Minocycline and matrix metalloproteinase inhibition in acute intracerebral hemorrhage: a pilot study.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) is a devastating cerebrovascular disorder with high morbidity and mortality. Minocycline is a matrix metalloproteinase-9 (MMP-9) inhibitor that may attenuate secondary mechanisms of injury in ICH. The feasibility and safety of minocycline in ICH patients were evaluated in a pilot, double-blinded, placebo-controlled randomized clinical trial. METHODS: Patients with acute onset (<12 h from symptom onset) ICH and small initial hematoma volume (<30 ml) were randomized to high-dose (10 mg/kg) intravenous minocycline or placebo. The outcome events included adverse events, change in serial National Institutes of Health Stroke Scale score assessments, hematoma volume and MMP-9 measurements, 3-month functional outcome (modified Rankin score) and mortality. RESULTS: A total of 20 patients were randomized to minocycline (n = 10) or placebo (n = 10). The two groups did not differ in terms of baseline characteristics. No serious adverse events or complications were noted with minocycline infusion. The two groups did not differ in any of the clinical and radiological outcomes. Day 5 serum MMP-9 levels tended to be lower in the minocycline group (372 ± 216 ng/ml vs. 472 ± 235 ng/ml; P = 0.052). Multiple linear regression analysis showed that minocycline was associated with a 217.65 (95% confidence interval -425.21 to -10.10, P = 0.041) decrease in MMP-9 levels between days 1 and 5. CONCLUSIONS: High-dose intravenous minocycline can be safely administered to patients with ICH. Larger randomized clinical trials evaluating the efficacy of minocycline and MMP-9 inhibition in ICH patients are required.

Walking in circles: navigation deficits from Parkinson's disease but not from cerebellar ataxia.

Little is known on the role of neuronal structures for spatial navigation. Our goal was to examine how Parkinson's disease (PD) and cerebellar ataxia, as human lesion models of the basal ganglia and cerebellum, affect spatial navigation round a circular walking path, blindfolded. Twelve subjects with idiopathic PD (ON and OFF medication), eight subjects with cerebellar ataxia and a control group of 20 age-matched healthy subjects participated. All groups performed well when walking around the circle with eyes open. In the eyes-closed condition, control subjects overshot the outlined trajectory but returned to their initial position, thus walking a further distance with eyes closed than with eyes open. When OFF medication, PD subjects navigated a larger radius than controls with eyes closed. When ON levodopa, PD subjects walked a similar distance as controls but with even larger errors in endpoint. Surprisingly, cerebellar patients navigated the circular walking task in the eyes closed condition with even more accuracy (i.e. following the outlined circle) than control and PD subjects. We conclude that blindfolded navigation around a previously seen circle requires intact basal ganglia, but not cerebellar input.

Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of beta-thalassaemia: a pilot study.

BACKGROUND: Trophectoderm biopsy at the blastocyst stage is an emerging approach in preimplantation genetic diagnosis (PGD). This study aimed to compare genotyping success and implantation rates in PGD cycles for beta-thalassaemia following biopsy at the cleavage versus the blastocyst stage, with transfer of blastocysts. METHODS: This pilot study included 20 cycles: Group A: 10 cycles, day 3 blastomere biopsy, day 5 transfer; Group B: 10 cycles, day 5 trophectoderm biopsy, day 6 transfer. Standard-assisted reproduction and laser biopsy procedures were used. Biopsied cells were genotyped using real-time PCR multiplexed with fluorescent microsatellite analysis. RESULTS: In Group A, 131 fertilized eggs developed to 101 embryos suitable for single blastomere biopsy; 76/101 blastomeres were diagnosed (75.2%), 30 unaffected blastocysts were transferred resulting in six pregnancies (eight fetal hearts, 26.7% implantation rate). In Group B, 128 fertilized eggs developed to 53 blastocysts for trophectoderm biopsy (four to five cells), with 50/53 blastocysts diagnosed (94.3%), 21 unaffected blastocysts transferred and 6 pregnancies initiated (10 fetal hearts, 47.6% implantation rate). Overall, nine pregnancies reached >10 weeks gestation and were confirmed unaffected by prenatal diagnosis, with 12 healthy babies born. CONCLUSIONS: This pilot study suggests that trophectoderm biopsy and blastocyst transfer may be more advantageous than cleavage stage biopsy with respect to outcome of PGD for monogenic diseases.

