Efficient Designer Nuclease-Based Homologous Recombination Enables Direct PCR Screening for Footprintless Targeted Human Pluripotent Stem Cells

SummaryGenetic engineering of human induced pluripotent stem cells (hiPSCs) via customized designer nucleases has been shown to be significantly more efficient than conventional gene targeting, but still typically depends on the introduction of additional genetic selection elements. In our study, we demonstrate the efficient nonviral and selection-independent gene targeting in human pluripotent stem cells (hPSCs). Our high efficiencies of up to 1.6% of gene-targeted hiPSCs, accompanied by a low background of randomly inserted transgenes, eliminated the need for antibiotic or fluorescence-activated cell sorting selection, and allowed the use of short donor oligonucleotides for footprintless gene editing. Gene-targeted hiPSC clones were established simply by direct PCR screening. This optimized approach allows targeted transgene integration into safe harbor sites for more predictable and robust expression and enables the straightforward generation of disease-corrected, patient-derived iPSC lines for research purposes and, ultimately, for future clinical applications

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Reply to Mankowska et al. Comment on “Muth et al. Assessing Critical Flicker Fusion Frequency: Which Confounders? A Narrative Review. <i>Medicina</i> 2023, <i>59</i>, 800”

First and foremost, we like to express our gratitude for the praise bestowed upon our narrative review [...

TALEN-mediated functional correction of X-linked chronic granulomatous disease in patient-derived induced pluripotent stem cells

X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes

Observation of burst activity from SGR1935+2154 associated to first galactic FRB with H.E.S.S.

Fast radio bursts (FRB) are enigmatic powerful single radio pulses with durations of several milliseconds and high brightness temperatures suggesting coherent emission mechanism. For the time being a number of extragalactic FRBs have been detected in the high-frequency radio band including repeating ones. The most plausible explanation for these phenomena is magnetar hyperflares. The first observational evidence of this scenario was obtained in April 2020 when an FRB was detected from the direction of the Galactic magnetar and soft gamma repeater SGR1935+2154. The FRB was preceded with a number of soft gamma-ray bursts observed by Swift-BAT satellite, which triggered the follow-up program of the H.E.S.S. imaging atmospheric Cherenkov telescopes (IACTs). H.E.S.S. has observed SGR1935+2154 over a 2 hour window few hours prior to the FRB detection by STARE2 and CHIME. The observations overlapped with other X-ray bursts from the magnetar detected by INTEGRAL and Swift-BAT, thus providing first observations of a magnetar in a flaring state in the very-high energy domain. We present the analysis of these observations, discuss the obtained results and prospects of the H.E.S.S. follow-up program for soft gamma repeaters and anomalous X-ray pulsars

Deep observations of Kepler's SNR with H.E.S.S.

Kepler’s supernova remnant (SNR) which is produced by the most recent naked-eye supernova in our Galaxy is one of the best studied SNRs, but its gamma-ray detection has eluded us so far. Observations with modern imaging atmospheric Cherenkov telescopes (IACT) have enlarged the knowledge about nearby SNRs with ages younger than 500 years by establishing Cassiopeia A and Tycho’s SNRs as very high energy (VHE) gamma-ray sources and setting a lower limit on the distance to Kepler’s SNR. This SNR is significantly more distant than the other two and expected to be one of the faintest gamma-ray sources within reach of the IACT arrays of this generation. We report strong evidence for a VHE signal from Kepler’s SNR based on deep observations of the High Energy Stereoscopic System (H.E.S.S.) with an exposure of 152 hours, including 122 hours accumulated in 2017-2020. We further discuss implications of this result for cosmic-ray acceleration in young SNRs