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  • 1
    Language: English
    In: The Journal of Membrane Biology, 2013, Vol.246(11), pp.861-867
    Description: The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli . In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA.
    Keywords: Alkaline lysis ; Electroextraction ; Plasmid DNA ; Escherichia coli
    ISSN: 0022-2631
    E-ISSN: 1432-1424
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  • 2
    Language: English
    In: Journal of Chromatography B, 15 October 2016, Vol.1033-1034, pp.91-96
    Description: Protein pegylation is a process of covalent attachment of a polyethylene glycol (PEG) group to the protein tertiary structure that can “mask” the agent from the immune system and also increases the hydrodynamic size of the agent. Usually the pegylation prolongs the protein stability in the organism due to reduced renal clearance and provides superior water solubility to hydrophobic molecules. The mono-pegylated form of protein is usually prefered for medical applications. Different conditions with different PEG reagents have to be tested to find optimal pegylation procedure with specific protein. The goal of this study was to prepare screening method for separation of random mono-pegylated protein. Cytochrome C and beta lactoglobulin were pegylated with four reagents and a complete screening of several chromatographic monoliths in ion exchange mode with different buffers was performed to optimaly separate each mono-pegylated protein. The screening method was developed that produces optimal separation of target pegylated protein on CIM monoliths. Because of short chromatographic run time, CIM monoliths are perfect candidates to test alot of parameters. The results obtained show that each protein has its own unique separation parameters (pH, ionexchange ligand, buffer type). Two biopharmaceuticals were isolated using protocol: super human leptin antagonist (SHLA) was purified from inclusion bodies and mono-pegylated super mouse leptin antagonist (SMLA) from pegylated mixture. During study it was observed that the convective interaction media (CIM) monoliths additionally discriminate between protein isoforms pegylated on different sites in 3D structure of the protein.
    Keywords: Monolith ; Ion Exchange ; Pegylating ; Purification ; Anatomy & Physiology
    ISSN: 1570-0232
    E-ISSN: 1873-376X
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  • 3
    Language: English
    In: Vaccine, 01 May 2014, Vol.32(21), pp.2487-2492
    Description: We explored the possibilities for purification of various ΔNS1 live, replication deficient influenza viruses on ion exchange methacrylate monoliths. Influenza A ΔNS1-H1N1, ΔNS1-H3N2, ΔNS1-H5N1 and ΔNS1-influenza B viruses were propagated in Vero cells and concentrated by tangential flow filtration. All four virus strains adsorbed well to CIM QA and CIM DEAE anion exchangers, with CIM QA producing higher recoveries than CIM DEAE. ΔNS1-influenza A viruses adsorbed well also to CIM SO3 cation exchanger at the same pH, while ΔNS1-influenza B virus adsorption to CIM SO3 was not complete. Dynamic binding capacity (DBC) for CIM QA, DEAE and SO3 methacrylate monoliths for influenza A ΔNS1-H1N1 virus were 1.9E + 10 TCID /ml, 1.0E + 10 TCID /ml and 8.9E + 08 TCID /ml, respectively. Purification of ΔNS1 viruses on CIM QA was scaled up and reproducibility was confirmed. Yields of infectious virus on CIM QA were between 70.8 ± 32.3% and 87 ± 30.8%. Total protein removal varied from 93.3 ± 0.4% to 98.6 ± 0.2% and host cell DNA removal efficiency was ranging from 76.4% to 99.9% and strongly depended on pretreatment with deoxyribonuclease.
    Keywords: Influenza Viruses ; Vaccines ; Purification ; Chromatography ; Monoliths ; Medicine ; Biology ; Veterinary Medicine ; Pharmacy, Therapeutics, & Pharmacology
    ISSN: 0264-410X
    E-ISSN: 1873-2518
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  • 4
    Book
    Book
    Ljubljana: Mladinska knjiga
    Description: 152 p.
