Co-targeting strategy for precise,scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)—in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.

A transgene-free strategy allows precise editing of genes lacking a selectable phenotype by electroporation of CRISPR/Cas9 ribonucleoproteins and single-stranded oligodeoxynucleotide templates.     

Effects of new ubiquinone-imidazo[2,1-b]thiazoles on mitochondrial complex I (NADH-ubiquinone reductase) and on mitochondrial permeability transition pore

In this work we describe the synthesis of a series of imidazo[2,1-b]thiazoles and 2,3-dihydroimidazo[2,1-b]thiazoles connected by means of a methylene bridge to CoQ(0). These compounds were tested as specific inhibitors of the NADH:ubiquinone reductase activity in mitochondrial membranes. The imidazothiazole system when bound to the quinone ring in place of the isoprenoid lateral side chain, may increase the inhibitory effect (with an IC(50) for NADH-Q(1) activity ranging between 0.25 and 0.96 microM) whereas the benzoquinone moiety seems to lose the capability to accept electrons from complex I as indicated by very low maximal velocity elicited by the compounds tested. Moreover the low rotenone sensitivity for almost all of these compounds suggests that they are only partially able to interact with the physiological ubiquinone-reduction site. The compounds were investigated for the capability of increasing the permeability transition of the inner mitochondrial membrane in isolated mitochondria. Unlike CoQ(0), which is considered a mitochondrial membrane permeability transition inhibitor, the new compounds were inducers.     

3-hydroxy-quinazoline-2,4-dione as a useful scaffold to obtain selective Gly/NMDA and AMPA receptor antagonists

The synthesis and Gly/NMDA, AMPA and KA receptor binding activities of some 3-hydroxy-quinazoline-2,4-dione derivatives are reported. The binding data, together with functional antagonism studies, showed that the 3-hydroxy-quinazoline-2,4-dione moiety can be considered a useful scaffold to obtain selective Gly/NMDA and AMPA receptor antagonists. In fact, introduction of chlorine atom(s) on precise position(s) of the benzofused moiety yielded Gly/NMDA selective antagonists, while the presence of the 6-(1,2,4-triazol-4-yl) group shifted the affinity and selectivity towards the AMPA receptor.     

The myelin-associated oligodendrocytic basic protein region MOBP15-36 encompasses the immunodominant major encephalitogenic epitope(s) for SJL/J mice and predicted epitope(s) for multiple sclerosis-associated HLA-DRB1*1501

Autoimmune response to the myelin-associated oligodendrocytic basic protein (MOBP), a CNS-specific myelin constituent, was recently suggested to play a role in the pathogenesis of multiple sclerosis (MS). The pathogenic autoimmune response to MOBP and the associated pathology in the CNS have not yet been fully investigated. In this study, we have characterized the clinical manifestations, pathology, T cell epitope-specificity, and TCRs associated with experimental autoimmune encephalomyelitis (EAE) induced in SJL/J mice with recombinant mouse MOBP (long isoform, 170 aa). Analysis of encephalitogenic MOBP-reactive T cells for reactivity to overlapping MOBP peptides defined MOBP15-36 as their major immunodominant epitope. Accordingly, MOBP15-36 was demonstrated to be the major encephalitogenic MOBP epitope for SJL/J mice, inducing severe/chronic clinical EAE associated with intense perivascular and parenchymal infiltrations, widespread demyelination, axonal loss, and remarkable optic neuritis. Molecular modeling of the interaction of I-A(s) with MOBP15-36, together with analysis of the MOBP15-36-specific T cell response to truncated peptides, suggests MOBP20-28 as the core sequence for I-A(s)-restricted recognition of the encephalitogenic region MOBP15-36. Although highly focused in their epitope specificity, the encephalitogenic MOBP-reactive T cells displayed a widespread usage of TCR Vbeta genes. These results would therefore favor epitope-directed, rather than TCR-targeted, approaches to therapy of MOBP-associated pathogenic autoimmunity. Localization by molecular modeling of a potential HLA-DRB1*1501-associated MOBP epitope within the encephalitogenic MOBP15-36 sequence suggests the potential relevance of T cell reactivity against MOBP15-36 to MS. The reactivity to MOBP15-36 detected in MS shown here and in another study further emphasizes the potential significance of this epitope for MS.     

Cell Volume-Induced Hormone Secretion: Signal Transduction and Specificity

Maintenance of the cell volume within physiological limits under anisosmotic conditions is an important prerequisite for survival and functioning of the cell. Cell volume alterations are also involved in numerous cellular events and are recently considered to be integrated into a physiological signal transduction network. Cell swelling induced by anisosmotic environment, hormones, oxidative stress, or substrate uptake evokes an immediate secretory burst of the material (peptide hormones, enzymes) stored in secretory vesicles from various types of cells (endocrine cells, neurons, leukocytes, exocrine pancreatic cells). The dynamics of this secretion are indistinguishable from those induced by specific secretagogues. This regulated secretion does not require a rise in the intracellular Ca²⁺. Using various tissues (pituitary, pancreatic islets, brain structures), hormones (prolactin, insulin, thyrotropin - releasing hormone - TRH, oxytocin), and inhibitors, we found that hormone secretion induced by cell swelling is not depressed by inhibition of stretch-activated channels (GdCl₃), mercury-sensitive aquaporins, protein kinase C (bisindolylmaleimide), microtubules and microfilaments (colchicine, cytochalasin)and does not involve arachidonic acid metabolites, prostaglandins and leukotrienes (indomethacin, NDGA). The blockade of Na⁺-K⁺-dependent ATPase, that of Na⁺ channels, or that of K⁺ channels exerted no effect on hyposmolarity-induced hormone secretion in pituitary cells. Norepinephrine, a physiological inhibitor of secretion of insulin, did not inhibit hypotonicity-induced secretion from pancreatic islets. The participation of such a general biophysical phenomenon in physiological reactions raises a question of its specificity. Cell swelling induced by an isosmotic ethanol-containing medium evoked release of TRH from hypothalamic paraventricular nucleus and posterior pituitary, while oxytocin (known to be engaged in the water and salt regulation) release was not stimulated. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 177–180, March–April, 2005.     

