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The virus is the smallest known replicative unit, usually in nanometer-range sizes. The most simple and sensitive detection assay involves molecular amplification of nucleic acids. This work shows a novel, straightforward detection based on the interaction of viral particles with fluorescent nanoconstructs without using enzymatic amplification, washing or separation steps. Fluorescent nanoconstructs are prepared with individual quantum dots of different emitting green and red fluorescence as a core. They are decorated with aptamers developed to recognise the receptor-binding region of the SARS-CoV-2 spike protein. Nanoconstructs can recognise SARS-CoV-2 viral particles fixed onto a coverglass generating aggregates. Meanwhile, SARS-CoV-2 viral particles/nanoconstructs complexes in solution yield aggregates and complexes, which a fluorescence microscope can visualise. The multiple molecular recognition allowed the detection of SARS-CoV-2 viral particles from a few microliters of patient swabs. This specific SARS-CoV-2/nanoconstructs interaction generates insoluble and precipitating aggregates. By using a mixture of green and red fluorescent nanoconstructs, upon the viral particle interaction, they yield heterochromatic green, red and yellow spectral fluorescence, easily identifiable by a fluorescence microscope. Washing and separation steps are not required, and aggregates allow one to easily recognise them, offering a sensitive, simple, and cheap alternative for viral detection. • Nanoconstructs were created by decorating quantum dots with aptamers against RBD. • Nanoconstructs generate aggregates in the presence of SARS-CoV-2 viral particles. • SARS-CoV-2/nanoconstructs aggregates are easily observed under fluorescence microscopy. • The nanoconstructs recognize SARS-CoV-2 viral particles from human swab samples. • The system is cheap, simple, highly sensitive and comparable with current diagnostic tests. [ABSTRACT FROM AUTHOR]

A novel functional micropipette tip for DNA purification, called DNAtip, is described based on a novel coating process. The functional layer is prepared by an internal coating with silica particles supported into a polyvinyl alcohol (PVA) film. The coating layer is stabilized by simultaneous radiation-induced crosslinking to avoid its dissolution. Three types of silica particles of different diameters: micron, sub-micron, and nano sizes are studied. In addition, pH of the coating solution and concentration of the silica particles were optimized. Tips coated with fumed silica showed the highest absorption capacity of a plasmid DNA with an acceptable purity. Different linear dsDNA fragments were used for evaluating the absorption capacity and size discrimination of fragments. The recovery in the range of 700 ng and 200 ng per tip was measured for dsDNA fragments in the range of 400 bp to 2 kbp and 50 bp to 500 bp respectively. DNA PCR fragments contaminated with BSA were successfully purified with sufficient quality to be sequenced or enzymatically digested. This disposable tip device, useful for plasmids or DNA fragments purification, is a novel lab-on-a-tip device, which can effectively improve automate genetic engineering procedures. [ABSTRACT FROM AUTHOR]

Abstract: The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-β-actin enhancer promoter control] gene plasmid (50 ng/μL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 μm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production. [Copyright &y& Elsevier]

Presents a letter to the editor on the safety of retroviral gene marking with a truncated nerve growth factor receptor, published in the journal 'Nature Medicine,' as of April 1, 2003. Safety factors associated with the use of nerve growth factor receptor.

A letter to the editor is presented which describes a case of a 26-year old male patient with successful GPi stimulation in genetic Parkinson's disease cause by mosaicism of alpha-synuclein gene duplication.

Abstract: Although transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis system using I-SceI meganuclease (intron-encoded endonuclease from S. cerevisiae) was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgene was measured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rates were higher (P < 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injection with pIS plus I-SceI after IVF increased frequency (P < 0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injection with I-SceI increased (P < 0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%, 11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos. [Copyright &y& Elsevier]