Precision measurement of the weak mixing angle in Møller scattering.

We report on a precision measurement of the parity-violating asymmetry in fixed target electron-electron (Møller) scattering: A(PV) = [-131 +/- 14(stat) +/- 10(syst)] x 10(-9), leading to the determination of the weak mixing angle sin2(thetaW(eff) = 0.2397 +/- 0.0010(stat) +/- 0.0008(syst), evaluated at Q2 = 0.026 GeV2. Combining this result with the measurements of sin2(thetaW(eff) at the Z0 pole, the running of the weak mixing angle is observed with over 6sigma significance. The measurement sets constraints on new physics effects at the TeV scale.

Financial incentive to control paratuberculosis (Johne's disease) on dairy farms in the United Kingdom.

This paper estimates the financial incentive to control paratuberculosis on dairy farms by establishing the level of expenditure that would minimise the total cost of the disease (output losses plus control expenditure). Given the late onset of the clinical signs and the lack of treatments, control was focused on minimising the financial impact of paratuberculosis by adjusting the dairy cow replacement policy. The optimum replacement policies for disease-free herds and infected herds were compared by using dynamic programming. At the standard settings, the disease justified adjusting the culling policy; under constant bioeconomic assumptions, it reduced the expected annuity from milk production under the optimal replacement policy by about 10 per cent (27 pounds sterling per cow annually), a considerably lower figure than for other major endemic diseases that affect dairy cows in the uk. The effect was even less at lower milk prices, suggesting that there is at present little incentive for dairy farmers to put more resources into controlling the disease. However, the incentive could be increased if more information were available about how best to manage the disease under specific farm circumstances. Any effect that paratuberculosis may have on the future demand for milk and hence on milk prices would also be an important consideration.

Birth of a healthy infant following trophectoderm biopsy from blastocysts for PGD of beta-thalassaemia major.

PGD is a well accepted reproductive choice for couples at genetic risk and involves the diagnosis and transfer of unaffected IVF embryos. PGD for monogenetic diseases is most commonly accomplished by the biopsy of one or two blastomeres from cleavage stage embryos, followed by PCR-based protocols. However, PCR-based DNA analysis of one or two cells is subject to several problems, including total PCR failure, or failure of one allele to amplify. Trophectoderm biopsy at the blastocyst stage enables the removal of more than two cells for diagnosis while being non-invasive to the inner cell mass which is destined for fetal development. The aim of this study was to develop a safe, reliable technique for the biopsy of trophectoderm cells from human blastocysts. This case report demonstrates that removal of trophectoderm cells prior to blastocyst transfer is compatible with implantation and development to term. Here we report successful PGD for beta-thalassaemia following trophectoderm cell biopsy from blastocysts and the birth of a healthy infant.

Observation of parity nonconservation in møller scattering.

We report a measurement of the parity-violating asymmetry in fixed target electron-electron (Møller) scattering: A(PV)=[-175+/-30(stat)+/-20(syst)] x 10(-9). This first direct observation of parity nonconservation in Møller scattering leads to a measurement of the electron's weak charge at low energy Q(e)(W)=-0.053+/-0.011. This is consistent with the standard model expectation at the current level of precision: sin((2)theta(W)(M(Z))((-)MS)=0.2293+/-0.0024(stat)+/-0.0016(syst)+/-0.0006(theory).

The 'GO' system--a novel method of microculture for in vitro development of mouse zygotes to the blastocyst stage.