    ISBN: 8611146816
    ISBN: 9788611146812
    Source: HathiTrust
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  • 5
    Language: English
    In: Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie, October 2005, Vol.243(10), pp.1018-27
    Description: Fundus autofluorescence (AF) in some patients with retinitis pigmentosa is characterized by a parafoveal ring of increased AF which surrounds the centre as hypofluorescent changes appear at the periphery. The aim of this study was to evaluate the AF patterns in relation to retinal function measured by electroretinography and visual fields. Thirty-two patients with RP were included in the study. AF imaging of the macular area was performed with the scanning laser ophthalmoscope. Patients were divided in two groups according to their fundus AF patterns. All patients from group 1 had a ring of increased AF of different size but no atrophic areas inside vascular arcades. Patients with a ring of increased AF and round atrophic changes at different eccentricities from their fovea were selected in group 2. Visual fields were tested with kinetic, automated perimetry and microperimetry; the radius of the hyperfluorescent ring and the smallest distance of hypofluorescent areas from the fovea were compared to visual fields, PERG P50 and N95 and mfERG P1 amplitudes of the inner three rings. A linear relationship was found in group 1 between the radius of the ring of increased AF and both the automated (r=0.82) and kinetic perimetry (r=0.80). The radius of the AF ring correlated highly with the PERG P50 (r=0.72) and N95 (r=0.74) amplitudes. In all patients, mfERG responses were reduced at all retinal locations, more pronounced at periphery. There was a good correlation between the ring of increased AF and the P1 amplitude of ring 2 of mfERG (r=0.62). Patients from group 2 had significantly reduced or non-recordable PERGs and mfERGs. The eccentricities of hypofluorescent changes did not correlate with any type of perimetry. Our results show that in stages of retinitis pigmentosa, before atrophic lesions spread inside the vascular arcades, the pattern of fundus autofluorescence correlates well with functional tests such as perimetry and electroretinography. The ring of increased AF appears to represent the border between functional and dysfunctional retina. This shows that autofluorescence, as a quick and non-invasive imaging tool, may be related to retinal function as well.
    Keywords: Fluorescence ; Retina -- Physiopathology ; Retinitis Pigmentosa -- Pathology
    ISSN: 0721-832X
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  • 6
    Language: English
    In: Vision Research, Dec 15, 2012, Vol.75, p.60(11)
    Description: To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.visres.2012.08.017 Byline: Ana Fakin (a), Martina Jarc-Vidmar (a), Damjan GlavaA (b), Crystel Bonnet (c), Christine Petit (c)(d)(e), Marko Hawlina (a) Keywords: Usher syndrome; Retinitis pigmentosa; Fundus autofluorescence; Hyperautofluorescent ring; Foveal patch; Optical coherence tomography Abbreviations: FAF, fundus autofluorescence; OCT, optical coherence tomography; ISe, inner segment ellipsoid (also known as IS/OS junction); ELM, external (outer) limiting membrane; RP, retinitis pigmentosa; USH, Usher; VA, visual acuity; ISH, Ishihara color vision test; OPL, outer plexiform layer; IPL, inner plexiform layer; ONL, outer nuclear layer Abstract: . Display Omitted Author Affiliation: (a) Eye Hospital, University Medical Centre Ljubljana, GrabloviAeva 46, 1000 Ljubljana, Slovenia (b) Institute of Pathology, Medical Faculty, University of Ljubljana, ZaloA ka 4, 1000 Ljubljana, Slovenia (c) Laboratory of Genetics and Physiology of Hearing, Inserm UMRS 587, Institut de la Vision, UPMC, 75012 Paris, France (d) Institut Pasteur, 75015 Paris, France (e) College de France, 75005 Paris, France Article History: Received 15 June 2012; Revised 23 August 2012
    Keywords: Universities And Colleges ; Fluorescence ; Tomography
    ISSN: 0042-6989
    Source: Cengage Learning, Inc.
