Improving biological relevancy of transcriptional biomarkers experiments by applying the MIQE guidelines to pre-clinical and clinical trials.

The "Minimum Information for the Publication of qPCR Experiments" (MIQE [3]) guidelines are very much targeted at basic research experiments and have to our knowledge not been applied to qPCR assays carried out in the context of clinical trials. This report details the use of the MIQE qPCR app for iPhone (App Store, Apple) to assess the MIQE compliance of one clinical and five pre-clinical trials. This resulted in the need to include 14 modifications that make the guidelines more relevant for the assessment of this special type of application. We also discuss the need for flexibility, since while some parameters increase experimental quality, they also require more reagents and more time, which is not always feasible in a clinical setting.

In vitro degradation of lysine by ruminal fluid-based fermentations and by Fusobacterium necrophorum.

The objective of these studies was to characterize some factors affecting lysine degradation by mixed ruminal bacteria and by ruminal Fusobacterium necrophorum. Mixed ruminal bacteria degraded lysine, and addition of pure cultures of F. necrophorum did not increase lysine degradation. Addition of acetic or propionic acid strikingly reduced NH(3) production from lysine by mixed ruminal bacteria at pH 6, but not at pH 7. Although typical ruminal environments with acidic pH and normal concentrations of volatile fatty acids might inhibit lysine degradation by F. necrophorum, ruminal fluid contained enough bacteria with a lysine-degrading capacity to ferment 50 mM lysine in vitro. Of 7 strains of ruminal F. necrophorum tested, all grew on both lactate and lysine as the primary energy source. Both subspecies of ruminal F. necrophorum (necrophorum and funduliforme) used lysine as a primary C and energy source. Lysine and glutamic acid were effectively fermented by F. necrophorum, but alanine and tryptophan were not, and histidine and methionine were fermented only to a minor extent. The end products of lactate fermentation by F. necrophorum were propionate and acetate, and those of lysine degradation were butyrate and acetate. Fermentation of glutamic acid by F. necrophorum yielded acetate and butyrate in a ratio near to 2:1. The minimum inhibitory concentration of tylosin for F. necrophorum was not dependent on whether bacteria were grown with lactate or lysine, but F. necrophorum was more susceptible to monensin when grown on lysine than on lactate. Although F. necrophorum is generally resistant to monensin, the ionophore may reduce lysine degradation by F. necrophorum in the rumen. The essential oil components limonene, at 20 or 100 µg/mL, and thymol, at 100 µg/mL, inhibited F. necrophorum growth, whereas eugenol, guaiacol, and vanillin had no effect. Our findings may lead to ways to minimize ruminal lysine degradation and thus increase its availability to the animal.

Hydroxymethyl lysine is a source of bioavailable lysine for ruminants.

Experiments were conducted to evaluate the availability to ruminants of lysine from hydroxymethyl lysine, a product potentially resistant to ruminal degradation yet able to release free lysine when subjected to the acidic environment of the abomasum. An in vitro ruminal fermentation assay that led to ammonia production from free lysine was used for initial assessments, but the hydroxymethyl lysine was inhibitory to lysine degradation at the concentrations tested in vitro; therefore, an in vivo assay with sheep, using plasma lysine concentrations as the response criterion, was used for assessment. twelve mature sheep were fed graded amounts of lysine from either a commercially available ruminally protected lysine product with known availability or from hydroxymethyl lysine. the protected lysine product provided 3 or 6 g/d of metabolizable lysine, whereas the hydroxymethyl lysine provided 3 or 6 g/d of total lysine. Plasma lysine concentrations increased linearly in response to both the ruminally protected lysine product and hydroxymethyl lysine. by slope ratio analysis, the bioavailability of lysine in hydroxymethyl lysine was estimated to be 94% of that for the commercially available product. We concluded that hydroxymethyl lysine may be used as an effective means of supplementing lysine to ruminants.

Effects of the hypo-osmotic swelling test on the outcome of intracytoplasmic sperm injection for patients with only nonmotile spermatozoa available for injection: a prospective randomized trial.

