CRISPR RNA-guided FokI nucleases repair a PAH variant in a phenylketonuria model

The CRISPR/Cas9 system is a recently developed genome editing technique. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9) to correct the most common variant (allele frequency 21.4%) in the phenylalanine hydroxylase (PAH) gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed. Correction of the disease-causing variants could rescue residual PAH activity and restore normal function. Co-expression of a single guide RNA plasmid, a FokI-dCas9-zsGreen1 plasmid, and the presence of a single-stranded oligodeoxynucleotide in PAH_c.1222C>T COS-7 cells - an in vitro model for PKU - corrected the PAH variant and restored PAH activity. Also in this system, the HDR enhancer RS-1 improved correction efficiency. This proof-of-concept indicates the potential of the FokI-dCas9 system for precision medicine, in particular for targeting PKU and other monogenic metabolic diseases

Mass Measurements of Neutron-Deficient Yb Isotopes and Nuclear Structure at the Extreme Proton-Rich Side of the N=82 Shell

International audienceHigh-accuracy mass measurements of neutron-deficient Yb isotopes have been performed at TRIUMF using TITAN’s multiple-reflection time-of-flight mass spectrometer (MR-TOF-MS). For the first time, an MR-TOF-MS was used on line simultaneously as an isobar separator and as a mass spectrometer, extending the measurements to two isotopes further away from stability than otherwise possible. The ground state masses of Yb150,153 and the excitation energy of Ybm151 were measured for the first time. As a result, the persistence of the N=82 shell with almost unmodified shell gap energies is established up to the proton drip line. Furthermore, the puzzling systematics of the h11/2-excited isomeric states of the N=81 isotones are unraveled using state-of-the-art mean field calculation

Transcriptional profiling of HERV-K(HML-2) in amyotrophic lateral sclerosis and potential implications for expression of HML-2 proteins

Abstract Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. About 90% of ALS cases are without a known genetic cause. The human endogenous retrovirus multi-copy HERV-K(HML-2) group was recently reported to potentially contribute to neurodegeneration and disease pathogenesis in ALS because of transcriptional upregulation and toxic effects of HML-2 Envelope (Env) protein. Env and other proteins are encoded by some transcriptionally active HML-2 loci. However, more detailed information is required regarding which HML-2 loci are transcribed in ALS, which of their proteins are expressed, and differences between the disease and non-disease states. Methods For brain and spinal cord tissue samples from ALS patients and controls, we identified transcribed HML-2 loci by generating and mapping HML-2-specific cDNA sequences. We predicted expression of HML-2 env gene-derived proteins based on the observed cDNA sequences. Furthermore, we determined overall HML-2 transcript levels by RT-qPCR and investigated presence of HML-2 Env protein in ALS and control tissue samples by Western blotting. Results We identified 24 different transcribed HML-2 loci. Some of those loci are transcribed at relatively high levels. However, significant differences in HML-2 loci transcriptional activities were not seen when comparing ALS and controls. Likewise, overall HML-2 transcript levels, as determined by RT-qPCR, were not significantly different between ALS and controls. Indeed, we were unable to detect full-length HML-2 Env protein in ALS and control tissue samples despite reasonable sensitivity. Rather our analyses suggest that a number of HML-2 protein variants other than full-length Env may potentially be expressed in ALS patients. Conclusions Our results expand and refine recent publications on HERV-K(HML-2) and ALS. Some of our results are in conflict with recent findings and call for further specific analyses. Our profiling of HML-2 transcription in ALS opens up the possibility that HML-2 proteins other than canonical full-length Env may have to be considered when studying the role of HML-2 in ALS disease

Dimensionless quantities in discrete element method: powder model parameterization for additive manufacturing

Powder bed-based additive manufacturing processes offer an extended freedom in design and enable the processing of metals, ceramics, and polymers with a high level of relative density. The latter is a prevalent measure of process and component quality, which depends on various input variables. A key point in this context is the condition of powder beds. To enhance comprehension of their particle-level formation and facilitate process optimization, simulations based on the Discrete Element Method are increasingly employed in research. To generate qualitatively as well as quantitatively reliable simulation results, an adaptation of the contact model parameterization is necessary. However, current adaptation methods often require the implementation of models that significantly increase computational effort, therefore limiting their applicability. To counteract this obstacle, a sophisticated formula-based adaptation and evaluation method is presented in this research. Additionally, the developed method enables accelerated parameter determination with limited experimental effort. Thus, it represents an integrative component, which supports further research efforts based on the Discrete Element Method by significantly reducing the parameterization effort. The universal nature of deducting this method also allows its adaptation to similar parameterization problems and its implementation in other fields of research

Association between Rumination Times Detected by an Ear Tag-Based Accelerometer System and Rumen Physiology in Dairy Cows