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  • 7
    Language: English
    In: Vision Research, 15 December 2012, Vol.75, pp.60-70
    Description: . ► Fifty-four patients with Usher syndrome of different subtypes were studied. ► Age at onset of nyctalopia was lower in USH1 than USH2 patients. ► Disease expression in retina of different Usher subtypes was similar. ► Hyperautofluorescent ring was seen in up to 30 years after onset of nyctalopia. ► Hyperautofluorescent patch was seen after 30 years with concurrent loss of vision. Purpose of this study was to characterize retinal disease in Usher syndrome using fundus autofluorescence and optical coherence tomography. Study included 54 patients (26 male, 28 female) aged 7–70 years. There were 18 (33%) USH1 and 36 (67%) USH2 patients. 49/52 (94%) patients were found to carry at least one mutation in Usher genes. Ophthalmological examination included assessment of Snellen visual acuity, color vision with Ishihara tables, Goldmann visual fields (targets II/1–4 and V/4), microperimetry, fundus autofluorescence imaging and optical coherence tomography. Average age at disease onset (nyctalopia) was significantly lower in USH1 than USH2 patients (average 9 vs. 17 years, respectively; 〈 0.01); however no significant differences were found regarding type of autofluorescence patterns, frequency of foveal lesions and CME, rate of disease progression and age at legal blindness. All representative eyes had abnormal fundus autofluorescence of either hyperautofluorescent ring (55%), hyperautofluorescent foveal patch (35%) or foveal atrophy (10%). Disease duration of more than 30 years was associated with a high incidence of abnormal central fundus autofluorescence (patch or atrophy) and visual acuity loss.
    Keywords: Usher Syndrome ; Retinitis Pigmentosa ; Fundus Autofluorescence ; Hyperautofluorescent Ring ; Foveal Patch ; Optical Coherence Tomography ; Medicine ; Anatomy & Physiology
    ISSN: 0042-6989
    E-ISSN: 1878-5646
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  • 8
    Language: English
    In: Documenta Ophthalmologica, 2015, Vol.130(3), pp.179-187
    Description: Byline: Martina Jarc-Vidmar (1), Mojca Tajnik (2), Jelka Brecelj (1), Ana Fakin (1), Maja Sustar (1), Mateja Naji (3), Branka Stirn-Kranjc (1), Damjan Glavac (2), Marko Hawlina (1) Keywords: LHON; Leber hereditary optic neuropathy; Electrophysiology; PERG; VEP Abstract: Background To report clinical and electrophysiology findings in Slovene patients with Leber hereditary optic neuropathy (LHON). Methods Eight patients with LHON (11--26 years one female and seven males) were examined in acute stages and at follow-up visits by means of Snellen visual acuity, Ishihara color vision, Goldmann or Octopus G2TOP perimetry, fluorescein angiography (FAG), pattern electroretinogram (PERG), visual evoked potentials (VEP) and genetic testing. Results Patients presented with typical LHON phenotype with bilateral visual acuity loss, abnormal color vision, central scotoma and hyperemic discs with no leakage on FAG. In the acute stage, electrophysiology was performed in 7/8 patients. The PERG P50 component was normal in 14/14 eyes, while the N95 component was reduced in 7/14 eyes. VEP wave P100 was reduced and delayed in 14/14 eyes. In this stage, temporal pallor of the optic disc was visible in 4/7 eyes with reduced PERG N95. At follow-up (1--11 months after), a reduced PERG N95 component was seen in 13/14 eyes and severely affected VEP in all eyes. In the only eye with a normal PERG N95, hyperemic optic disc was seen 5 months after visual acuity loss, while it was atrophic in all the others. Known mutations (14484T〉C, 3460G〉A) were found in 2/8 patients, while in others high-throughput sequencing identified new potentially pathogenic mutations. Conclusions In Leber hereditary optic neuropathy, a reduced N95 component of PERG and severely reduced VEP P100 may be present already in the acute stage of disease, before optic disc pallor appears, suggesting primary dysfunction of retinal ganglion cells. Author Affiliation: (1) Eye Hospital, University Medical Centre, Grabloviceva 46, 1000, Ljubljana, Slovenia (2) Department of Molecular Genetics, Faculty of Medicine, University of Ljubljana, Korytkova, 1000, Ljubljana, Slovenia (3) University Medical Center Maribor, Ljubljanska ulica 5, 2000, Maribor, Slovenia Article History: Registration Date: 09/02/2015 Received Date: 24/11/2014 Accepted Date: 09/02/2015 Online Date: 19/02/2015 Article note: Part of this work was presented at 12th European Neuro-Ophthalmology Society, April 10--13, 2013, Oxford UK and at ISCEV July 2014, Boston USA. Martina Jarc-Vidmar and Mojca Tajnik have contributed equally to this work.
