OCT ANGIOGRAPHY AND DOPPLER ULTRASOUND IN HYPERTENSION GLAUCOMA.

AIMS: The main aim of this work was to find out if there is a correlation between vessel density (VD) and results of measured perfusion values in ophthalmic artery and in central retinal artery of the same eye in a group with hypertension glaucoma (HTG). MATERIALS AND METHODS: The file included 20 patients with HTG, thereof 13 women of average age 68.7 years (49-80 years) and 7 men of average age 58.4 years (27-81 years). Criteria for inclusion in the study: visual acuity 1,0 with possible correction less than ±3 diopters, approximately the same changes in visual fields in every patient, intraocular pressure (IOP) less than 18 mmHg, no other ocular or neurological diseases. VD was measured by Avanti RTVue XR by Optovue firm, perfusion parameters were measured using Doppler ultrasound with Affinity 70G machine by Philips firm. The peak systolic velocity (PSV) and end diastolic velocity (EDV) and resistance index (RI) were measured both in ophthalmic artery (AO) and in central retinal artery (CRA). Visual field (VF) was examined by quick threshold glaucoma program by Medmont M 700 machine. The sum of sensitivities in apostilbs (abs) was evaluated in the range 0-22 degrees of visual field. The results of sensitivities in visual field were compared to VD and perfusion parameters in CRA and AO of the same eye. RESULTS: Pearsons correlation coefficient (p = 0,05) was used to assess the dependency between chosen parameters. By comparing VF and VD from measured areas, strong correlation (r = 0.64, resp. 0.65) was revealed. It was then proved that VD (WI-VDs) correlates with RICRA weakly (r = -0.35) and moderately strongly (WI-VDa r = -0.4, PP-VDs r = -0.43 and PP-VDa r = -0.45). This means that with increasing resistance index in CRA the density in VD decreases. The other correlations between VD and perfusion parameters (PSV and EDV) in CRA and AO were not significant. CONCLUSION: Measured values showed that the vascular component of VD has a huge impact on the changes in visual fields in HTG. Weak to moderate influence exists between VD and RI in CRA. OCTA has proven to be more suitable than Doppler ultrasound for determining the condition of blood circulation in the eye.

OCT ANGIOGRAPHY AND DOPPLER SONOGRAPHY IN NORMAL-TENSION GLAUCOMA.

AIMS: To investigate the dependence of blood vessel density and velocity in ophthalmic artery and arteria centralis retinae of the same eye in patients with normotensive glaucoma. METHODS: The sample consisted of 20 patients with normotensive glaucoma (NTG). There were 17 women (mean age 56.1) and 3 men (mean age 60 years). Inclusion criteria for study: visual acuity 1.0 with correction up to ±3 dioptres, approximately equal changes in the visual field, whereby it was incipient NTG and diagnosis was confirmed by electrophysiological examination, without further ocular or neurological disease. Parameters of vessel density (VD) were evaluated by Avanti RTVue XR (Optovue). Perfusion parameters such as peak systolic velocity (PSV), end diastolic velocity (EDV) and resistive index (RI) were evaluated for ophthalmic artery (AO) and arteria centralis retinae (ACR) using Doppler sonography (Affinity 70G Philips, probe 5-12 MHz). Visual field (VF) was evaluated by automated perimeter (Medmont M700) using fast threshold glaucoma strategy test. The sum of sensitivity levels in apostilb (asb) were evaluated in range 0-22 degrees of visual field. Resulting values of VF were compared with VD and perfusion parameters in AO and ACR at the same eye. RESULTS: Pearsons correlation coefficient was used to evaluate the dependence. Data shows, that changes in visual fields are mainly caused by peripapillary VD of small and all vessels, and vessels throughout measured image area also. Correlation of small vessels throughout measured image area was weak (r = 0.23). Moderate negative correlation was found for PSV in AO and peripapillary small VD (r = -0.46), all peripapillary VD (r = -0.49), VD in whole area (r = -0.45), then between EDV in AO and VD in whole area (r = -0.42). Other correlations between VD and perfusion parameter were insignificant. CONCLUSIONS: Study confirms, that changes of visual field in NTG patients are mainly caused by VD rather than perfusion parameters, especially in AO. Perfusion parameters in ACR are not significantly correlated with changes of VF in NTG patients.

ALKB-8, a 2-Oxoglutarate-Dependent Dioxygenase and S-Adenosine Methionine-Dependent Methyltransferase Modulates Metabolic Events Linked to Lysosome-Related Organelles and Aging in C. elegans.