A novel system of in vitro culture termed the 'glass oviduct' or 'GO' culture system is described. Mouse zygotes were cultured in pairs to the blastocyst stage in open-ended 1 microl glass capillaries. 'GO' culture supported the development of significantly more hatching or hatched blastocysts than did a standard microdroplet (10 zygotes per 20 microl) control culture (48.3 versus 3.3%, respectively). 'GO' bslastocysts contained significantly larger populations of cells (92+/-3 versus 75+/-3), and inner cell mass (25+/-1 versus 21+/-1) and trophectoderm (68+/-2 versus 53+/-3) subpopulations, compared with microdroplet-derived blastocysts. Before blastulation, 'GO'-derived morulae were found to contain significantly more cells than microdroplet-derived morulae (27+/-0.7 versus 14+/-0.5). After implantation, 'GO' blastocysts formed fetuses at a similar rate to microdroplet-derived blastocysts (55 versus 62%), but at a lower rate than blastocysts derived in vivo (80%). 'GO'- and microdroplet-derived fetuses were similar in wet weight to each other (0.412 and 0.415 g, respectively) but were heavier than fetuses derived from flushed blastocysts (0.390 g). An additional experiment investigated whether the beneficial effect of 'GO' culture was due to the significantly increased embryo density. Proportions of hatching or hatched blastocysts after 'GO' culture (50%) were higher than after standard microdroplet culture (7.6%), but were not different from culture in high embryo density microdroplets (20 zygotes per 10 microl; 42%). 'GO' blastocysts contained more cells (79.6+/-2.1) than did standard microdroplet-derived blastocysts (68.7+/-2.0), but were similar to high density microdroplet-derived blastocysts (85.8+/-2.7). Similarly, 'GO' blastocysts contained more trophectoderm cells (62.2+/-2.0) than did standard microdroplet-derived blastocysts (52.7+/-1.7), but were similar to the high density microdroplet blastocysts (68.8+/-2.5). Numbers of inner cell mass cells ('GO', standard microdroplet and high density microdroplet culture) were not different from each other (17.4+/-0.5, 16+/-0.5 and 17+/-0.4, respectively). In conclusion, the 'GO' culture system represents an alternative method to the microdroplet system for small numbers of preimplantation embryos, without detriment to implantation potential.

Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos.

Over the past 30 years many cryopreservation procedures have been applied to oocytes, embryos, sperm, ovarian and testicular tissue. Over this time many, often specialized, terms have been developed for all aspects of these procedures. This can make it difficult for readers who are not familiar with the terminology or protocols to compare and evaluate different procedures. This paper describes the main cryopreservation procedures, the terminology associated with them, and briefly explains the underlying physical, chemical and biological processes. The aim is to help readers understand and interpret other papers on slow cooling, rapid cooling, ultrarapid cooling and vitrification.

Q2 evolution of the generalized Gerasimov-Drell-Hearn integral for the neutron using a 3He target.

We present data on the inclusive scattering of polarized electrons from a polarized 3He target at energies from 0.862 to 5.06 GeV, obtained at a scattering angle of 15.5 degrees. Our data include measurements from the quasielastic peak, through the nucleon resonance region, and beyond, and were used to determine the virtual photon cross-section difference sigma(1/2)-sigma(3/2). We extract the extended Gerasimov-Drell-Hearn integral for the neutron in the range of four-momentum transfer squared Q2 of 0.1-0.9 GeV2.

The effect of extracellular matrix molecules on mouse preimplantation embryo development in vitro.

The extracellular matrix (ECM) molecules, laminin (LN), chondroitin sulfate (CS), fibronectin (FN), hyaluronic acid (HA), mucin (MUC) and heparan sulfate proteoglycan (HS), were investigated as supplements to culture medium to improve the in vitro development of mouse 1-cell zygotes to blastocysts. Development was also compared with that in medium supplemented with bovine serum albumin (BSA) to determine the potential for ECM molecules as suitable alternatives to serum albumin in culture medium. Supplementation of sequential culture media with LN at all concentrations examined failed to result in more than 70% of zygotes developing to blastocysts; therefore, LN was considered unsuitable as a replacement for BSA and was not examined further. The optimal concentration of the remaining ECM molecules was used to supplement sequential culture media and the effect on blastocyst quality was assessed by determining the differential cell numbers of blastocysts grown in BSA-supplemented medium. Development to blastocyst was similar, regardless of the macromolecule used. The number of inner cell mass cells was significantly higher in HS-supplemented medium compared with controls. Trophectoderm cell numbers were similar to control values for all ECM molecules examined except CS for which there were fewer trophectoderm cells. It is concluded that ECM molecules, FN, HA, MUC and HS may be used as substitutes for serum protein supplementation of culture media EG0/G2 for mouse preimplantation embryo development. Heparan sulfate proteoglycan increases inner cell mass numbers and this may be due to interactions with the growth factors fibroblast growth factor 4 (FGF-4) and granulocyte-macrophage colony-stimulating factor.

