Native homing endonucleases can target conserved genes in humans and in animal models

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assay

Native homing endonucleases can target conserved genes in humans and in animal models

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays

FIGURES 65–70 in The oak gall wasps of Israel (Hymenoptera, Cynipidae, Cynipini) - diversity, distribution and life history

FIGURES 65–70. Galls on various Quercus species. 65. Andricus sp. nr. quercusradicis, sexual generation on leaf of Quercus ithaburensis; 66. Neuroterus anthracinus, asexual generation on Quercus ithaburensis; 67. Andricus multiplicatus, sexual generation on Quercus libani; 68. Andricus melikai, sexual generation on Quercus libani; 69. Andricus sp. nr. amenti, sexual generation on Quercus libani; 70. Cerroneuroterus sp. nr. lanuginosus, asexual generation on Quercus libani.Published as part of Shachar, Einat, Melika, George, Inbar, Moshe & Dorchin, Netta, 2018, The oak gall wasps of Israel (Hymenoptera, Cynipidae, Cynipini) - diversity, distribution and life history, pp. 451-498 in Zootaxa 4521 (4) on page 493, DOI: 10.11646/zootaxa.4521.4.1, http://zenodo.org/record/261002

The oak gall wasps of Israel (Hymenoptera, Cynipidae, Cynipini) - diversity, distribution and life history

Shachar, Einat, Melika, George, Inbar, Moshe, Dorchin, Netta (2018): The oak gall wasps of Israel (Hymenoptera, Cynipidae, Cynipini) - diversity, distribution and life history. Zootaxa 4521 (4): 451-498, DOI: 10.11646/zootaxa.4521.4.

FIGURES 29–34 in The oak gall wasps of Israel (Hymenoptera, Cynipidae, Cynipini) - diversity, distribution and life history

FIGURES 29–34. Galls on Quercus boissieri. 29. Cynips cornifex, asexual generation; 30. Neuroterus numismalis, sexual generation; 31. Neuroterus anthracinus, asexual generation; 32. Cynips quercus, asexual generation; 33. Cynips divisa, asexual generation; 34. Neuroterus albipes, asexual generation

FIGURES 1–4 in The oak gall wasps of Israel (Hymenoptera, Cynipidae, Cynipini) - diversity, distribution and life history

FIGURES 1–4. Cynipid collection sites on oaks in Israel. 1. Q. libani and Q. cerris. 2. Q. boissieri. 3. Q. ithaburensis. 4. Q. calliprinos. Numbers correspond to the following collection sites: 1. Mt. Hermon, 1780m; 2. Mt. Hermon, 1500m; 3. Mt. Kahal; 4. Odem Forest; 5. En Zivan; 6. Allone HaBashan; 7. Tel Hazeqa; 8. Yehudiyya; 9. Mezar; 10. Mt. Meron; 11. Pa'ar cave; 12. Mt. Addir; 13. Nahal Rakefet; 14. Alonim; 15. Hosha'aya; 16. Bet Keshet Forest; 17. Rehan Forest; 18. HaSharon Forest; 19. Zur Hadassa

Altered expression and distribution of cathepsins in neuronopathic forms of Gaucher disease and in other sphingolipidoses

The neuronopathic forms of the human inherited metabolic disorder, Gaucher disease (GD), are characterized by severe neuronal loss, astrogliosis and microglial proliferation, but the cellular and molecular pathways causing these changes are not known. Recently, a mouse model of neuronopathic GD was generated in which glucocerebrosidase deficiency is limited to neural and glial progenitor cells. We now show significant changes in the levels and in the distribution of cathepsins in the brain of this mouse model. Cathepsin mRNA expression was significantly elevated by up to similar to 10-fold, with the time-course of the increase correlating with the progression of disease severity. Cathepsin activity and protein levels were also elevated. Significant changes in cathepsin D distribution in the brain were detected, with cathepsin D elevated in areas where neuronal loss, astrogliosis and microgliosis were observed, such as in layer V of the cerebral cortex, the lateral globus pallidus and in various nuclei in the thalamus, brain regions known to be affected in the disease. Cathepsin D elevation was greatest in microglia and also noticeable in astrocytes. The distribution of cathepsin D was altered in neurons in a manner consistent with its release from the lysosome to the cytosol. Remarkably, ibubrofen treatment significantly reduced cathepsin D mRNA levels in the cortex of Gaucher mice. Finally, cathepsin levels were also altered in mouse models of a number of other sphingolipidoses. Our findings suggest the involvement of cathepsins in the neuropathology of neuronal forms of GD and of other lysosomal storage diseases, and are consistent with a crucial role for reactive microglia in neuronal degeneration in these diseases