trajectories obtained with UCNP labels are no longer limited by the photophysics of the label but only by the dynamics of the system and, in particular, the movement of the label out of the field of view and/or focal plane.

Thienoguanosine (thG) is an isomorphic fluorescent guanosine (G) surrogate, which almost perfectly mimics the natural G in DNA duplexes and may therefore be used to sensitively investigate for example protein-induced local conformational changes. To fully exploit the information given by the probe, we carefully re-investigated the thG spectroscopic properties in 12-bp duplexes, when the Set and Ring Associated (SRA) domain of UHRF1 flips its 5' flanking methylcytosine (mC). The SRA-induced flipping of mC was found to strongly increase the fluorescence intensity of thG, but this increase was much larger when thG was flanked in 3' by a C residue as compared to an A residue. Surprisingly, the quantum yield and fluorescence lifetime values of thG were nearly constant, regardless of the presence of SRA and the nature of the 3' flanking residue, suggesting that the differences in fluorescence intensities might be related to changes in absorption properties. We evidenced that thG lowest energy absorption band in the duplexes can be deconvoluted into two bands peaking at ∼350 nm and ∼310 nm, respectively red-shifted and blue-shifted, compared to the spectrum of thG monomer. Using quantum mechanical calculations, we attributed the former to a nearly pure ππ * excitation localized on thG and the latter to excited states with charge transfer character. The amplitude of thG red-shifted band strongly increased when its 3' flanking C residue was replaced by an A residue in the free duplex, or when its 5' flanking mC residue was flipped by SRA. As only the species associated with the red-shifted band were found to be emissive, the highly unusual finding of this work is that the brightness of thG in free duplexes as well as its changes on SRA-induced mC flipping almost entirely depend on the relative population and/or absorption coefficient of the red-shifted absorbing species., (© 2022 IOP Publishing Ltd.)

Background: A total of 3,419 new HIV diagnoses were reported in Germany in 2016, with migrants from sub-Saharan Africa (misSA) accounting for 14.1%. To understand the driving factors behind the epidemiological situation, we conducted a quantitative cross-sectional survey on knowledge, attitudes, behavior, and practices regarding HIV and sexually transmitted infections (STIs) among misSA living in six German cities utilizing participatory health research., Methods: Participants were recruited by peer researchers. Levels of knowledge, information needs, and preferred methods of information dissemination were analyzed to inform future prevention planning. Additionally, we analyzed sexual behavior and other risk factors for contracting HIV and STIs. The results may facilitate the formulation of targeted prevention messages in the future., Results: We included 2,432 participants in the analysis. General knowledge about HIV was adequate, as 86.9% were aware of the presented information. Statements about HIV co-infections were prior knowledge for 53.4% of the participants and about German HIV policies and HIV testing for 54.7%. Knowledge about other STIs differed, ranging from 69.6% who have ever heard of gonorrhea to 23.8% who have ever heard of genital warts. Groups with particular knowledge gaps were i) younger misSA, ii) recent migrants, iii) misSA without regular access to the German health care system, iv) misSA of lower socioeconomic status, and v) misSA with Muslim religious affiliation. The majority of participants reported information needs (72.8%), and 71.3% wanted to obtain this information from health professionals. Male misSA were more likely to report five or more sexual partners compared to females. Less than half of participants reported always using condoms with non-steady sexual partners (46.8%). Reasons for not using condoms differed between males and females. A considerable proportion of females (16.3%) and males (6.8%) experienced sexualized violence. More than one fourth of women (26.9%) were affected by female genital mutilation/cutting., Discussion: Future prevention planning should focus on sub-groups with particular knowledge gaps, recognizing their preferred methods of information dissemination. Prevention messages for male misSA should focus on their own risk perception and for female misSA on empowerment, e.g. to negotiate condom use., Competing Interests: The authors have declared that no competing interests exist.

