Generation of a TMEM43 knockout human induced pluripotent stem cell line (HDZi003-A-1) using CRISPR/Cas9.

TMEM43 (LUMA) is a ubiquitously expressed protein with unknown function. The protein is phylogenetically highly conserved and also found in Drosophila melanogaster (Klinke et al., 2022). TMEM43-p.S358L is a rare, fully penetrant mutation that leads to arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5). To understand the function of the ARVC5-associated mutation it is first important to understand the function of the TMEM43 protein. Therefore, a TMEM43 knockout induced pluripotent stem cell (iPSC) line was generated using the CRISPR/Cas9 genome editing system. The resulting cell line had a deficiency of TMEM43 and showed normal morphology and a stable karyotype. The colonies were positive for pluripotency markers and could be differentiated into the three germ layers.

A Versatile Toolset for Genetic Manipulation of the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is an ascomycetous yeast that frequently dominates the population in the first two days of wine fermentations. It contributes to the production of many beneficial as well as detrimental aroma compounds. While the genome sequence of the diploid type strain DSM 2768 has been largely elucidated, transformation by electroporation was only recently achieved. We here provide an elaborate toolset for the genetic manipulation of this yeast. A chromosomal replication origin was isolated and used for the construction of episomal, self-replicating cloning vectors. Moreover, homozygous auxotrophic deletion markers (Huura3, Huhis3, Huleu2, Huade2) have been obtained in the diploid genome as future recipients and a proof of principle for the application of PCR-based one-step gene deletion strategies. Besides a hygromycin resistance cassette, a kanamycin resistance gene was established as a dominant marker for selection on G418. Recyclable deletion cassettes flanked by loxP-sites and the corresponding Cre-recombinase expression vectors were tailored. Moreover, we report on a chemical transformation procedure with the use of freeze-competent cells. Together, these techniques and constructs pave the way for efficient and targeted manipulations of H. uvarum.

The use of magnetic resonance imaging to examine ossification of the proximal tibial epiphysis for forensic age estimation in living individuals.

The establishment of radiation-free examination procedures in the field of forensic age diagnostics in living persons is to be considered of special scientific interest so as to minimize necessary exposure to X-rays while facilitating additional assessment of skeletal development in all cases. To this end, the advantages offered by magnetic resonance imaging in securing a practical application which is as unrestricted and complication-free as possible should be among the methods exploited in investigating such indicators of skeletal maturity. Within the framework of a retrospective study, we investigated the ossification status of the proximal tibial epiphysis on the MRI scans of 124 females and 166 males aged between 10 and 30 years. All the images had been generated on a 3.0 T scanner using a T1-weighted turbo spin-echo sequence. When evaluating the ossification stage, a combination of modified classifications proposed by Schmeling et al. and by Kellinghaus et al. was used. The statistical evaluation included calculation of a variety of measures to describe specific ossification stages as well as kappa coefficients to assess intra- and inter-observer agreement on diagnoses of individual stages. In forensic contexts, completion of the 14th year of life can be adequately evidenced in females with an ossification stage IV according to Schmeling et al. and in males with an ossification stage III c according to Kellinghaus et al. or an ossification stage IV according to Schmeling et al. In forensic contexts, the presence of an ossification stage IV according to Schmeling et al. can prove that the age of 16 years has been exceeded only in the male sex, whereby for age estimation purposes the diagnosis should be in line with other skeletal maturity indicators. The results available displayed a high degree of intra- and inter-observer agreement. Examination of the ossification status of the proximal tibial epiphysis using magnetic resonance imaging represents an effective additional tool for use in radiation-free forensic age diagnostics in living persons.

Forensic age estimation in living individuals using 3.0 T MRI of the distal femur.

To increase the reliability of forensic age estimations in living adolescents and young adults where there is no legitimation for X-ray examinations, it would be desirable to establish radiation-free imaging methods. In the present study, the ossification stage of the distal femoral epiphysis was determined retrospectively in 166 male and 124 female individuals in the age group 10 to 30 years using 3.0T MRI. When evaluating the ossification stage, a combination of the classifications proposed by Schmeling et al. and by Kellinghaus et al. was used. Within the study population, stages 3b, 3c and 4 did not occur in the male sex before the 14th birthday, although the validity of the results is distinctly limited relative to stage 3b in view of the small number of cases. In females, stages 3c and 4 did not occur before the 14th birthday. Stage 4 did not occur in males before the 18th birthday. Should this result be confirmed in future studies, there would be an additional criterion providing evidence that the age of 18 years has been completed.