Forward versus backward walking: transfer of podokinetic adaptation.

We asked whether podokinetic adaptation to walking on a circular treadmill transfers to different forms of locomotion. Subjects were blindfolded and asked to walk straight across the floor, in the forward and backward directions, following podokinetic (PK) stimulation that consisted of 30 min of forward walking-in-place on the perimeter of a disk rotating in the clockwise direction. During both forward and backward walking following forward-walking PK stimulation, subjects involuntarily walked along curved trajectories at angular velocities well above vestibular threshold, although they perceived that they were walking along straight paths. The curved paths of forward and backward walking were indistinguishable from one another. Transfer of PK adaptations acquired during forward walking on the turntable to backward walking trials suggests that the PK system controls general locomotor trajectory. Adaptation of the system thus influences forms of locomotion other than that used during acquisition of the adaptation. This transfer also supports the concept that forward and backward walking are controlled by neural networks that share common elements. An interesting feature of the transfer of PK adaptation is that for both forward and backward walking, subjects turned in a counterclockwise direction. As such, the direction of relative rotation between the trunk and feet was maintained for both forward and backward walking. However, the relationship of the lower extremities to the center of rotation was not preserved. The left limb was the inner leg during PK stimulation and forward walking after adaptation, but the left leg was the outer leg during backward walking. These results suggest that PK adaptation affects general locomotor trajectory via a remodeling of the rotational relationship between the trunk and the feet.

Sex ratio and birth weights of infants born as a result of blastocyst transfers compared with early cleavage stage embryo transfers.

OBJECTIVE: To analyze the birth weights and sex ratio of infants born as a result of blastocyst transfer and compare them with data resulting from the transfer of early-cleavage stage embryos. DESIGN: Retrospective analysis. SETTING: Monash IVF (private in vitro fertilization clinic). PATIENTS(S): One hundred twenty-five infertile patients who became pregnant after IVF procedures involving blastocyst transfer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sex ratio and birth weights of infants born after blastocyst transfer. RESULT(S): The sex ratio of 129.6 for infants born after blastocyst transfer was not significantly different from the sex ratio calculated from data compiled by NPSU for births resulting from early cleavage stage embryo transfers at Monash IVF (100.6) and all other assisted conception units in Australia and New Zealand (97.9). No differences were observed in the combined mean birth weight of male and female infants born as a result of blastocyst transfers and early-cleavage stage embryo transfers. CONCLUSION(S): There is no evidence of abnormal fetal growth or a shift in the sex ratio for infants born as a result of blastocyst transfer when compared with the case of births resulting from early cleavage stage embryo transfers within our unit or all other assisted conception units in Australia and New Zealand.

Ontogeny of hepatic enzymes involved in serine- and folate-dependent one-carbon metabolism in rabbits.

Serine occupies a central position in folate-dependent, one-carbon metabolism through 5,10-methylenetetrahydrofolate (MTHF) and 5-formyltetrahydrofolate (FTHF). We characterized the ontogeny of the specific activity of key enzymes involved in serine, 5,10-MTHF, and 5-FTHF metabolism: methenyltetrahydrofolate synthetase (MTHFS), MTHF reductase (MTHFR), the glycine cleavage system (GCS), methionine synthase (MS), and serine hydroxymethyltransferase (SHMT) in rabbit liver, placenta, brain, and kidney. In liver, MTHFS activity is low in the fetus (0.36 +/- 0.07 nmol. min(-1). mg protein(-1)), peaks at 3 wk (1.48 +/- 0.50 nmol. min(-1). mg protein(-1)), and then decreases to adult levels (1.13 +/- 0.32 nmol. min(-1). mg protein(-1)). MTHFR activity is highest early in gestation (24.9 +/- 2.4 nmol. h(-1). mg protein(-1)) and declines rapidly by birth (4.7 +/- 1.3 nmol. h(-1). mg protein(-1)). MS is highest during fetal life and declines after birth. Cytosolic SHMT activity does not vary during development, but mitochondrial SHMT peaks at 23 days. GCS activity is high in the fetus and the neonate, declining after weaning. In placenta and brain, all activities are low throughout gestation. Cytosolic and mitochondrial SHMT activities are low in kidney and rise after weaning, whereas MTHFS is low throughout development. These data suggest that the liver is the primary site of activity for these enzymes. Throughout development, there are multiple potential sources for production of 5,10-MTHF, but early in gestation high MTHFR activity and low MTHFS activity could reduce 5,10-MTHF availability.

