MRNIP is a replication fork protection factor.

The remodeling of stalled replication forks to form four-way DNA junctions is an important component of the replication stress response. Nascent DNA at the regressed arms of these reversed forks is protected by RAD51 and the tumor suppressors BRCA1/2, and when this function is compromised, stalled forks undergo pathological MRE11-dependent degradation, leading to chromosomal instability. However, the mechanisms regulating MRE11 functions at reversed forks are currently unclear. Here, we identify the MRE11-binding protein MRNIP as a novel fork protection factor that directly binds to MRE11 and specifically represses its exonuclease activity. The loss of MRNIP results in impaired replication fork progression, MRE11 exonuclease-dependent degradation of reversed forks, persistence of underreplicated genomic regions, chemosensitivity, and chromosome instability. Our findings identify MRNIP as a novel regulator of MRE11 at reversed forks and provide evidence that regulation of specific MRE11 nuclease activities ensures protection of nascent DNA and thereby genome integrity.

Intra-articular injection of tranexamic acid reduce blood loss in cemented total knee arthroplasty.

The purpose of this study was to compare the efficacy of intravenous and topical tranexamic acid (TXA) versus control group for reduction in blood loss following primary total knee arthroplasty (TKA). A total of 90 patients were prospectively allocated to each of three groups (control, intravenous IV and intra-articular) and underwent unilateral total knee arthroplasty. In the IV group, patients received one dose of TXA of 15 mg/kg before deflation of the tourniquet, while in the intra-articular group patients received 2 g TXA via the drain retrogradely after closure of the wound. The mean drained blood loss in control, IV and intra-articular groups was 415 ± 24, 192 ± 21 and 121 ± 17 ml, respectively. About 43 % (control), 23 % (IV) and 17 % (intra-articular) of each group required transfusion, and the mean transfusion was 338, 168 and 79 ml, respectively. Preoperative hemoglobin values decreased at 24 h by 2.80 ± 0.14, 2.24 ± 0.17 and 2.26 ± 0.18 mg/dl, respectively. TXA reduced blood loss and transfusion requirement. Compared with one-dose intravenous administration, intra-articular administration of TXA seems to be more effective in terms of reducing drained blood loss and transfusion frequency. We recommend administration of topical TXA in primary TKA in healthy patients to decrease perioperative blood loss.

Dynamics of gene expression patterns during early development of the European seabass (Dicentrarchus labrax).

Larval and embryonic stages are the most critical period in the life cycle of marine fish. Key developmental events occur early in development and are influenced by external parameters like stress, temperature, salinity, and photoperiodism. Any failure may cause malformations, developmental delays, poor growth, and massive mortalities. Advanced understanding of molecular processes underlying marine larval development may lead to superior larval rearing conditions. Today, the new sequencing and bioinformatic methods allow transcriptome screens comprising messenger (mRNA) and microRNA (miRNA) with the scope of detecting differential expression for any species of interest. In the present study, we applied Illumina technology to investigate the transcriptome of early developmental stages of the European seabass (Dicentrarchus labrax). The European seabass, in its natural environment, is a euryhaline species and has shown high adaptation processes in early life phases. During its embryonic and larval phases the European seabass lives in a marine environment and as a juvenile it migrates to coastal zones, estuaries, and lagoons. Investigating the dynamics of gene expression in its early development may shed light on factors promoting phenotypic plasticity and may also contribute to the improvement and advancement of rearing methods of the European seabass, a species of high economic importance in European and Mediterranean aquaculture. We present the identification, characterization, and expression of mRNA and miRNA, comprising paralogous genes and differentially spliced transcripts from early developmental stages of the European seabass. We further investigated the detection of possible interactions of miRNA with mRNA.

Effects of testosterone on gonadotropins, testes, and plasma 17alpha,20beta-dihydroxy-4-pregnene-3-one levels in postbreeding mature Atlantic salmon, Salmo salar, male parr.