ALKB-8 is a 2-oxoglutarate-dependent dioxygenase homologous to bacterial AlkB, which oxidatively demethylates DNA substrates. The mammalian AlkB family contains AlkB homologues denominated ALKBH1 to 8 and FTO. The C. elegans genome includes five AlkB-related genes, homologues of ALKBH1, 4, 6, 7, and 8, but lacks homologues of ALKBH2, 3, and 5 and FTO. ALKBH8 orthologues differ from other AlkB family members by possessing an additional methyltransferase module and an RNA binding N-terminal module. The ALKBH8 methyltransferase domain generates the wobble nucleoside 5-methoxycarbonylmethyluridine from its precursor 5-carboxymethyluridine and its (R)- and (S)-5-methoxycarbonylhydroxymethyluridine hydroxylated forms in tRNA Arg/UCG and tRNA Gly/UCC. The ALKBH8/ALKB-8 methyltransferase domain is highly similar to yeast TRM9, which selectively modulates translation of mRNAs enriched with AGA and GAA codons under both normal and stress conditions. In this report, we studied the role of alkb-8 in C. elegans. We show that downregulation of alkb-8 increases detection of lysosome-related organelles visualized by Nile red in vivo. Reversely, forced expression of alkb-8 strongly decreases the detection of this compartment. In addition, overexpression of alkb-8 applied in a pulse during the L1 larval stage increases the C. elegans lifespan.

Valproic Acid Decreases the Nuclear Localization of MDT-28, the Nematode Orthologue of MED28.

Mediator is a multiprotein complex that connects regulation mediated by transcription factors with RNA polymerase II transcriptional machinery and integrates signals from the cell regulatory cascades with gene expression. One of the Mediator subunits, Mediator complex subunit 28 (MED28), has a dual nuclear and cytoplasmic localization and function. In the nucleus, MED28 functions as part of Mediator and in the cytoplasm, it interacts with cytoskeletal proteins and is part of the regulatory cascades including that of Grb2. MED28 thus has the potential to bring cytoplasmic regulatory interactions towards the centre of gene expression regulation. In this study, we identified MDT-28, the nematode orthologue of MED28, as a likely target of lysine acetylation using bioinformatic prediction of posttranslational modifications. Lysine acetylation was experimentally confirmed using anti-acetyl lysine antibody on immunoprecipitated GFP::MDT-28 expressed in synchronized C. elegans. Valproic acid (VPA), a known inhibitor of lysine deacetylases, enhanced the lysine acetylation of GFP::MDT-28. At the subcellular level, VPA decreased the nuclear localization of GFP::MDT-28 detected by fluorescencelifetime imaging microscopy (FLIM). This indicates that the nuclear pool of MDT-28 is regulated by a mechanism sensitive to VPA and provides an indirect support for a variable relative proportion of MED28 orthologues with other Mediator subunits.

Spectral properties of ionic benzotristhiazole based donor-acceptor NLO-phores in polymer matrices and their one- and two-photon cellular imaging ability.

A series of ionic benzotristhiazolium (BTT) push-pull chromophores, with different nitrogen donor groups and different lengths of conjugated bridges, was successfully doped in polar polymer matrices (PVC and PSS). The spectral (photophysical) properties of their low concentration thin polymeric films are compared with those in solution and are discussed in terms of matrix polarity/viscosity influence, specific polymer-chromophore interaction, structure-spectral property relationship and Twisted Intramolecular Charge-Transfer (TICT) state formation. The elimination of a non-emissive phantom and TICT state formation by restricted intramolecular rotations in the polymer matrix or viscous solvent results in a relatively high ΦF of all the investigated NLO-phores; particularly for near-infrared NIR molecular rotors bearing diphenylamino and julolidine donor groups. Because of cationic characteristics, small molecular weight, calculated high second hyperpolarizability and significant emission efficiency dependence on surroundings' viscosity (rigidochromic effect), two dyes were chosen as candidates for potential fluorescent probes for one-photon (1P) and two photon (2P) cellular imaging. The selected BTT NLO-phore with a julolidine donor is promising as a mitochondria-specific fluorescent small molecular probe for live cell super-resolution imaging with low cytotoxicity and good photostability, and is also potentially suitable for super-resolution STED imaging.

Synergistic action of doxorubicin bound to the polymeric carrier based on N-(2-hydroxypropyl)methacrylamide copolymers through an amide or hydrazone bond.

