576 Genetik und Evolution
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- DNA typing (3)
- DNA damage (2)
- Short tandem repeat (STR) (2)
- Whole genome amplification (2)
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- ACacylcarnitines (1)
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Cyanobacteria are gaining considerable interest as a method of supporting the long-term presence of humans on the Moon and settlements on Mars due to their ability to produce oxygen and their potential as bio-factories for space biotechnology/synthetic biology and other applications. Since many unknowns remain in our knowledge to bridge the gap and move cyanobacterial bioprocesses from Earth to space, we investigated cell division resumption on the rehydration of dried Chroococcidiopsis sp. CCMEE 029 accumulated DNA damage while exposed to space vacuum, Mars-like conditions, and Fe-ion radiation. Upon rehydration, the monitoring of the ftsZ gene showed that cell division was arrested until DNA damage was repaired, which took 48 h under laboratory conditions. During the recovery, a progressive DNA repair lasting 48 h of rehydration was revealed by PCR-stop assay. This was followed by overexpression of the ftsZ gene, ranging from 7.5- to 9-fold compared to the non-hydrated samples. Knowing the time required for DNA repair and cell division resumption is mandatory for deep-space experiments that are designed to unravel the effects of reduced/microgravity on this process. It is also necessary to meet mission requirements for dried-sample implementation and real-time monitoring upon recovery. Future experiments as part of the lunar exploration mission Artemis and the lunar gateway station will undoubtedly help to move cyanobacterial bioprocesses beyond low Earth orbit. From an astrobiological perspective, these experiments will further our understanding of microbial responses to deep-space conditions.
Survival of patients with pediatric acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-SCT) is mainly compromised by leukemia relapse, carrying dismal prognosis. As novel individualized therapeutic approaches are urgently needed, we performed whole-exome sequencing of leukemic blasts of 10 children with post–allo-SCT relapses with the aim of thoroughly characterizing the mutational landscape and identifying druggable mutations. We found that post–allo-SCT ALL relapses display highly diverse and mostly patient-individual genetic lesions. Moreover, mutational cluster analysis showed substantial clonal dynamics during leukemia progression from initial diagnosis to relapse after allo-SCT. Only very few alterations stayed constant over time. This dynamic clonality was exemplified by the detection of thiopurine resistance-mediating mutations in the nucleotidase NT5C2 in 3 patients’ first relapses, which disappeared in the post–allo-SCT relapses on relief of selective pressure of maintenance chemotherapy. Moreover, we identified TP53 mutations in 4 of 10 patients after allo-SCT, reflecting acquired chemoresistance associated with selective pressure of prior antineoplastic treatment. Finally, in 9 of 10 children’s post–allo-SCT relapse, we found alterations in genes for which targeted therapies with novel agents are readily available. We could show efficient targeting of leukemic blasts by APR-246 in 2 patients carrying TP53 mutations. Our findings shed light on the genetic basis of post–allo-SCT relapse and may pave the way for unraveling novel therapeutic strategies in this challenging situation.
Modern PCR-based analytical techniques have reached sensitivity levels that allow for obtaining complete forensic DNA profiles from even tiny traces containing genomic DNA amounts as small as 125 pg. Yet these techniques have reached their limits when it comes to the analysis of traces such as fingerprints or single cells. One suggestion to overcome these limits has been the usage of whole genome amplification (WGA) methods. These methods aim at increasing the copy number of genomic DNA and by this means generate more template DNA for subsequent analyses. Their application in forensic contexts has so far remained mostly an academic exercise, and results have not shown significant improvements and even have raised additional analytical problems. Until very recently, based on these disappointments, the forensic application of WGA seems to have largely been abandoned. In the meantime, however, novel improved methods are pointing towards a perspective for WGA in specific forensic applications. This review article tries to summarize current knowledge about WGA in forensics and suggests the forensic analysis of single-donor bioparticles and of single cells as promising applications.
The development of whole-genome amplification (WGA) techniques has opened up new avenues for genetic analysis and genome research, in particular by facilitating the genome-wide analysis of few or even single copies of genomic DNA, such as from single cells (prokaryotic or eukaryotic) or virions. Using WGA, the few copies of genomic DNA obtained from such entities are unspecifically amplified using PCR or PCR-related processes in order to obtain higher DNA quantities that can then be successfully analysed further.
Polymerase Chain Reaction
(2021)
DNA Sequencing
(2021)
Background: Atypical myopathy (AM), an acquired multiple acyl-CoA dehydrogenase deficiency (MADD) in horses, induce changes in mitochondrial metabolism. Only few veterinary laboratories offer diagnostic testing for this disease. Inborn and acquired MADD exist in humans, therefore determination of organic acids (OA) in urine and acylcarnitines (AC) in blood by assays available in medical laboratories can serve as AM diagnostics. The evolution of OA and AC profiles in surviving horses is unreported.
Methods: AC profiles using electrospray ionization tandem mass spectrometry (ESI-MS/MS) and OA in urine using gas chromatography mass spectrometry (GC–MS) were determined in dried blot spots (DBS, n = 7) and urine samples (n = 5) of horses with AM (n = 7) at disease presentation and in longitudinal samples from 3/4 survivors and compared to DBS (n = 16) and urine samples (n = 7) from control horses using the Wilcoxon test.
Results: All short- (C2-C5) and medium-chain (C6-C12) AC in blood differed significantly (p < 0.008) between horses with AM and controls, except for C5:1 (p = 0.45) and C5OH + C4DC (p = 0.06). In AM survivors the AC concentrations decreased over time but were still partially elevated after 7 days. 14/62 (23%) of OA differed significantly between horses with AM and control horses. Concentrations of ethylmalonic acid, 2-hydroxyglutaric acid and the acylglycines (butyryl-, valeryl-, and hexanoylglycine) were highly elevated in the urine of all horses with AM at the day of disease presentation. In AM survivors, concentrations of those metabolites were initially lower and decreased during remission to approach normalization after 7 days.
Conclusion: OA and AC profiling by specialized human medical laboratories was used to diagnose AM in horses. Elevation of specific metabolites were still evident several days after disease presentation, allowing diagnosis via analysis of samples from convalescent animals.
Polymerase Chain Reaction
(2011)
Trade of wild-caught animals is illegal for many taxa and in many countries. Common regulatory procedures involve documentation and marking techniques. However, these procedures are subject to fraud and thus should be complemented by routine genetic testing in order to authenticate the captive-bred origin of animals intended for trade. A suitable class of genetic markers are SNPSTRs that combine a short tandem repeat (STR) and single nucleotide polymorphisms (SNPs) within one amplicon. This combined marker type can be used for genetic identification and for parentage analyses and in addition, provides insight into haplotype history. As a proof of principle, this study establishes a set of 20 SNPSTR markers for Athene noctua, one of the most trafficked owls in CITES Appendix II. These markers can be coamplified in a single multiplex reaction. Based on population data, the percentage of observed and expected heterozygosities of the markers ranged from 0.400 to 1.000 and 0.545 to 0.850, respectively. A combined probability of identity of 5.3*10-23 was achieved with the whole set, and combined parentage exclusion probabilities reached over 99.99%, even if the genotype of one parent was missing. A direct comparison of an owl family and an unrelated owl demonstrated the applicability of the SNPSTR set in parentage testing. The established SNPSTR set thus proved to be highly useful for identifying individuals and analysing parentage to determine wild or captive origin. We propose to implement SNPSTR-based routine certification in wildlife trade as a way to reveal animal laundering and misdeclaration of wild-caught animals.