Inducible mutagenesis in TEPC 2372, a mouse plasmacytoma cell line that harbors the transgenic shuttle vector lambdaLIZ.

The plasmacytoma cell line, TEPC 2372, was derived from a malignant plasma cell tumor that developed in the peritoneal cavity of a BALB/c mouse that harbored the transgenic shuttle vector for the assessment of mutagenesis in vivo, lambdaLIZ. TEPC 2372 was found to display the typical features of a BALB/c plasmacytoma. It consisted of pleomorphic plasma cells that secreted a monoclonal immunoglobulin (IgG2b/lambda), was initially dependent on the presence of IL-6 to grow in cell culture, contained a hyperdiploid chromosome complement with a tendency to undergo tetraploidization, and harbored a constitutively active c-myc gene by virtue of a T(6;15) chromosomal translocation. TEPC 2372 was further characterized by the ability to respond to in vitro exposure with 4-NQO (4-nitroquinoline-1-oxide), an oxidative model mutagen, with a vigorous dose-dependent increase in mutagenesis that peaked at a 7.85-fold elevation of mutant rates in lambdaLIZ when compared to background mutant rates in untreated controls. Cotreatment with 4-NQO and BSO (buthionine sulfoximine), a glutathione-depleting compound that causes endogenous oxidative stress, resulted in a 9.03-fold increase in the mutant frequency in lambdaLIZ. These results demonstrated that TEPC 2372, the malignant plasma cell counterpart of the lambdaLIZ-based in vivo mutagenesis assay, may be useful as an in vitro reference point for the further elucidation of oxidative mutagenesis in lymphoid tissues.

Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts.

Histological staining and counting of blastocyst inner cell mass (ICM) and trophectoderm (TE) cells differentially with chromatin-specific dyes is a more accurate indicator of cultured blastocyst quality and normality than total cell number assessment. The aim of this study was to test the effectiveness of a simplified method of chemically-defined differential blastocyst staining. The TE of cultured mouse and bovine blastocysts of different developmental stages was stained when blastocysts were treated with a permeabilizing solution containing the ionic detergent Triton X-100 and the fluorochrome propidium iodide. Blastocysts were then incubated in a second solution containing 100% ethanol (for fixation) and the secondary fluorochrome bisbenzimide. Fixed and stained whole blastocysts were mounted and assessed for cell number using ultraviolet fluorescent microscopy. Using this method, in-vitro cultured mouse blastocysts (day 4.5) were shown to have an ICM:TE ratio of 1:2.63 with an average total cell count of 75.3 +/- 3. While day 7 and 8 in-vitro produced bovine blastocysts were shown to have an ICM:TE ratio of 1:3.42 and 1:3.36 with an average total cell count of 151.3 +/- 5.48 and 217.8 +/- 8.75 respectively. Blastocyst staining patterns indicate that this modified technique represents a simple and reliable alternative to current bichromatic blastocyst staining techniques for the differential assessment of cell numbers and may be useful for the assessment of blastocysts derived from in-vitro maturation, novel culture systems and advanced reproductive technologies such as cloning.

Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro.

