Efficient and precise CRISPR/Cas9-mediated MECP2 modifications in human-induced pluripotent stem cells

Patients with Rett syndrome (RTT) have severe mental and physical disabilities. The majority of RTT patients carry a heterozygous mutation in methyl-CpG binding protein 2 (MECP2), an X-linked gene encoding an epigenetic factor crucial for normal nerve cell function. No curative therapy for RTT syndrome exists, and cellular mechanisms are incompletely understood. Here, we developed a CRISPR/Cas9-mediated system that targets and corrects the disease relevant regions of the MECP2 exon 4 coding sequence. We achieved homologous recombination (HR) efficiencies of 20% to 30% in human cell lines and iPSCs. Furthermore, we successfully introduced a MECP2(R270X) mutation into the MECP2 gene in human induced pluripotent stem cells (iPSCs). Consequently, using CRISPR/Cas9, we were able to repair such mutations with high efficiency in human mutant iPSCs. In summary, we provide a new strategy for MECP2 gene targeting that can be potentially translated into gene therapy or for iPSCs-based disease modeling of RTT syndrome

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Unequal crossing-over between aldosterone synthase and 11 beta-hydroxylase genes causes congenital adrenal hyperplasia

Congenital adrenal hyperplasia is one of the most frequently inherited diseases. It is characterized by a severe decline in cortisol secretion, which results in a compensatory increase in ACTH and consequent adrenal growth (hyperplasia). Here we describe the first case of 11{beta}-hydroxylase deficiency that is caused by an unequal cross-over of the genes encoding aldosterone synthase (CYP11B2) and 11{beta}-hydroxylase (CYP11B1). CYP11B1 and CYP11B2 are located on chromosome 8q24 approximately 45 kb apart from each other. The investigated genetic recombination deleted the normal alleles of the two genes and created a chimeric fusion gene, which consists of the promotor and exons 1 through 4 of the aldosterone synthase gene plus intron 4 through exon 9 of the 11{beta}-hydroxylase gene. This recombination event subordinates any remaining 11β-hydroxylase activity of the chimeric enzyme to the control mechanisms of CYP11B2, the expression of which is mainly regulated by angiotensin II and K+. Normally the 11{beta}-hydroxylase activity is controlled by ACTH. The existence of the CYP11B2/CYP11B1 chimera was discovered by means of a PCR method and was confirmed with a Southern blot. Furthermore, by applying a minigene expression method we demonstrated a point mutation in intron 3 (IVS3 + 16G→T) of the patient's second 11{beta}-hydroxylase allele that radically diminishes proper splicing of the pre-mRNA by giving rise to a new, highly preferred donor splice site

On equations for the total suction and its matric and osmotic components

A clear fundamental understanding of suctions is crucial for the study of the behaviour of plastic cement mortar and concrete, including plastic shrinkage cracking. In this paper, the expression relating the change in free energy of the pore water with an isothermal change in pressure is first derived. Based upon definitions of suctions, it is then shown that total, matric, and osmotic suctions can all be expressed in the same thermodynamic form. The widely accepted, but not yet satisfactorily validated, assumption that the total suction comprises matric and osmotic components is then confirmed theoretically. The well-known Kelvin equation for matric suction, and Morse and van’t Hoff equations for osmotic suction are subsequently derived from the corresponding thermodynamic equations. The applicability of latter two equations in evaluating the osmotic suctions of cement mortar and concrete is highlighted

Synthesis of chromenoimidazocarbolines by a reaction of quaternary iminium salts with o-hydroxybenzaldehydes

[Figure not available: see fulltext.] It was shown that the interaction of N2-(cyanomethyl)-β-carbolinium bromide with o-hydroxybenzaldehydes in the presence of ammonium acetate upon refluxing in ethanol proceeded as a domino reaction and led to the formation of chromenoimidazocarbolines. © 2017, Springer Science+Business Media, LLC

Synthesis of chromenoimidazocarbolines by a reaction of quaternary iminium salts with o-hydroxybenzaldehydes

[Figure not available: see fulltext.] It was shown that the interaction of N2-(cyanomethyl)-β-carbolinium bromide with o-hydroxybenzaldehydes in the presence of ammonium acetate upon refluxing in ethanol proceeded as a domino reaction and led to the formation of chromenoimidazocarbolines. © 2017, Springer Science+Business Media, LLC

Unexpected formation of dinaphthoaza-17-crown-5 ether containing γ-aminopiperidine subunit

New dinaphthoaza-17-crown-5 derivative containing γ-aminopiperidine subunit was synthesized by the one-pot Petrenko– Kritschenko multicomponent condensation based on 2,2’-[3,6-dioxaoctane-1,8-diylbis(oxy)]di(1-naphthaldehyde) as the podand. The crystal structure of the new azacrown compound was elucidated by X-ray diffraction. © 201

Synthesis and cytotoxicity of novel γ-piperidone-containing dibenzo-1,7-diaza-14-crown-4 ethers

For the development of new antitumor agents, novel dibenzo-1,7-diaza-14-crown-4 ethers containing γ-piperidone moiety were synthesized by a domino condensation of new podands, ketones and ammonium acetate. The crystal structure of one Ts of them was studied by X-ray diffraction. Four crown compounds were evaluated in vitro for cytotoxic activity against 5 human cancer cell lines. © 201