Surgical treatment of space occupying edema and hemorrhage due to cerebral venous thrombosis during pregnancy.

BACKGROUND: During late pregnancy and the puerperium cerebral venous and sinus thrombosis (CVST) is a rare but important cause of stroke. Despite adequate anticoagulation some patients deteriorate, which may warrant the use of more aggressive treatment modalities. CASE REPORT: A 29-year-old pregnant woman (29th week of pregnancy) presented with diffuse headaches and a progressive left hemiparesis. MRI revealed a CVST involving the superior sagittal sinus, the left lateral sinus, and the rectal sinus. Furthermore, it showed a space occupying brain edema and a congestional bleeding within the frontal and parietal lobes on the right side. Despite immediate intravenous anticoagulation and treatment with mannitol the patient developed a progressive loss of consciousness and unilateral third nerve palsy as a result of a beginning transtentorial herniation. Due to the severe course of the CVST an urgent decompressive craniectomy and shortly thereafter a cesarean section were performed. The patient made an excellent recovery. CONCLUSION: While previous reports have demonstrated the feasibility of decompressive hemicraniectomy in selected patients with CVST and beginning herniation due to space occupying brain edema, venous infarction and congestional bleeding with mass effect, our rare case supports the notion that this procedure can also be life-saving in pregnant women.

Update on multidetector coronary CT angiography of coronary stents: in vitro evaluation of 29 different stent types with dual-source CT.

The aim of this study was to test a large sample of the latest coronary artery stents using four image reconstruction approaches with respect to lumen visualization, lumen attenuation, and image noise in dual-source multidetector row CT (DSCT) in vitro and to provide a CT catalogue of currently used coronary artery stents. Twenty-nine different coronary artery stents (19 steel, 6 cobalt-chromium, 2 tantalum, 1 iron, 1 magnesium) were examined in a coronary artery phantom (vessel diameter 3 mm, intravascular attenuation 250 HU, extravascular density -70 HU). Stents were imaged in axial orientation with standard parameters: 32 x 0.6 collimation, pitch 0.24, 400 mAs, 120 kV, rotation time 0.33 s. Image reconstructions were obtained with four different convolution kernels (soft, medium-soft, standard high-resolution, stent-dedicated). To evaluate visualization characteristics of the stent, the lumen diameter, intraluminal density, and noise were measured. The stent-dedicated kernel offered best average lumen visualization (54 +/- 8.3%) and most realistic lumen attenuation (222 +/- 44 HU) at the expense of increased noise (23.9 +/- 1.9 HU) compared with standard CTA protocols (p < 0.001 for all). The magnesium stent showed the least artifacts with a lumen visibility of 90%. The majority of stents (79%) exhibited a lumen visibility of 50-59%. Less than half of the stent lumen was visible in only six stents. Stent lumen visibility largely varies depending on the stent type. Magnesium is by far more favorable a stent material with regard to CT imaging when compared with the more common materials steel, cobalt-chromium, or tantalum. The magnesium stent exhibits a lumen visibility of 90%, whereas the majority of the other stents exhibit a lumen visibility of 50-59%.

Electrocardiogram-independent image reconstruction in cardiac multidetector computed tomography using retrospective motion synchronization.

OBJECTIVE: We sought to evaluate an electrocardiogram (ECG)-independent image reconstruction technique for coronary computed tomography (CT)-angiography based on cardiac motion. MATERIALS AND METHODS: The raw data from 20 patients was reconstructed with both an ECG-gated algorithm and a motion-dependent algorithm that calculates the cardiac motion-function directly from the CT raw data using a center of mass technique. Images were reconstructed in 5% steps over the R-R interval and the cardiac motion-cycle. For both approaches multiplanar reformations were created and the set of images with the least motion artifacts was used for the evaluation. Motion artifacts affecting the ascending aorta, the left main coronary artery and the entire course of the LAD, LCX and RCA were scored using a 5-point scale. RESULTS: The mean optimal reconstruction window was at 60% of the R-R interval and 30% of the cardiac motion cycle. A total of 73 of 100 vascular regions showed no motion artifacts in ECG-gated images, with the motion-synchronized algorithm only 41 regions were free of motion artifacts. The mean motion-score was 1.4 (+/-0.6) and 2.4 (+/-1.2) respectively (P < 0.05). CONCLUSION: In the currently implemented form the motion-gated algorithm is inferior to ECG-gated image reconstruction but can be used in patients with an incomplete or corrupt ECG-signal.