Detection of deleterious on-target effects after CRISPR-mediated genome editing in human induced pluripotent stem cells

The CRISPR/Cas9 system is an exceedingly powerful technology for precise genome editing. Its ease of use, high editing efficiency and an ever-growing CRISPR-based toolbox has provided researchers with novel possibilities to unravel the molecular and systemic consequences of changes in the genetic code. For this reason, CRISPR is now applied for editing in a wide range of different cell lines and organisms for basic and translational research. Here, accurate and precise editing is an indispensable prerequisite to generate reliable research models. However, a lot remains to be understood about the molecular mechanism of double-strand-breaks (DSBs) in the DNA as introduced by the Cas9 nuclease during editing. In fact, CRISPR editing can be accompanied by inadvertent genomic changes at the targeted locus (on-target) as well as other genomic sites (off-target). These can have drastic consequences on gene activity or expression and therefore need to be carefully investigated. Characterizing and avoiding unwanted off-target effects (OffTE) has been the focus of several studies and reliable tools for their detection have been developed. This is, however, not the case for on-target effects (OnTE) that have only been reported very recently. These can be large deletions, large insertions, complex rearrangements, or regions of copy-neutral loss of heterozygosity (LOH) around the target site. Several studies have described frequent occurrence of OnTEs in mice, but it has not been investigated if clinically relevant human cells, such as induced pluripotent stem cells (iPSCs) are also affected. The main problem with OnTEs is that they often remain unnoticed in standard quality controls like Sanger genotyping of the target locus, and additional checks are lacking in most CRISPR-based studies. This is also because there are no simple detection tools available. Therefore, in this study, we developed simple and reliable tools for OnTE detection after CRISPR genome editing: Structural alterations like large deletions, large insertions or complex rearrangements can be identified by quantifying the number of intact alleles at the edited locus using our new method called quantitative genotyping PCR (qgPCR). In addition, we validated genotyping of neighboring single nucleotide polymorphisms (SNPs) either by Sanger sequencing or SNP microarrays to reveal editing-induced regions of LOH. The entire workflow is broadly applicable to different cell lines and organisms after editing by the NHEJ or HDR pathway. We have applied our newly established detection technology to human iPSCs after HDR-mediated editing and demonstrate universal occurrence of OnTEs at multiple loci in up to 40% of edited single-cell clones. Furthermore, using an in vitro model of Alzheimer’s disease, we illustrate deleterious consequences of OnTEs on expression of the edited gene that may reduce pathogenic effects and therefore interfere with experimental findings. Overall, the threat of undetected OnTEs undermining the reliability of CRISPR-based studies has not received sufficient attention in the field so far. With this thesis, we hope to raise further awareness and propose that our simple and reliable on-target quality control workflow should be an essential part of all relevant genome editing experiments

Ethnomedicine of the Kagera Region, north western Tanzania. Part 3: plants used in traditional medicine in Kikuku village, Muleba District.

BACKGROUND\ud \ud The Kagera region of north western Tanzania has a rich culture of traditional medicine use and practice. Traditional medicines are the mainstay of healthcare in this region and are known to support the management of many illnesses such as malaria, bacterial infections, epilepsy, gynecological problems and others. However, most of the plants being used have either not been documented or evaluated for safety and efficacy or both. This study, the sixth of an ongoing series, reports on the medicinal plants that are used at Kikuku village, Muleba District.\ud \ud METHODOLOGY\ud \ud A semi-structured questionnaire was used to collect information on the common/local names of the plants, parts of the plants used, diseases treated, methods of preparing the herbal remedies, dosage of the remedies administered, frequency and duration of treatment and toxicity of the medicines. A literature review was carried out for information on the ethnomedical uses of the reported plants.\ud \ud RESULTS\ud \ud A total of 49 plant species belonging to 47 genera and 24 plant families were documented. The family Euphorbiaceae and Asteraceae had the highest representation. The plants are used for the treatment of skin conditions (10 plants; 20%), bacterial infections and wounds (14 plants; 28.6%), malaria (14 plants; 28.6%), gastrointestinal disorders (11 plants; 22.4%), gynecological problems including infertility (8 plants; 16.3%), hypertension (5 plants; 10.2%), viral infections (7 plants; 14.3%), chest problems (5 plants; 10.2%), diabetes (3 plants; 6.1%), cancer (2 plants; 4.1%), inflammatory conditions (arthritis, rheumatism), HIV and AIDS, and hernia each treated by 1 plant (3 plants in total; 6.1%). Information obtained from the literature indicate that 25 (51.0%) of the therapeutic claims are supported by laboratory results or have similar claims of ethnomedical use from other countries.\ud \ud CONCLUSION\ud \ud Herbal remedies comprise an important and effective component of the healthcare system in Kikuku village with plants in the families Euphorbiaceae and Asteraceae comprising an important part of plants used in the indigenous healthcare management in the village. Malaria and bacterial infections dominate the list of diseases that are managed using traditional medicines