DLX transcription factors (TFs) are master regulators of the developing vertebrate brain, driving forebrain GABAergic neuronal differentiation. Ablation of Dlx1&2 alters expression of genes that are critical for forebrain GABAergic development. We integrated epigenomic and transcriptomic analyses, complemented with in situ hybridization (ISH), and in vivo and in vitro studies of regulatory element (RE) function. This revealed the DLX-organized gene regulatory network at genomic, cellular, and spatial levels in mouse embryonic basal ganglia. DLX TFs perform dual activating and repressing functions; the consequences of their binding were determined by the sequence and genomic context of target loci. Our results reveal and, in part, explain the paradox of widespread DLX binding contrasted with a limited subset of target loci that are sensitive at the epigenomic and transcriptomic level to Dlx1&2 ablation. The regulatory properties identified here for DLX TFs suggest general mechanisms by which TFs orchestrate dynamic expression programs underlying neurodevelopment., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Vaccination approaches have generally focused on the antigen rather than the resultant antibodies generated, which differ greatly in quality and function between individuals. The ability to replace the variable regions of the native B cell receptor (BCR) heavy and light chain loci with defined recombined sequences of a preferred monoclonal antibody could enable curative adoptive cell transfer. We report CRISPR-mediated homologous recombination (HR) into the BCR of primary human B cells. Ribonucleoprotein delivery enabled editing at the model CXCR4 locus, as demonstrated by T7E1 assay, flow cytometry, and TIDE analysis. Insertion via HR was confirmed by sequencing, cross-boundary PCR, and restriction digest. Optimized conditions were used to achieve HR at the BCR variable heavy and light chains. Insertion was confirmed at the DNA level, and transgene expression from the native BCR promoters was observed. Reprogramming the specificity of antibodies in the genomes of B cells could have clinical importance., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In 2015, 3,674 new HIV diagnoses were notified in Germany; 16% of those newly diagnosed cases originated from sub-Saharan Africa (sSA). One quarter of the newly diagnosed cases among migrants from sSA (MisSA) are notified as having acquired the HIV infection in Germany. In order to reach MisSA with HIV testing opportunities, we aimed to identify which determinants influence the uptake of HIV testing among MisSA in Germany., Methods: To identify those determinants, we conducted a quantitative cross-sectional survey among MisSA in Germany. The survey was designed in a participatory process that included MisSA and other stakeholders in HIV-prevention. Peer researchers recruited participants to complete standardized questionnaires on HIV knowledge and testing. We conducted multivariable analyses (MVA) to identify determinants associated with ever having attended voluntary HIV testing; and another MVA to identify determinant associated with having had the last voluntary HIV test in Germany., Results: Peer researchers recruited 2,782 participants eligible for inclusion in the MVA. Of these participants, 59.9% (1,667/2,782) previously had an HIV test. For each general statement about HIV that participants knew prior to participation in the study, the odds of having been tested increased by 19% (OR 1.19; 95%-CI: 1.11-1.27). Participants reporting that HIV is a topic that is discussed in their community had 92% higher odds of having been tested for HIV (OR 1.92; 95%-CI: 1.60-2.31). Migrants living in Germany for less than a year had the lowest odds of having had their last HIV test in Germany (OR 0.17; 95%-CI: 0.11-0.27). Additionally, MisSA 18 to 25 years (OR 0.55; 95%-CI: 0.42-0.73) and participants with varied sexual partners and inconsistent condom use (OR 0.75; 95%-CI: 0.44-0.97) had significantly lower odds of having had their last HIV test in Germany., Discussion: Through participatory research, we were able to show that knowledge about HIV and discussing HIV in communities increased the odds of having attended HIV testing among MisSA. However, recent migrants and young sexually active people are among the least reached by testing offers in Germany. Community-based interventions may present opportunities to reach such migrants and improve knowledge and increase discussion about HIV.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer fatalities in Western societies, characterized by high metastatic potential and resistance to chemotherapy. Critical molecular mechanisms of these phenotypical features still remain unknown, thus hampering the development of effective prognostic and therapeutic measures in PDAC. Here, we show that transcriptional co-factor Transducin beta-like (TBL) 1 was over-expressed in both human and murine PDAC. Inactivation of TBL1 in human and mouse pancreatic cancer cells reduced cellular proliferation and invasiveness, correlating with diminished glucose uptake, glycolytic flux, and oncogenic PI3 kinase signaling which in turn could rescue TBL1 deficiency-dependent phenotypes. TBL1 deficiency both prevented and reversed pancreatic tumor growth, mediated transcriptional PI3 kinase inhibition, and increased chemosensitivity of PDAC cells in vivo. As TBL1 mRNA levels were also found to correlate with PI3 kinase levels and overall survival in a cohort of human PDAC patients, TBL1 was identified as a checkpoint in the malignant behavior of pancreatic cancer and its expression may serve as a novel molecular target in the treatment of human PDAC., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Disturbances in lipid homeostasis are hallmarks of severe metabolic disorders and their long-term complications, including obesity, diabetes, and atherosclerosis. Whereas elevation of triglyceride (TG)-rich very-low-density lipoproteins (VLDL) has been identified as a risk factor for cardiovascular complications, high-density lipoprotein (HDL)-associated cholesterol confers atheroprotection under obese and/or diabetic conditions. Here we show that hepatocyte-specific deficiency of transcription factor transforming growth factor β 1-stimulated clone (TSC) 22 D1 led to a substantial reduction in HDL levels in both wild-type and obese mice, mediated through the transcriptional down-regulation of the HDL formation pathway in liver. Indeed, overexpression of TSC22D1 promoted high levels of HDL cholesterol in healthy animals, and hepatic expression of TSC22D1 was found to be aberrantly regulated in disease models of opposing energy availability. The hepatic TSC22D1 transcription factor complex may thus represent an attractive target in HDL raising strategies in obesity/diabetes-related dyslipidemia and atheroprotection.

Excessive genome damage activates the apoptosis response. Protein kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. Here, we deciphered the molecular mechanism of HIPK2 activation and show its relevance for DNA damage-induced apoptosis in cellulo and in vivo. HIPK2 autointeracts and site-specifically autophosphorylates upon DNA damage at Thr880/Ser882. Autophosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphorylation and apoptosis in cellulo. Moreover, HIPK2 autophosphorylation is conserved between human and zebrafish and is important for DNA damage-induced apoptosis in vivo. Mechanistically, autophosphorylation creates a binding signal for the phospho-specific isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by inhibiting HIPK2 polyubiquitination and modulating Siah-1-HIPK2 interaction. Concordantly, Pin1 is required for DNA damage-induced HIPK2 stabilization and p53 Ser46 phosphorylation and is essential for induction of apotosis both in cellulo and in zebrafish. Our results identify an evolutionary conserved mechanism regulating DNA damage-induced apoptosis.