Differences during the first lactation between cows cloned by somatic cell nuclear transfer and noncloned cows.

Lactation performance is dependent on both the genetic characteristics and the environmental conditions surrounding lactating cows. However, individual variations can still be observed within a given breed under similar environmental conditions. The role of the environment between birth and lactation could be better appreciated in cloned cows, which are presumed to be genetically identical, but differences in lactation performance between cloned and noncloned cows first need to be clearly evaluated. Conflicting results have been described in the literature, so our aim was to clarify this situation. Nine cloned Prim' Holstein cows were produced by the transfer of nuclei from a single fibroblast cell line after cell fusion with enucleated oocytes. The cloned cows and 9 noncloned counterparts were raised under similar conditions. Milk production and composition were recorded monthly from calving until 200d in milk. At 67d in milk, biopsies were sampled from the rear quarter of the udder, their mammary epithelial cell content was evaluated, and mammary cell renewal, RNA, and DNA were then analyzed in relevant samples. The results showed that milk production did not differ significantly between cloned and noncloned cows, but milk protein and fat contents were less variable in cloned cows. Furthermore, milk fat yield and contents were lower in cloned cows during early lactation. At around 67 DIM, milk fat and protein yields, as well as milk fat, protein, and lactose contents, were also lower in cloned cows. These lower yields could be linked to the higher apoptotic rate observed in cloned cows. Apoptosis is triggered by insulin-like factor growth binding protein 5 (IGFBP5) and plasminogen activator inhibitor (PAI), which both interact with CSN1S2. During our experiments, CSN1S2 transcript levels were lower in the mammary gland of cloned cows. The mammary cell apoptotic rate observed in cloned cows may have been related to the higher levels of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) transcripts, coding for products that maintain the epigenetic status of cells. We conclude, therefore, that milk production in cloned cows differs slightly from that of noncloned cows. These differences may be due, in part, to a higher incidence of subclinical mastitis. They were associated with differences in cell apoptosis and linked to variations in DNMT1 mRNA. However, milk protein and fat contents were more similar among cloned cows than among noncloned cows.

Interface effects and the evolution of ferromagnetism in La2/3Sr1/3MnO3 ultrathin films.

Pulse laser deposited La2/3Sr1/3MnO3 ultrathin films on SrTiO3 substrates were characterized by polar and longitudinal Kerr magneto-optical spectroscopy. Experimental data were confronted with theoretical simulations based on the transfer matrix formalism. An excellent agreement was achieved for a 10.7 nm thick film, while a distinction in the Kerr effect amplitudes was obtained for a 5 nm thick film. This demonstrated the suppression of ferromagnetism due to the layer/substrate interface effects. A revised, depth-sensitive theoretical model with monolayer resolution described the experimental data well, and provided clear cross-section information about the evolution of ferromagnetism inside the film. It was found that the full restoration of the double-exchange mechanism, responsible for the ferromagnetic ordering in La2/3Sr1/3MnO3, occurs within the first nine monolayers of the film. Moreover, all the studied films exhibited magneto-optical properties similar to bulk crystals and thick films. This confirmed a fully developed perovskite structure down to 5 nm.

A euglycaemic/non-diabetic perinatal environment does not alleviate early beta cell maldevelopment and type 2 diabetes risk in the GK/Par rat model.