Atlantic salmon, Salmo salar, mature male parr were implanted with testosterone (T) in small (T3) or large (T10) Silastic capsules in the breeding season or at its end in November or December, in order to find out whether the postbreeding decline in 17alpha, 20beta-dihydroxy-4-pregnene-3-one (17,20beta-P) and milt production is a consequence of declining T levels. In the first of three experiments, fish were sampled the following January and March, whereas in the second and in the third they were sampled in April. Pituitary and plasma concentrations of gonadotropic hormone (GTHs) I and II and plasma levels of T, 11-ketotestosterone (11-KT) and 17, 20beta-P were measured by radioimmunoassays, and the testes were examined histologically. Administration of T prolonged the period in which running milt was present, suppressed Sertoli cells, and prevented the postbreeding decline in testes weight in experiment two. The postbreeding decline in plasma 17,20beta-P levels was diminished by T10 in experiment one, and by both T3 and T10 in experiment two. The similar decline in 11-KT levels was not influenced by T treatment (only studied in experiment one). T treatment also prevented a decline in pituitary GTH II content (in experiments two and three) and in plasma GTH II levels (only studied in experiment three). However, pituitary GTH I content was not influenced (experiment two and three), whereas plasma GTH I levels (only studied in experiment three) were suppressed by T. To summarize, T treatment prevents postbreeding decline in 17,20beta-P levels, probably via a stimulation of GTH II secretion. J. Exp. Zool. 284:425-436, 1999.

Feedback control of gonadotropins in Atlantic salmon, Salmo salar, male parr.II. Aromatase inhibitor and androgen effects.

Both positive and negative feedback on the (hypothalmus)- pituitary-gonad axis occur in salmonids. The aim of the present study was to investigate the role of different androgens, and in particular the involvement of aromatization of androgens to estrogens in feedback mechanisms. Previously mature Atlantic salmon, Salmo salar, male parr were studied in two experiments. In the first experiment, intact fish were implanted with Silastic capsules filled with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) in spring and sampled in the autumn when the rematuring males were starting to display running milt. In the second experiment, castrated males were implanted with capsules containing ATD, the androgens testosterone (T) and 11-ketoandrostenedione (11KA), or ATD and T combined in spring. These fish were sampled in the summer when rematuring fish were starting to show signs of gonadal growth. Pituitary and plasma gonadotropins (GTH I and GTH II) were studied using radioimmunoassay. In autumn, ATD treatment reduced pituitary and plasma GTH II levels. In summer, GTH II was consistently nondetectable in plasma. Castration diminished pituitary GTH II content. Treatment with T increased pituitary GTH II content, an effect that was attenuated when T treatment was combined with ATD. All these results are consistent with the presence of an aromatase-dependent positive feedback of T on GTH II. 11KA also had a stimulatory effect on GTH II, although weaker than that of T. Testicular size and spermiation was reduced by ATD in autumn; the latter of these results is likely to be due to the inhibitory effect of ATD on GTH II. Positive effects of ATD on plasma GTH I were found in autumn, indicative of an aromatase-dependent negative feedback in this phase. On the other hand, castration increased plasma and pituitary GTH I levels in summer, indicating that the gonads in this phase exert a predominantely negative control of GTH I. In summer, negative effects of T on GTH I pituitary levels were not suppressed, but were rather enhanced, by the combined treatment with ATD. Furthermore, plasma GTH I levels were lower after treatment with T in combination with ATD than with T or ATD separately. These negative effects of T were not diminished by ATD, so that they are nonaromatase-dependent. Furthermore, since they were actually more pronounced in the presence of ATD it is suggested that there is also a positive aromatase-dependent feedback component in this phase. In addition, 11KA had a negative effect on plasma and pituitary GTH I in castrated previously mature males. Thus, GTH I secretion is controlled by both aromatase-dependent and nonaromatase-dependent feedback effects, of which at least the former may be positive or negative depending on season. In summary, the feedback control of GTH I appears to be more complex than that of GTH II.

Feedback control of gonadotropins in Atlantic salmon, Salmo salar, male parr.I. Castration effects in rematuring and nonrematuring fish.