Pronuclear microinjection and intracytoplasmic sperm injection-mediated gene transfer (ICSI-mgt) are useful techniques to obtain transgenic animals. Nevertheless, a high frequency of mosaic expression is observed in embryos and offspring produced by these techniques. A possible explanation is that the transgene integrates in the embryo genome after the first cell division. Our main objective was to develop a new technique to generate transgenic bovine embryos without mosaic expression and with high efficiency. We hypothesize that fertilizing metaphase II (MII) oocytes with transgenic androgenetic haploidblastomeres (AHB) (from mosaic embryos) would result in non-mosaic transgenic embryos. To this aim, in the first experiment we generated AHB by enucleating IVM MII oocytes, before or after injecting with a single spermatozoon incubated with pCX-EGFP (enhanced green fluorescent protein) plasmid. These treatments were analyzed by Fisher test (P< 0.05). The rate of cleavage of the androgenetic transgenic embryos enucleated before and after ICSI-mgt was 35.1% (34/97) and 61.2% (71/116), respectively (P< 0.05). These embryos showed expression of EGFPof 11.8% (4/34) and 42.3% (30/71) (P< 0.05) with 0% (0/34) and 9.9% (7/71) of non-mosaic expression. The haploid condition of the androgenetic embryos was confirmed by karyotype analysis. After this first approach, we chose the procedure of enucleation after ICSI for successive experiments. In the second experiment, the haploid androgenetic embryos (4 to 16 cells) were disaggregated, and the AHB obtained were used to fertilize MII oocytes. Fertilization was carried out by fusing a single AHB to a zona-free MII oocyte, followed by chemical activation. Presumptive zygotes were cultured in SOF medium in the well of the well (WOW) system. To confirm fertilization, single AHB produced with sexed Y spermatozoa and embryos generated with them were checked by PCR using Y- and X-specific sequence primers. PCR analysis confirmed Y-specific sequences in all the AHB and XY-specific sequences in each of the analyzed embryos. FISH analysis on blastocysts was performed with a specific probe for a Y chromosome sequence, confirming the sexed sperm genome in all blastocyst cells. Additionally, the expression pattern of Oct-4 (pluripotent marker gene) was examined in the blastocysts by inmunocytochemistry with a confocal microscope. Blastocysts displayed a pattern of Oct-4 expression similar to that of IVF embryos, indicating efficient nuclear reprogramming. Finally, we fertilized MII oocytes with EGFP-AHB to produce transgenic bovine embryos without mosaic expression. The development reached 85.1% of cleavage and 9.0% of blastocysts (n= 84). One hundred percent of the embryos showed EGFP expression, with 90.1% non-mosaic expression. In conclusion, our results proved that it is possible to use AHB for fertilization of MII oocyte, and that fertilization with transgenic AHB is a highly efficient technique for the generation of transgenic non-mosaic bovine embryos. [ABSTRACT FROM AUTHOR]

As animal transgenesis is an essential tool in medicine and agriculture, it is necessary to understand the mechanisms in order to develop novel methods of transgenesis. We intended to determine if the injection of cells or their parts into metaphase II (MII) oocytes after incubating with exogenous DNA can induce transgenesis in embryos. Sperm cells for intracytoplasmic sperm injection (ICSI) in ovine and cumulus cells for NT in bovine were incubated with pCX-EGFP plasmid (5 to 50 ng μL-1) for 5 min in 2.8% Na citrate at 0°C before transfer into a 10% polyvinylpyrrolidone (PVP) droplet and injection into MII oocytes (previously enucleated in NT). In both species, oolemma-ooplasmic vesicles (OOV) of 9 μm diameter obtained from MII oocytes by microsurgery were directly incubated in PVP droplet with same pCX-EGFP concentration. As a control group, pCX-EGFP suspension from PVP droplet was injected into MII oocytes. The NT bovine zygotes were activated in 5 μM ionomycin (Io) for 4 min followed by 1.9 mMDMAP immediately for 3 h. In ICSI ovine, the treatment with DMAP was applied 3 h later. Injected oocytes of OOV and controls were activated as NT in bovine and as ICSI in ovine. Expression of EGFP was determined with fluorescence microscopy under blue light (488 nm) at Days 4 to 7, and data were analyzed by Fisher test (P= 0.05). A group of NT, ICSI, OOV, and control presumptive zygotes were treated with FITC-labeled bovine fragments (100-300 bb) DNA in order to determine the binding sites with exogenous DNA by laser confocal microscope analysis. Quantitative PCR (qPCR) was performed to determine pCX-EGFP copy number at 0, 8, 16, and 24 h after Io in all ovine treatments. Embryos expressing EGDP from all techniques were subjected to FISH with rhodamine-labeled pCX-EGFP plasmid as a probe. In ovine, ICSI and OOV injection green embryos at Day 4 [58% (61/105) v.21.5% (8/38); P< 0.05] and green blastocysts at Day 7 [71.8% (23/32) v.66.6% (2/3)] were obtained, respectively. In bovine, green embryos [49.2% (34/69) v.29.7% (14/47); P< 0.05] and green blastocysts [95.8% (23/24) v.25.0% (2/8); P< 0.05] were produced by NT and OOV injection, respectively. In controls, no green embryos were obtained in ovine (0/47) and only low rates were observed in bovine [3.0% (2/65)]. Confocal images of zygotes showed specific signal only in cumulus cells, spermatozoa, and OOV The qPCR analysis showed similar plasmid copy number/zygote between treatment and times in ovine (range 30 000-300,000). Embryo FISH images showed 1 to 2 specific signals in ICSI and NT interphases of both species and in OOV ovine metaphases, the latter being direct evidence of transgene integration. These results suggest that the injected cells or cellular parts (OOV) dramatically increase transgenesis in ovine and bovine embryos. Until now, the generation of NT and OOV embryos after short exposure to the DNA construction has not been reported. We are performing embryo transfer and at the moment we have a pregnancy derived from ICSI in ewes. In conclusion, the cellular parts/transgene complex may affect exogenous DNA delivery or its interaction with embryo DNA, facilitating the mechanism of transgenesis in mammals. [ABSTRACT FROM AUTHOR]