The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits

Protein synthesis is initiated on ribosomal subunits. However, it is not known how 70S ribosomes are dissociated into small and large subunits. Here we show that 70S ribosomes, as well as the model post-termination complexes, are dissociated into stable subunits by cooperative action of three translation factors: ribosome recycling factor (RRF), elongation factor G (EF-G), and initiation factor 3 (IF3). The subunit dissociation is stable enough to be detected by conventional sucrose density gradient centrifugation (SDGC). GTP, but not nonhydrolyzable GTP analog, is essential in this process. We found that RRF and EF-G alone transiently dissociate 70S ribosomes. However, the transient dissociation cannot be detected by SDGC. IF3 stabilizes the dissociation by binding to the transiently formed 30S subunits, preventing re-association back to 70S ribosomes. The three-factor-dependent stable dissociation of ribosomes into subunits completes the ribosome cycle and the resulting subunits are ready for the next round of translation.     

1-Substituted pyrazolo[1,5-c]quinazolines as novel Gly/NMDA receptor antagonists: synthesis, biological evaluation, and molecular modeling study

A new set of 5,6-dihydro-pyrazolo[1,5-c]quinazoline-2-carboxylates (2-18), bearing different substituents (COOEt, Cl, Br, CH(3), and COOH) at position-1, were synthesized in order to investigate the influence of various groups at this specific position on Gly/NMDA receptor affinity and/or selectivity. All the herein reported compounds were evaluated for their binding at the Gly/NMDA, AMPA, and KA receptors. Some selected compounds were also tested for their functional antagonistic activity at both the AMPA and NMDA receptor-ion channels. The results obtained in this study have highlighted that a C-1 lipophilic substituent on the pyrazolo[1,5-c]quinazoline-2-carboxylate core shifts selectivity toward the Gly/NMDA receptor, while a C-1 anionic carboxylate residue is able to increase affinity toward this receptor subtype. In particular, the 2-carboxylic acids 15 and 16, bearing a chlorine atom at position-1, are not only potent (K(i)=0.18 and 0.16muM, respectively), but also highly Gly/NMDA versus AMPA selective (selectivity ratio>500). Furthermore, the 1,2-dicarboxylic acids 13 and 14 are endowed with the highest Gly/NMDA receptor binding activity (K(i)=0.09 and 0.059muM, respectively), among the pyrazoloquinazoline series of derivatives. A molecular modeling study has been carried out to better understand receptor affinity and selectivity of these new pyrazoloquinazoline derivatives.     

Molecular and functional characterization of NKG2D, NKp80, and NKG2C triggering NK cell receptors in rhesus and cynomolgus macaques: monitoring of NK cell function during simian HIV infection

An involvement of innate immunity and of NK cells during the priming of adaptive immune responses has been recently suggested in normal and disease conditions such as HIV infection and acute myelogenous leukemia. The analysis of NK cell-triggering receptor expression has been so far restricted to only NKp46 and NKp30 in Macaca fascicularis. In this study, we extended the molecular and functional characterization to the various NK cell-triggering receptors using PBMC and to the in vitro-derived NK cell populations by cytofluorometry and by cytolytic activity assays. In addition, RT-PCR strategy, cDNA cloning/sequencing, and transient transfections were used to identify and characterize NKp80, NKG2D, CD94/NKG2C, and CD94/NKG2A in M. fascicularis and Macaca mulatta as well as in the signal transducing polypeptide DNAX-activating protein DAP-10. Both M. fascicularis and M. mulatta NK cells express NKp80, NKG2D, and NKG2C molecules, which displayed a high degree of sequence homology with their human counterpart. Analysis of NK cells in simian HIV-infected M. fascicularis revealed reduced surface expression of selected NK cell-triggering receptors associated with a decreased NK cell function only in some animals. Overall surface density of NK cell-triggering receptors on peripheral blood cells and their triggering function on NK cell populations derived in vitro was not decreased compared with uninfected animals. Thus, triggering NK cell receptor monitoring on macaque NK cells is possible and could provide a valuable tool for assessing NK cell function during experimental infections and for exploring possible differences in immune correlates of protection in humans compared with cynomolgus and rhesus macaques undergoing different vaccination strategies.     

A Passive Exoskeleton Can Push Your Life Up: Application on Multiple Sclerosis Patients

In the present study, we report the benefits of a passive and fully articulated exoskeleton on multiple sclerosis patients by means of behavioral and electrophysiological measures, paying particular attention to the prefrontal cortex activity. Multiple sclerosis is a neurological condition characterized by lesions of the myelin sheaths that encapsulate the neurons of the brain, spine and optic nerve, and it causes transient or progressive symptoms and impairments in gait and posture. Up to 50% of multiple sclerosis patients require walking aids and 10% are wheelchair-bound 15 years following the initial diagnosis. We tested the ability of a new orthosis, the “Human Body Posturizer”, designed to improve the structural and functional symmetry of the body through proprioception, in multiple sclerosis patients. We observed that a single Human Body Posturizer application improved mobility, ambulation and response accuracy, in all of the tested patients. Most importantly, we associated these clinical observations and behavioral effects to changes in brain activity, particularly in the prefrontal cortex.