Cell-penetrating peptide secures an efficient endosomal escape of an intact cargo upon a brief photo-induction

Since their discovery, cell-penetrating peptides (CPPs) have provided a novel, efficient, and non-invasive mode of transport for various (bioactive) cargos into cells. Despite the ever-growing number of successful implications of the CPP-mediated delivery, issues concerning their intracellular trafficking, significant targeting to degradative organelles, and limited endosomal escape are still hindering their widespread use. To overcome these obstacles, we have utilized a potent photo-induction technique with a fluorescently labeled protein cargo attached to an efficient CPP, TP10. In this study we have determined some key requirements behind this induced escape (e.g., dependence on peptide-to-cargo ratio, time and cargo), and have semi-quantitatively assessed the characteristics of the endosomes that become leaky upon this treatment. Furthermore, we provide evidence that the photo-released cargo remains intact and functional. Altogether, we can conclude that the photo-induced endosomes are specific large complexes-condensed non-acidic vesicles, where the released cargo remains in its native intact form. The latter was confirmed with tubulin as the cargo, which upon photo-induction was incorporated into microtubules. Because of this, we propose that combining the CPP-mediated delivery with photo-activation technique could provide a simple method for overcoming major limitations faced today and serve as a basis for enhanced delivery efficiency and a subsequent elevated cellular response of different bioactive cargo molecules.     

Mutations in SEC63 cause autosomal dominant polycystic liver disease

Mutations in PRKCSH, encoding the beta-subunit of glucosidase II, an N-linked glycan-processing enzyme in the endoplasmic reticulum (ER), cause autosomal dominant polycystic liver disease. We found that mutations in SEC63, encoding a component of the protein translocation machinery in the ER, also cause this disease. These findings are suggestive of a role for cotranslational protein-processing pathways in maintaining epithelial luminal structure and implicate noncilial ER proteins in human polycystic disease.     

The mosaic that is our genome

The discovery of the basis of genetic variation has opened inroads to understanding our history as a species. It has revealed the remarkable genetic similarity we share with other individuals as well as with our closest primate relatives. To understand what make us unique, both as individuals and as a species, we need to consider the genome as a mosaic of discrete segments, each with its own unique history and relatedness to different contemporary and ancestral individuals.     

A genome-wide comparison of recent chimpanzee and human segmental duplications

We present a global comparison of differences in content of segmental duplication between human and chimpanzee, and determine that 33% of human duplications (> 94% sequence identity) are not duplicated in chimpanzee, including some human disease-causing duplications. Combining experimental and computational approaches, we estimate a genomic duplication rate of 4-5 megabases per million years since divergence. These changes have resulted in gene expression differences between the species. In terms of numbers of base pairs affected, we determine that de novo duplication has contributed most significantly to differences between the species, followed by deletion of ancestral duplications. Post-speciation gene conversion accounts for less than 10% of recent segmental duplication. Chimpanzee-specific hyperexpansion (> 100 copies) of particular segments of DNA have resulted in marked quantitative differences and alterations in the genome landscape between chimpanzee and human. Almost all of the most extreme differences relate to changes in chromosome structure, including the emergence of African great ape subterminal heterochromatin. Nevertheless, base per base, large segmental duplication events have had a greater impact (2.7%) in altering the genomic landscape of these two species than single-base-pair substitution (1.2%).     

Analysis of one million base pairs of Neanderthal DNA

Neanderthals are the extinct hominid group most closely related to contemporary humans, so their genome offers a unique opportunity to identify genetic changes specific to anatomically fully modern humans. We have identified a 38,000-year-old Neanderthal fossil that is exceptionally free of contamination from modern human DNA. Direct high-throughput sequencing of a DNA extract from this fossil has thus far yielded over one million base pairs of hominoid nuclear DNA sequences. Comparison with the human and chimpanzee genomes reveals that modern human and Neanderthal DNA sequences diverged on average about 500,000 years ago. Existing technology and fossil resources are now sufficient to initiate a Neanderthal genome-sequencing effort.     

Ancient DNA: Methodological challenges

The study of ancient DNA offers the possibility of following genetic change over time. However, the field is plagued by a problem which is unique in molecular biology-the difficulty of verifying results by reproduction. Some of the reasons for this are technical and derive from the low copy number and damaged state of ancient DNA molecules. Other reasons are the unique nature of many of the objects from which DNA is extracted. We describe methodological approaches with which these problems can be alleviated in order to ensure that results are scientific in the sense that they can be reproduced by others.     

Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome

To gain insight into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase occupancy and histone modifications at 16,000 promoters in primary human somatic and germline cells. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in male gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes are methylated in somatic cells, suggesting additional functional selection. These results show that promoter sequence and gene function are major predictors of promoter methylation states. Moreover, we observe that inactive unmethylated CpG island promoters show elevated levels of dimethylation of Lys4 of histone H3, suggesting that this chromatin mark may protect DNA from methylation.