Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples

Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.     

Simplifying the digital workflow of facial prostheses manufacturing using a three-dimensional (3D) database: setup,development, and aspects of virtual data validation for reproduction

PurposeTo set up the digital database (DDB) of various anatomical parts, skin details and retention elements in order to simplify the digital workflow of facial prostheses manufacturing; and to quantify the reproduction of skin wrinkles on the prostheses prototypes with stereolithography (SLA) and direct light processing (DLP) methods.MethodsTwo structured light scanners were used to obtain the nasal and auricle forms of 50 probands. Furthermore, the ala nasi and scapha areas were captured with the digital single lens reflex camera and saved in jpeg format. The four magnetic retention elements were remodeled in computer aided design (CAD) software. The 14 test blocks with embossed wrinkles of 0.05–0.8 mm were printed with SLA and DLP methods and afterwards analyzed by means of profilometry and confocal microscopy.ResultsThe introduced DDB allows for production of customized facial prosthesis and makes it possible to consider the integration of concrete retention elements on the CAD stage, which makes the prosthesis modelling more predictable and efficient. The obtained skin structures can be applied onto the prosthesis surface for customization. The reproduction of wrinkles from 0.1 to 0.8 mm in depth may be associated with the loss of 4.5%–11% of its profile with SLA or DLP respectively. Besides, the reproduction of 0.05 mm wrinkles may be met with up to 40% profile increasement.ConclusionsThe utilization of DDB may simplify the digital workflow of facial prostheses manufacturing. The transfer of digitally applied skin wrinkles till the prostheses’ prototypes may be associated with deviations from 11 to 40%.     

Alanine Aminotransferase Elevation in Obese Infants and Children: A Marker of Early Onset Non Alcoholic Fatty Liver Disease

Background:

Elevated aminotransferases serve as surrogate markers of non-alcoholic fatty liver disease, a feature commonly associated with the metabolic syndrome. Studies on the prevalence of fatty liver disease in obese children comprise small patient samples or focus on those patients with liver enzyme elevation.

Objectives:

We have prospectively analyzed liver enzymes in all overweight and obese children coming to our tertiary care centre.

Patients and Methods:

In a prospective study 224 healthy, overweight or obese children aged 1 - 12 years were examined. Body Mass Index-Standard Deviation Score, alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl-transpeptidase were measured.

Results:

Elevated alanine aminotransferase was observed in 29% of children. 26 % of obese and 30 % of overweight children had liver enzyme elevations. Obese children had significantly higher alanine aminotransferase levels than overweight children (0.9 vs. 0.7 times the Upper Limit of Normal; P = 0.04).

Conclusions:

Elevation of liver enzymes appears in 29 % obese children in a tertiary care centre. Absolute alanine aminotransferase levels are significantly higher in obese than in overweight children. Even obese children with normal liver enzymes show signs of fatty liver disease as demonstrated by liver enzymes at the upper limit of normal.     

Time-course of the electrophysiological maturation and integration of transplanted cardiomyocytes

Electrophysiological maturation and integration of transplanted cardiomyocytes are essential to enhance safety and efficiency of cell replacement therapy. Yet, little is known about these important processes. The aim of our study was to perform a detailed analysis of electrophysiological maturation and integration of transplanted cardiomyocytes. Fetal cardiomyocytes expressing enhanced green fluorescent protein were transplanted into cryoinjured mouse hearts. At 6, 9 and 12days after transplantation, viable slices of recipient hearts were prepared and action potentials of transplanted and host cardiomyocytes within the slices were recorded by microelectrodes. In transplanted cells embedded in healthy host myocardium, action potential duration at 50% repolarization (APD50) decreased from 32.2±3.3ms at day 6 to 27.9±2.6ms at day 9 and 19.6±1.6ms at day 12. The latter value matched the APD50 of host cells (20.5±3.2ms, P=0.78). Integration improved in the course of time: 26% of cells at day 6 and 53% at day 12 revealed no conduction blocks up to a stimulation frequency of 10Hz. APD50 was inversely correlated to the quality of electrical integration. In transplanted cells embedded into the cryoinjury, which showed no electrical integration, APD50 was 49.2±4.3ms at day 12. Fetal cardiomyocytes transplanted into healthy myocardium integrate electrically and mature after transplantation, their action potential properties after 12days are comparable to those of host cardiomyocytes. Quality of electrical integration improves over time, but conduction blocks still occur at day 12 after transplantation. The pace of maturation correlates with the quality of electrical integration. Transplanted cells embedded in cryoinjured tissue still possess immature electrophysiological properties after 12days.