Sexual maturation of Atlantic salmon (Salmo salar) male parr is a seasonally recurrent "all or none" response; either a fish matures fully or it does not mature. To study whether gonadal feedback on gonadotropic hormones, GTH I and GTH II, is involved in the control of maturation, previously mature Atlantic salmon male parr were either sham-operated or castrated in spring. They were then sampled during the onset of gonadal growth (late June-early July) or shortly before the breeding season (late September). In autumn, sham-operated males separated into two groups: nonrematuring males with low pituitary and plasma levels of both GTH I and GTH II, and those rematuring with high levels of gonadotropins. Castrated males had low GTH I and GTH II plasma and pituitary levels, similar to those of the nonrematuring fish, suggesting positive feedback mechanisms, separating the sham-operated fish into low and high GTH level groups. In the summer, plasma GTH II was nondetectable in all fish. Pituitary GTH II content was lower in nonrematuring, than in rematuring males and was even lower in castrated fish. In contrast, castration increased pituitary and plasma levels of GTH I in the summer, suggesting a negative feedback at this reproductive stage. There were no significant differences in immunoassayable levels of GTH I in plasma in rematuring and nonrematuring sham-operated males at this time. The control of rematuration is complex and may involve factors other than circulating GTH I levels, possibly with differences in gonadal sensitivity to GTH I.

Effects of gonadectomy and androgen treatments on pituitary and plasma levels of gonadotropins in mature male Atlantic salmon, Salmo salar, parr--positive feedback control of both gonadotropins.

The aim of the present study was to clarify the role of feedback control on the (brain)-pituitary-gonadal axis in regulating FSH (gonadotropic hormone, GTH I) and LH (GTH II) in natural maturation in salmonids. In two experiments, 2-yr-old previously mature Atlantic salmon male parr were castrated or sham operated in the spring following their first reproductive season. In one of the experiments, castrated fish were also implanted with silicone elastomer capsules containing testosterone (T) or 11-ketoandrostenedione (11KA). The fish were sampled in July, September, and November (spawning period). Pituitary and plasma LH and FSH levels were measured using RIA and were lower in castrated than in sham-operated fish, indicating positive feedback on both FSH and LH. T, and to a lesser extent 11KA, increased pituitary LH content in castrated fish. The 11KA increased plasma and pituitary FSH levels, whereas T suppressed FSH in July and stimulated it in November. Plasma FSH levels peak earlier than LH, and it is suggested that if one or more feedback effects are involved in controlling the "all-or-nothing response," i.e., whether a fish will mature or not, a feedback effect on FSH is the most likely candidate.

Effects of aromatase inhibitors on sexual maturation in Atlantic salmon, Salmo salar, male parr.

Atlantic salmon, Salmo salar, male parr were implanted with Silastic capsules filled with different aromatase inhibitors: 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4OH), and the non-steroidal CGS16949 A, 4-benzonitrile monohydrochloride (CGS). Aromatization in brain homogenates were lower in salmon implanted with CGS and ATD than in controls. This was not the case for 4OH, but administration of 4OH to brain homogenates reduced the aromatase activity. All three aromatase inhibitors had effected gonadal weights in fish sampled in the summer, but the effects were markedly different among inhibitors. Plasma levels of the androgen 11-ketotestosterone (11KT) and the progestin 17α, 20ß-dihydroxy-4-pregnen-3-one (17,20P) were measured by means of radioimmunoassay. CGS and ATD, but not 4OH, significantly decreased the plasma 17,20P levels in the autumn. Plasma levels of 11 KT were not influenced by ATD or CGS treatment, but 4OH had a lowering effect in one autumn sampling. ATD and 4OH (CGS not tested) increased the proportion of maturing males.These findings suggest that aromatization is of physiological importance in different mechanisms controlling reproduction in salmon.

Chromosome breakage in individuals with single-cell structural aberrations and habitual abortions.

The rate of spontaneous and methotrexate (MTX)-induced chromosome breakage was studied in individuals with a history of habitual abortions in whom a structural chromosomal aberration was found in a single cell during routine cytogenetic analysis. Twelve such individuals were selected because they were not under the influence of any known mutagenic factor such as smoking, alcohol, medication and apparent irradiation; they were compared to 12 age- and sex-matched control parents. A detailed statistical analysis revealed that the spontaneous and MTX-induced chromosome breakage was significantly increased in the abortion group. The MTX-induced breakage rate was especially elevated in the women of the abortion group.