Monitoring rumination activity is considered a useful indicator for the early detection of diseases and metabolic disorders. Accelerometer-based sensor systems provide health alerts based on individual thresholds of rumination times in dairy cows. Detailed knowledge of the relationship between sensor-based rumination times and rumen physiology would help detect conspicuous animals and evaluate the treatment’s success. This study aimed to investigate the association between sensor-based health alerts and rumen fluid characteristics in Holstein-Friesian cows at different stages of lactation. Rumen fluid was collected via a stomach tube from 63 pairs of cows with and without health alerts (ALRT vs NALRT). Pairs were matched based on the day of lactation, the number of lactations, and health criteria. Rumen fluid was collected during and after health alerts. The parameters of color, odor, consistency, pH, redox potential, sedimentation flotation time, and the number of protozoa were examined. Results showed differences between both groups in odor, rumen pH, sedimentation flotation time, and protozoan count at the first rumen fluid collection. Within the groups, greater variations in rumen fluid parameters were found for ALRT cows compared to NALRT cows. The interaction between health alert and stage of lactation did not affect the rumen fluid parameters

Systematic Evaluation of Different Fresh Cow Monitoring Procedures

Establishing fresh cow monitoring procedures is considered beneficial for cow health, welfare, and productivity. However, they are time consuming and require the cows to be locked up, which restricts their natural behavior. In this study, different fresh cow monitoring procedures were evaluated. Two experiments were conducted to determine: (1) the duration of various examinations and treatments; (2) the time cows remain locked up in headlocks; and (3) the proportion of examination and treatment times relative to the total headlock time. In advance, standard operating procedures were established. Three veterinarians conducted the examinations and treatments based on changes in milk yield, clinical symptoms, and alarms by an accelerometer system. The headlock time was evaluated for three workflow strategies, which differed in the order of examinations and treatments. To determine the duration, cameras were installed, and the video footage was analyzed. The examinations lasted between 1 and 227 s, and the cows were locked up in headlocks between 0.01 and 1.76 h. The lock-up times differed significantly among the three strategies, as well as the proportion. This study provides information that can be used as a basis for the development of time-efficient strategies, and to minimize the impact on cows’ time budgets

Generation of an iPSC line from a patient with infantile liver failure syndrome 2 due to mutations in NBAS: DHMCi004-A

Fibroblasts of a patient with Infantile Liver Failure Syndrome 2 (OMIM #616483) due to a homozygous missense variant in the neuroblastoma amplified sequence gene (NBAS; c.[2708T>G]; c.[2708T>G]/p.[Leu903Arg]; p.[Leu903Arg]) were reprogrammed to iPSCs using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) delivering the reprogramming factors Oct3/4, Sox2, c-Myc and Klf4. Cells showed a normal karyotype. Pluripotency of DHMCi004-A was proven using immunohistochemistry, RT-PCR analysis, flow cytometry and differentiation into all three germ layers using the STEMdiff™ Trilineage Differentiation Kit (Stemcell Technologies). DHMCi004-A represents the first iPS-based cell model system to elucidate the pathomechanism underlying this disease

Association between Rumination Times Detected by an Ear Tag-Based Accelerometer System and Rumen Physiology in Dairy Cows

Monitoring rumination activity is considered a useful indicator for the early detection of diseases and metabolic disorders. Accelerometer-based sensor systems provide health alerts based on individual thresholds of rumination times in dairy cows. Detailed knowledge of the relationship between sensor-based rumination times and rumen physiology would help detect conspicuous animals and evaluate the treatment\u27s success. This study aimed to investigate the association between sensor-based health alerts and rumen fluid characteristics in Holstein-Friesian cows at different stages of lactation. Rumen fluid was collected via a stomach tube from 63 pairs of cows with and without health alerts (ALRT vs NALRT). Pairs were matched based on the day of lactation, the number of lactations, and health criteria. Rumen fluid was collected during and after health alerts. The parameters of color, odor, consistency, pH, redox potential, sedimentation flotation time, and the number of protozoa were examined. Results showed differences between both groups in odor, rumen pH, sedimentation flotation time, and protozoan count at the first rumen fluid collection. Within the groups, greater variations in rumen fluid parameters were found for ALRT cows compared to NALRT cows. The interaction between health alert and stage of lactation did not affect the rumen fluid parameters

Heterogeneous knowledge: Trends in German discourse analysis against an international background

This contribution maps the complex field of discourse analysis in Germany by situating its major currents and putting them in historical perspective. In a first step, it presents the major intellectual sources, such as (post-)structuralism, pragmatism/interactionism as well as hermeneutics, which have served as a backdrop for the establishment of discourse analysis as an interdisciplinary field since the 1980s. In a second step, it takes a closer look at the intellectual conjunctures in the social sciences such as Critical Theory and systems theory before turning to the discourse analytical tendencies that have emerged since the 1980s in the light of Foucault's reception in Germany. Finally, it discusses the features of heterogeneous knowledge discourses. As against top-down studies of political discourse in France and bottom-up investigations of everyday discourse in the US, many discourse analysts in Germany focus on knowledge production as a multi-leveled process involving texts and contexts. Therefore, discourse is seen as a heterogeneous object constructed in the interplay of language, praxis and knowledge