AIMS/HYPOTHESIS: We used the GK/Par rat, a spontaneous model of type 2 diabetes with early defective beta cell neogenesis, to determine whether the development of GK/Par offspring in a non-diabetic intrauterine/postnatal environment would prevent the alteration of fetal beta cell mass (BCM) and ultimately decrease the risk of diabetes later in adult life. METHODS: We used an embryo-transfer approach, with fertilised GK/Par ovocytes (oGK) being transferred into pregnant Wistar (W) or GK/Par females (pW and pGK). Offspring were phenotyped at fetal age E18.5 and at 10 weeks of age, for BCM, expression of genes of pancreatic progenitor cell regulators (Igf2, Igf1r, Sox9, Pdx1 and Ngn3), glucose tolerance and insulin secretion. RESULTS: (1) Alterations in neogenesis markers/regulators and BCM were as severe in the oGK/pW E18.5 fetuses as in the oGK/pGK group. (2) Adult offspring from oGK transfers into GK (oGK/pGK/sGK) had the expected diabetic phenotype compared with unmanipulated GK rats. (3) Adult offspring from oGK reared in pW mothers and milked by GK foster mothers had reduced BCM, basal hyperglycaemia, glucose intolerance and low insulin, to an extent similar to that of oGK/pGK/sGK offspring. (4) In adult offspring from oGK transferred into pW mothers and milked by their W mothers (oGK/pW/sW), the phenotype was similar to that in oGK/pGK/sGK or oGK/pW/sGK offspring. CONCLUSIONS/INTERPRETATION: These data support the conclusion that early BCM alteration and subsequent diabetes risk in the GK/Par model are not removed despite normalisation of the prenatal and postnatal environments, either isolated or combined.

Trends in prescriptions for glaucoma and ocular hypertension in France between 2014 and 2019.

PURPOSE: To explore the national trend in prescriptions for glaucoma and ocular hypertension (OHT) in France between 2014 and 2019. METHODS: This is a retrospective descriptive study based on prescription data from the Primary Health Insurance Fund databases. All patients with a social security number who received one or more glaucoma/OHT prescriptions between 2014 and 2019 were identified. Figures for 2020 are not yet available as of the date of submission of this article. Demographic characteristics from Common Classification of Medical Acts information and from National Institute of Statistics and Economic Studies were analyzed. The data analysis was carried out using the R version 3.6.2.software from the available databases of the Information Systems Medicalization Program. RESULTS: Our results suggest an increase in the number of patients treated with glaucoma drugs, which cannot be explained simply by demographic growth. There is also a change in drug prescription habits, both in the class of medication used and in the use of fixed combinations. We also note the increasing use of SLT (Selective Laser Trabeculoplasty), a relatively newer tool in the therapeutic arsenal. Over the same time period, demographic characteristics remained stable; age and sex distribution for each year remained constant. In addition, the phenomenon of poor therapeutic compliance, which we attempted to explore, remained stable. DISCUSSION: This study updates the French epidemiologic data available on prescriptions for glaucoma and ocular hypertension, a true public health concern. CONCLUSION: On the one hand, prescribing practices have evolved over the study period. On the other hand, the number of patients treated has increased faster than the growth of the French population over the same period. These findings are consistent with trends observed in previous studies.

Revealing the dynamics of gene expression during embryonic genome activation and first differentiation in the rabbit embryo with a dedicated array screening.

Early mammalian development is characterized by extensive changes in nuclear functions that result from epigenetic modifications of the newly formed embryonic genome. While the first embryonic cells are totipotent, this status spans only a few cell cycles. At the blastocyst stage, the embryo already contains differentiated trophectoderm cells and pluripotent inner cell mass cells. Concomitantly, the embryonic genome becomes progressively transcriptionally active. During this unique period of development, the gene expression pattern has been mainly characterized in the mouse, in which embryonic genome activation (EGA) spans a single cell cycle after abrupt epigenetic modifications. To further characterize this period, we chose to analyze it in the rabbit, in which, as in most mammals, EGA is more progressive and occurs closer to the first cell differentiation events. In this species, for which no transcriptomic arrays were available, we focused on genes expressed at EGA and first differentiation and established a 2,000-gene dedicated cDNA array. Screening this with pre-EGA, early post-EGA, and blastocyst embryos divided genes into seven clusters of expression according to their regulation during this period and revealed their dynamics of expression during EGA and first differentiation. Our results point to transient properties of embryo transcriptome at EGA, due not only to the transition between maternal and embryonic transcripts but also to the transient expression of a subset of embryonic genes whose functions remained largely uncharacterized. They also provide a first view of the functional consequences of the changes in gene expression program.

