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SummaryRecently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

This study aimed to evaluate laser fluorescence (LF) for monitoring the initial stage of subsurface de- and remineralization (<150 μm depth). Ninety-six sound blocks of bovine enamel, selected according to surface hardness (SH) and LF were used in two experimental studies, in vitro and in situ. In vitro, blocks were exposed to a demineralizing solution, then remineralized by pH cycling for 6 days. In situ, 10 volunteers wore acrylic palatal appliances, each containing 4 dental enamel blocks that were demineralized for 14 days by exposure to 20% sucrose solution. Following this treatment, blocks were submitted to remineralization for 1 week with fluoride dentifrice (1,100 μg F/g). In both experiments, SH and LH were measured after demineralization and after remineralization. Further, enamel blocks were selected after the demineralization/remineralization steps for measurement of cross-sectional hardness and integrated loss of subsurface hardness (ΔKHN). SH and ΔKHN showed significant differences among the phases in each study. LF values for sound, demineralized and remineralized enamel were: 5.2 ± 1.1, 8.1 ± 1.2 and 5.6 ± 0.8, respectively, in the in vitro study, and 5.3 ± 0.3, 16.5 ± 4.7 and 6.5 ± 2.5, respectively, in the in situ study, values for demineralized enamel being significantly higher than for sound and remineralized enamel in both studies. However, LF was correlated with ΔKHN only in situ. LF was capable of monitoring de- and remineralization in early lesions in situ, when bacteria are presumably present in the caries lesion body, but is not correlated with mineral changes in bacteria-free systems.

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Endogenous retroviruses present in the human genome provide a rich record of ancient infections. All presently recognized elements, including the youngest and most intact proviruses of the human endogenous retrovirus K(HML-2) [HERV-K(HML-2)] family, have suffered postinsertional mutations during their time of chromosomal residence, and genes encoding the envelope glycoprotein (Env) have not been spared these mutations. In this study, we have, for the first time, reconstituted an authentic Env of a HERV-K(HML-2) provirus by back mutation of putative postinsertional amino acid changes of the protein encoded by HERV-K113. Aided by codon-optimized expression, we demonstrate that the reconstituted Env regained its ability to be incorporated into retroviral particles and to mediate entry. The original ancient HERV-K113 Env was synthesized as a moderately glycosylated gp95 precursor protein cleaved into surface and transmembrane (TM) subunits. Of the nine N-linked oligosaccharides, four are part of the TM subunit, contributing 15 kDa to its apparent molecular mass of 41 kDa. The carbohydrates, as well as the cytoplasmic tail, are critical for efficient intracellular trafficking, processing, stability, and particle incorporation. Whereas deletions of the carboxy-terminal 6 residues completely abrogated cleavage and virion association, more extensive truncations slightly enhanced incorporation but dramatically increased the ability to mediate entry of pseudotyped lentiviruses. Although the first HERV-K(HML-2) elements infected human ancestors about 30 million years ago, our findings indicate that their glycoproteins are in most respects remarkably similar to those of classical contemporary retroviruses and can still mediate efficient entry into mammalian cells.

Tissue sodium content (TSC) is a sensitive measure of pathological changes and can be detected non-invasively by MRI. For the absolute quantification of TSC, B1 inhomogeneities must be corrected, which is not well established beyond research applications. An in-depth analysis of B1 mapping methods which are suitable for application in TSC quantification is presented. On the basis of these results, a method for simultaneous B1 mapping and imaging is proposed in order to enhance accuracy and to reduce measurement time at clinical field strengths. The B1 mapping techniques used were phase-sensitive (PS), Bloch-Siegert shift (BSS), double-angle (DAM) and actual flip-angle imaging (AFI) methods. Experimental and theoretical comparisons demonstrated that the PS technique yields the most accurate field profiles and exhibits the highest signal-to-noise ratio (SNR). Simultaneous B1 mapping and imaging was performed for the PS method, employing both degrees of freedom of the MR signal: the B1 field is encoded into signal phase and the amplitude provides the concentration information. In comparison with the more established DAM, a 13% higher SNR was obtained and field effects could be corrected more accurately without the need for additional measurement time. The protocol developed was applied to measure TSC in the healthy human head at an isotropic resolution of 4 mm. TSC was determined to be 35 ± 1 mM in white matter and 134 ± 3 mM in vitreous humor. By employing the proposed simultaneous characterization of the B1 field and acquisition of the spin density-weighted sodium signal, the accuracy of the non-invasive measurement of TSC is enhanced and the measurement time is reduced. This should allow (23)Na MRI to be better incorporated into clinical studies and routine.

Background: Inquiry-based science education (IBSE) is suitable to teach scientific contents as well as to foster scientific skills. Similar conclusions are drawn by studies with respect to scientific literacy, motivational aspects, vocabulary knowledge, conceptual understandings, critical thinking, and attitudes toward science. Nevertheless, IBSE is rarely adopted in schools. Often barriers for teachers account for this lack, with the result that even good teachers struggle to teach science as inquiry. More importantly, studies indicate that several barriers and constraints could be ascribed to problems teacher students have at the university stage. Purpose: The purpose of this explorative investigation is to examine the problems teacher students have when teaching science through inquiry. In order to draw a holistic picture of these problems, we identified problems from three different points of view leading to the research question: What problems regarding IBSE do teacher students have from an objective, a subjective, and a self-reflective perspective? Design & method: Using video analysis and observation tools as well as qualitative content analysis and open questionnaires we identified problems from each perspective. Results: The objectively stated problems comprise the lack of essential features of IBSE especially concerning 'Supporting pupils' own investigations' and 'Guiding analysis and conclusions.' The subjectively perceived problems comprise concerns about 'Teachers' abilities' and 'Pupils' abilities,' 'Differentiated instruction' and institutional frame 'Conditions' while the self-reflectively noticed problems mainly comprise concerns about 'Allowing inquiry,' 'Instructional Aspects,' and 'Pupils' behavior.' Conclusions: Each of the three different perspectives provides plenty of problems, partially overlapping, partially complementing one another, and partially revealing completely new problems. Consequently, teacher educators have to consider these three perspectives.

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