In all, 143 human embryos obtained 3 days (day 3) after insemination or intracytoplasmic sperm injection (ICSI) were biopsied and a single nucleated cell removed for identification of aneuploidy by fluorescent in-situ hybridization (FISH) for chromosomes X, Y, 13, 16, 18 and 21. Fifty-one per cent of embryos were aneuploid and significantly more aneuploid embryos blocked in further development to morulae and blastocysts than euploid embryos (59 versus 34%; P < 0.001). Chromosomal analysis of the generated blastocysts revealed 40% were aneuploid (16 of 40 generated blastocysts). Re-examination of cells by FISH for the same chromosome probes of the inner cell mass (ICM) of expanded and hatching blastocysts derived from the aneuploid embryos revealed a high incidence of mosaicism of ICM cell lineages that were usually predictable from observations of day 3 single-cell biopsies. These data would not support the hypothesis of a preferential allocation of euploid cells to the ICM and aneuploid cells to the trophectoderm. A high concordance between day 3 aneuploidy diagnosis and ICM cell lineages was observed with trisomies (97%), and multiple complex chromosome numerical abnormalities (100%). A reduced concordance was observed with monosomies (65%) and haploidy (18%). Concomitantly, the proportion of ICM cell lineages was increased in blastocysts whose chromosomal condition was diagnosed as haploid (21%) or with complex numerical abnormalities (50%).

Effect of recombinant human gonadotrophins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro.

The response of murine, bovine and human oocytes to pure recombinant preparations of human follicle stimulating hormone (rFSH) and luteinizing hormone (rLH) for meiotic maturation and subsequent developmental competence in vitro were examined in the present experiments. Maturation of immature bovine oocytes to the metaphase II stage was significantly increased by the addition of 1 IU/ml of rFSH in combination with either 1 IU/ml rLH or 10 IU/ml rLH. Similarly, embryonic development to the blastocyst stage was improved in bovine oocytes treated with a 1:10 combination of rFSH:rLH. However, no significant difference was observed in the number of inner cell mass or trophectoderm cells of the resulting blastocysts. Although the increased maturation to metaphase II was not significant, human embryonic developmental competence was improved by maturing oocytes in the presence of a 1:10 ratio of rFSH:rLH as only those oocytes exposed to a 1:10 ratio of rFSH: rLH during maturation showed normal cleavage patterns beyond day 2. In addition, 1 IU/ml rFSH and 1 IU/ml rLH increased the expression of oocyte proteins in human oocytes. The inclusion of recombinant gonadotrophins, either singly or in combination, had no significant effect on the maturation, fertilization or embryonic development of in-vitro matured mouse oocytes. These data provide support for the responsiveness of human and bovine oocytes to gonadotrophins in vitro and the need to consider variations in the relative concentrations for optimization of oocyte developmental competence.

Mechanisms of toxicity, clinical features, and management of diquat poisoning: a review.

USES: Diquat (1,1'-ethylene-2,2'-bipyridilium) is a nonselective bipyridyl herbicide, related structurally to paraquat, which is used both as a contact herbicide and a preharvest desiccant. In comparison to paraquat, diquat is used much less widely in agriculture. MECHANISMS OF TOXICITY: Diquat is a potent redox cycler and is readily converted to a free radical which, in reaction with molecular oxygen, generates superoxide anions and subsequently other redox products. These products can induce lipid peroxidation in cell membranes, and potentially cause cell death. FEATURES: Over the period 1968-1999, only 30 cases of diquat poisoning were reported in detail in the literature, of which 13 (43%) were fatal. Local and systemic effects have been reported following diquat exposure, with systemic features being invariably associated with ingestion. In severe and usually fatal cases, gastrointestinal mucosal ulceration, paralytic ileus, hypovolemic shock, acute renal failure, and coma have been reported. MANAGEMENT: After rapid confirmation of the diagnosis using a qualitative urine test, gut decontamination may be considered in patients who present within 1 hour of a life-threatening ingestion (>6 g). Supportive measures including fluid and electrolyte replacement should then be employed. Although hemofiltration and hemodialysis are of proven value if renal failure supervenes, there is no clinical evidence that hemodialysis or hemoperfusion removes toxicologically significant amounts of diquat, thereby reducing the risk of organ failure and preventing a fatal outcome in severe cases.