Studies toward the total synthesis of mitrephorone A

This Ph.D. thesis describes progress toward the total syntheses of the ent-trachylobane diterpenoid mitrephorone A. Mitrephorone A was isolated from the bark of Mitrephora glabra, an Indonesian custard apple tree. The natural product possesses an interesting molecular scaffold, comprising a hexacyclic ring system with eight stereocenters, an adjacent ketone moiety and an oxetane ring. Mitrephorone A shows moderate cytotoxic activities against tumour cell lines, which makes it a potential chemotherapeutic agent. The first part of this thesis describes our efforts to develop an enantioselective and convergent synthetic route to the core structure of mitrephorone A. The elaborated route commences with the preparation of enone I via an enantioselective Diels–Alder reaction. Conversion to alkyne II is realized in a six-step sequence involving an intramolecular Diels–Alder reaction to build up the caged structure found in the natural product. Next, the two building blocks II and III are joined via a Sonogashira cross coupling. An asymmetric dearomative cyclization of V closes the last carbon ring of mitrephorone A and sets the right quaternary stereochemistry at C10. Moreover, studies to advance precursor VI to the natural product are presented. In the second part of this thesis, we present an alternative enantioselective synthesis of the complete mitrephorone A carbon skeleton. This robust synthetic sequence starts with literature-known building block VII. A Sharpless dihydroxylation and a Robinson annulation sequence gave enone VIII, which is further converted to IX by α-vinylation and a late stage intramolecular Diels–Alder reaction. The challenging final deprotection of carbonate IX afforded triol X. In summary, a versatile synthetic strategy which yields a decorated scaffold of the ent-trachylobanes under comparably mild conditions is presented

Studies toward the total synthesis of mitrephorone A

This Ph.D. thesis describes progress toward the total syntheses of the ent-trachylobane diterpenoid mitrephorone A. Mitrephorone A was isolated from the bark of Mitrephora glabra, an Indonesian custard apple tree. The natural product possesses an interesting molecular scaffold, comprising a hexacyclic ring system with eight stereocenters, an adjacent ketone moiety and an oxetane ring. Mitrephorone A shows moderate cytotoxic activities against tumour cell lines, which makes it a potential chemotherapeutic agent. The first part of this thesis describes our efforts to develop an enantioselective and convergent synthetic route to the core structure of mitrephorone A. The elaborated route commences with the preparation of enone I via an enantioselective Diels–Alder reaction. Conversion to alkyne II is realized in a six-step sequence involving an intramolecular Diels–Alder reaction to build up the caged structure found in the natural product. Next, the two building blocks II and III are joined via a Sonogashira cross coupling. An asymmetric dearomative cyclization of V closes the last carbon ring of mitrephorone A and sets the right quaternary stereochemistry at C10. Moreover, studies to advance precursor VI to the natural product are presented. In the second part of this thesis, we present an alternative enantioselective synthesis of the complete mitrephorone A carbon skeleton. This robust synthetic sequence starts with literature-known building block VII. A Sharpless dihydroxylation and a Robinson annulation sequence gave enone VIII, which is further converted to IX by α-vinylation and a late stage intramolecular Diels–Alder reaction. The challenging final deprotection of carbonate IX afforded triol X. In summary, a versatile synthetic strategy which yields a decorated scaffold of the ent-trachylobanes under comparably mild conditions is presented

Making Methamphetamine

In many parts of the U.S. methamphetamine production remains a serious problem despite efforts to restrict access to precursor chemicals. Methamphetamine laboratories are a particular issue in rural areas, where many essential chemicals are available and where a lower population density means that noxious fumes are less likely to be noticed. This paper shows how the problem emerged and changed over time and includes: Early production in the U.S., the spread of meth production, the process of making meth, precursor control efforts, superlabs versus small local operations, health risks, contaminated lab sites, and children found in methamphetamine lab sites

A review of What You Are Getting Wrong about Appalachia by Elizabeth Catte

No abstract available

Influence of the active nucleus on the multiphase interstellar medium in NGC 1068

The luminous spiral NGC 1068 has now been imaged from x-ray to radio wavelengths at comparably high resolution (approximately less than 5 in. FWHM). The bolometric luminosity of this well-known Seyfert is shared almost equally between the active nucleus and an extended 'starburst' disk. In an ongoing study, we are investigating the relative importance of the nucleus and the disk in powering the wide range of energetic activity observed throughout the galaxy. Our detailed analysis brings together a wealth of data: ROSAT HRI observations, VLA lambda lambda 6-20 cu cm and OVRO interferometry, lambda lambda 0.4-10.8 micron imaging, and Fabry-Perot spectrophotometry