The cytostatic effects of polymeric conjugates based on N-(2-hydroxypropyl)methacrylamide copolymers (PHPMA) and containing doxorubicin bound through amide and hydrazone bonds (mixed conjugates) were compared with the cytostatic effects of monoconjugates containing drug bound through an amide or hydrazone bond. One group of mixed conjugates was formed from two comonomers containing doxorubicin bound to the methacryloyl group through a spacer and an amide (DOX(AM)) or hydrazone (DOX(HYD)) bond via copolymerization with HPMA. A second group of mixed conjugates was formed from two different interconnected HPMA copolymers, one containing DOX(AM) and the other DOX(HYD), forming a high-molecular-weight branched structure. The third mixed polymeric system was a simple mixture of monoconjugates DOX(AM)-PHPMA and DOX(HYD)-PHPMA. Simultaneous treatment with all mixed forms of the polymeric derivatives of doxorubicin significantly increased antitumor efficacy after application of monoconjugates, suggesting a synergizing effect that could be used in designing new doxorubicin-containing therapeutic systems.

Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility.

A series of experiments was conducted to examine potential toxic effects of cryoprotectants on motility of human spermatozoa. The data indicated that exposure of spermatozoa to cryoprotectant medium for as little as 15 minutes at room temperature caused a reduction in motility. This reduction in motility was caused by glycerol. Lowering glycerol concentrations from 7.5% to 5.0% improved sperm motility at 24 hours post-thaw. Sperm motility was not affected by either slow or abrupt cooling rates above -5 degrees C. Motility was greater in cryopreserved sperm at 24 hours post-thaw when glycerol was added at -5 degrees C rather than at room temperature. These data suggest that avoiding glycerol toxicity either by reducing the concentration used or by adding glycerol at a lower temperature, or both, may improve human sperm cryosurvival rates.

Factors affecting the cryosurvival of mouse two-cell embryos.

A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryopreservation of human spermatozoa. II. Postthaw chronology of motility and of zona-free hamster ova penetration.

Postthaw dynamics of motility maintenance and ability to penetrate zona-free hamster ova were examined with human sperm. Ten semen samples were each divided into two equal volumes; one was cryopreserved while the other half remained untreated. Frozen samples were thawed, and initial evaluations for motility and hamster egg penetration were made on both untreated and frozen-thawed samples. The time difference between the initial evaluations for the two treatment groups was approximately 30 minutes as a result of the time required to freeze and thaw aliquots. Subsequent evaluations were made 6, 12, 24, and 48 hours later. Over all times both the motility and fertilizability of cryopreserved spermatozoa were significantly reduced (P less than 0.05) when compared with those of untreated sperm. The pattern of motility loss over time was similar between untreated and frozen-thawed sperm (P greater than 0.10). Conversely, differences between untreated and frozen-thawed sperm in fertilizability patterns were dramatic (P less than 0.05). This was evidenced by penetration rates for cryopreserved sperm highest at 0 hour and decreasing over time, whereas penetration by untreated spermatozoa was lowest at 0 hour, increasing to a maximum at 24 hours. These observations may be important in the development of laboratory protocols for freezing and clinical protocols for using frozen-thawed sperm.

Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability, and acrosome reaction.

Three experiments were conducted to evaluate effects of holding semen at +5.0 degrees C for 30 minutes or -5.0 degrees C for 10 minutes and ice crystal induction (seeding) on frozen-thawed human spermatozoa. In experiment 1, spermatozoa were frozen, and postthaw motility was evaluated immediately (0 hour) and 24 hours later. At both 0 and 24 hours, nonfrozen control samples had higher motility than all other treatment groups. At 0 hour postthaw, motility was higher in samples held at -5.0 degrees C for 10 minutes with no significant effect of seeding. At 24 hours, samples held at -5.0 degrees C for 10 minutes and seeded, but not samples held at -5.0 degrees C and not seeded, had higher motility than samples held at +5.0 degrees C. In experiment 2, semen samples were frozen, and fertilizability was evaluated in a zona-free hamster egg penetration assay. Seeded samples had a higher frequency of sperm penetration than either nonfrozen or nonseeded samples. In experiment 3, nonfrozen controls and frozen treatment groups were evaluated for the frequency of survival and acrosomal integrity. Seeded samples had higher frequencies of survival and loss of acrosomal integrity than nonseeded samples. All frozen-thawed samples had a lower frequency of survival and a higher frequency of loss of acrosomal integrity than nonfrozen controls. Although altered patterns of fertilizability and acrosomal integrity are induced, collectively these data suggest that incorporating a holding temperature of -5.0 degrees C for 10 minutes and seeding may result in a superior protocol for freezing human spermatozoa.