    Keywords: LHON ; Leber hereditary optic neuropathy ; Electrophysiology ; PERG ; VEP
    ISSN: 0012-4486
    E-ISSN: 1573-2622
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  • 9
    In: Acta Ophthalmologica, December 2016, Vol.94(8), pp.e786-e794
    Description: To purchase or authenticate to the full-text of this article, please visit this link: http://onlinelibrary.wiley.com/doi/10.1111/aos.13202/abstract Byline: Damjan GlavaA, Martina Jarc-Vidmar, Katarina Vrabec, Metka Ravnik-GlavaA, Ana Fakin, Marko Hawlina Keywords: best vitelliform macular dystrophy; BEST1 gene; clinical phenotype; genetic eye disease; mutation detection Abstract Purpose To determine the spectrum of BEST1 mutations and to study the phenotype in Slovenian families with Best vitelliform macular dystrophy (BVMD) to identify genotype-phenotype correlations. Methods Twenty patients from five families underwent the ophthalmological examination including electrooculogram (EOG; N = 17), fundus autofluorescence imaging (N = 16) and optical coherence tomography (N = 14). Mutational screening was performed by direct DNA sequencing of the BEST1 gene. Results Mutation c.43G〉C (p.Gly15Arg) was detected in three patients from family M presenting with different clinical stages of Best disease. Mutation c.313G〉C (p.Arg105Gly) was found in families K, ST, S, B and was associated with incomplete clinical penetrance and variable retinal changes, including extramacular and multifocal lesions. In three patients from family K, an atypical form of BVMD was observed; there were additional peripheral lesions outside of the vascular arcades in addition to the typical macular lesions. Multiple alterations between the vitelliruptive and pseudohypopyon stages over a period of 11 years were seen in one patient. Conclusion Two previously unreported disease-associated variants in the BEST1 gene (p.Gly15Arg and p.Arg105Gly) were found in Slovenian patients with Best disease. Our data expand the mutation spectrum of the BEST1 gene and further support the broad phenotypic variability observed clinically and with optical coherence tomography (OCT) and AF imaging. Article Note: We wish to thank all of the participating patients and family members for their co-operation in this study. This work was supported by the Slovenian Research Agency (ARRS P3-0333 and P3-054). These authors contributed equally to this work.
    Keywords: Best Vitelliform Macular Dystrophy ; Best 1 Gene ; Clinical Phenotype ; Genetic Eye Disease ; Mutation Detection
    ISSN: 1755-375X
    E-ISSN: 1755-3768
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  • 10
    Language: English
    In: Zdravniški Vestnik, 01 June 2012, Vol.82
    Description: Background: The aim of our study was to evaluate retinal function in children and young adults with Stargardt dystrophy by correlating retinal morphology (autofluorescence, OCT) with functional tests (visual acuity, microperimetry) and electroretinography (full-field ERG, pattern ERG, multifocal ERG). Patients and methods: 6 patients (3 F, 3 M, VA: 0.2 ± 0.1, average onset of problems at 10 yrs of age) were included in the study. Autofluorescence (AF) was recorded and the central 10 º visual fields were tested by microperimetry. The morphology of the retina was recorded by optical coherence tomography (OCT). Full-field ERG, pattern ERG (PERG) and multifocal ERG (mfERG) were recorded according to ISCEV standard in all the patients. Results: AF showed mottled central hypo-hyperautofluorescent areas with or without peripheral hyperautofuorescent flecks in 4 patients and a central hypoautofluorescent region with hyperautofluorescent ring in 2 patients. OCT showed transverse loss of the central photoreceptor layers (average OCT thickness: 38.9 ± 14.8μm, average OCT volume 6.1 ± 0.5 mm3). Microperimetry showed superior shift of fixation to the preferred retinal locus (PRL). PERG was abnormal in 8 eyes. Full-field ERG was normal in all except two patients with abnormal cone and 30 Hz responses. MfERG showed reduced responses in all five rings (ring 1: 22.6 % of mean normal value, ring 2: 27.3 %, ring 3: 38.9 %, ring 4: 57.9 %, ring 5: 64.2 %). Conclusions: In young patients with Stargardt dystrophy central retinal atrophy was shown by OCT and AF. The shift of fixation to the PRL was seen in all the patients. MfERG showed central cone dysfunction in all the patients.
    Keywords: Medicine
    ISSN: 1318-0347
    E-ISSN: 1581-0224
    Source: Directory of Open Access Journals (DOAJ)
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