OBJECTIVE: Hypo-osmotic swelling test (HOST) has been shown to be an effective method for the selection of live sperm. On-going pregnancies were obtained by using HOST-selected sperm. The aim of this study was to evaluate the effect of using HOST-selected "live" sperm versus nonselected sperm on the outcome of intracytoplasmic sperm injection cycles when only nonmotile sperm were available for injection. DESIGN: Prospective randomized study. SETTING: Governmental tertiary care hospital. PATIENT(S): Thirty ICSI cycles with no motile sperm were included in this study. INTERVENTION(S): For the HOST group, potentially live spermatozoa detected by hypo-osmotic reaction of the tail were injected into oocytes. For the No-HOST group, the sperm were randomly injected into the oocytes without checking the viability. MAIN OUTCOME MEASURE(S): The fertilization, cleavage, embryo quality, pregnancy, and implantation rates were assessed for the two groups. RESULT(S): Among 30 cycles, 15 fall into each group. Fertilization, cleavage rates, and the number of good quality embryos were similar between two groups. CONCLUSION(S): HOST-selected live spermatozoa can be safely used for intracytoplasmic sperm injection to establish pregnancies. There is a tendency for higher pregnancy and implantation rates to result, but it does not reach statistical significance.

Differential display analysis of mRNAs in chronic myelogenous leukaemia.

Analysis of expressed mRNAs with differential display-polymerase chain reaction (DD-PCR) is a powerful tool for the characterization of genes involved in malignant pathways and might identify markers for different phases of chronic myelogenous leukaemia (CML). We examined the presence of BCR-ABL transcripts in 25 CML patients in either the chronic phase or blast crisis. We then analysed the expression of leukocytic RNA transcripts in CML phases. DD-PCR technique was used to examine CML cases with BCR-ABL in comparison with CML cases lacking detectable BCR-ABL transcripts. Our results support the use of differential display not only for characterization of the CML differentially expressed genes but also to locate patterns that can be implemented as valuable fingerprints for each phase of CML.

Differential display analysis of mRNAs in chronic myelogenous leukaemia

Analysis of expressed mRNAs with differential display-polymerase chain reaction [DD-PCR] is a powerful tool for the characterization of genes involved in malignant pathways and might identify markers for different phases of chronic myelogenous leukaemia [CML]. We examined the presence of BCR-ABL transcripts in 25 CML patients in either the chronic phase or blast crisis. We then analysed the expression of leukocytic RNA transcripts in CML phases. DD-PCR technique was used to examine CML cases with BCR-ABL in comparison with CML cases lacking detectable BCR-ABL transcripts. Our results support the use of differential display not only for characterization of the CML differentially expressed genes but also to locate patterns that can be implemented as valuable fingerprints for each phase of CML

Pregnancy after transfer of embryos which were generated from in-vitro matured oocytes.

In-vitro maturation of human oocytes is an important technique in assisted reproduction due to its potential for reducing the use of fertility drugs. We offered this technique as an alternative to cancelling the cycle to a patient who was at risk of ovarian hyperstimulation syndrome (OHSS) after treatment with gonadotrophin-releasing hormone analogue (GnRHa) and human menopausal gonadotrophin (HMG). The patient had 40 visible antral follicles with a maximum diameter of 13 mm and an oestradiol concentration of 14,000 pmol/l on cycle day 12. Immature oocytes were aspirated transvaginally under ultrasound guidance. Ten cumulus-enclosed oocytes were harvested and nine of them completed nuclear maturation to metaphase II after 48 h in culture. By 18 h after an intracytoplasmic sperm injection (ICSI) procedure, seven of these metaphase II stage oocytes displayed two distinct pronuclei and two polar bodies. All fertilized oocytes but one underwent cleaveage; four of these were transferred 2 days later. Endometrial priming was initiated with 8 mg oestradiol valerate daily from the day of oocyte retrieval and 50 mg progesterone was injected i.m. daily starting 2 days after that. A single intrauterine sac was seen containing one fetus with positive fetal heart beat on ultrasound at 7 weeks of gestation. Unfortunately, the pregnancy ended at 24 weeks shortly after premature rupture of membranes; a live healthy-looking girl was delivered who died 18 days later.

Effect of immature Schistosoma mansoni worms on hamsters' plasma enzymes.

1. The pattern and activity of isocitrate, lactate and malate dehydrogenases and malic enzyme were studied in plasma of normal hamsters and hamsters at the 26th day of infection with S. mansoni. 2. Although the electrophoretic patterns of these enzymes were similar in normal and infected hamsters, their activities were higher in the latter than the former group of animals. The elevation in the enzymic activity indicates that there is tissue damage caused by the larvae at this stage.