Retrotransposon expression as a defining event of genome reprogramming in fertilized and cloned bovine embryos.

Genome reprogramming is the ability of a nucleus to modify its epigenetic characteristics and gene expression pattern when placed in a new environment. Low efficiency of mammalian cloning is attributed to the incomplete and aberrant nature of genome reprogramming after somatic cell nuclear transfer (SCNT) in oocytes. To date, the aspects of genome reprogramming critical for full-term development after SCNT remain poorly understood. To identify the key elements of this process, changes in gene expression during maternal-to-embryonic transition in normal bovine embryos and changes in gene expression between donor cells and SCNT embryos were compared using a new cDNA array dedicated to embryonic genome transcriptional activation in the bovine. Three groups of transcripts were mostly affected during somatic reprogramming: endogenous terminal repeat (LTR) retrotransposons and mitochondrial transcripts were up-regulated, while genes encoding ribosomal proteins were downregulated. These unexpected data demonstrate specific categories of transcripts most sensitive to somatic reprogramming and likely affecting viability of SCNT embryos. Importantly, massive transcriptional activation of LTR retrotransposons resulted in similar levels of their transcripts in SCNT and fertilized embryos. Taken together, these results open a new avenue in the quest to understand nuclear reprogramming driven by oocyte cytoplasm.

Glaucoma progression analysis by Spectral-Domain Optical Coherence Tomography (SD-OCT).

Spectral domain optical coherence tomography (SD-OCT) provides an objective quantification of the lesions of various target tissue structures in glaucoma, with unprecedented resolution, which has now demonstrated its interest in controlling the progression of glaucomatous neuropathy, from early stages to late stages. A certain number of well-established proofs state that a progressive modification in OCT is a common predictor of functional loss, and that patients with rapid OCT changes have an increased risk of developing glaucomatous scotoma. Follow-up of the progression goes through three stages. It consists first of all in detecting the evolution of damage to the retinal nerve fiber layer (RNFL), then that of the macular ganglion cell complex (GCC), in order to better define this progression of the damage to the target structures and, thirdly, to complete its analysis by integrating it with the analysis of the functional impairment. We note today that there is a greater risk of developing a future functional deficit of the visual field in subjects with a RNFL loss slope greater than -1/year, for all clinical stages of glaucoma. The characteristics of GCC progression are much better specified. Often earlier than that of the progress of the thinning of the RNFL and much faster in the subjects considered as "progressors", its cartography is better defined, with a particular interest for the follow-up of diversion maps and "wide field" acquisitions offering better visibility of deficits and their progression. To date, a certain number of suspicious indicators of short-term progress can be retained, highlighting the essential precaution of having two or more basic measures and a confirmation of the change on at least one new OCT acquisition. Finally, if the interpretation of the progression must always be based on clinical examination data, and the macula in particular, it remains crucial to confront the progression of the RNFL with that of the GCC and with that of the visual field.

Phylogenetic position of the saola (Pseudoryx nghetinhensis) inferred from cytogenetic analysis of eleven species of Bovidae.

Previous morphological and molecular analyses failed to resolve the phylogenetic position of the critically endangered saola (Pseudoryx nghetinhensis) with respect to its placement in Bovina (cattle, bison, and yak) or Bubalina (Asian and African buffaloes). In the present study, G- and C-banding, Ag-staining and FISH with 28S and telomeric probes was undertaken for 17 bovid species. An analysis of these data allowed us to identify 49 structural rearrangements that included autosomes, gonosomes and 17 different NOR sites. The combined data set was subjected to a cladistic analysis aimed at: (i) providing new insights on phylogenetic relationships of the saola and other species within the subfamily Bovinae, and (ii) testing the suitability of different classes of chromosomal characters for phylogenetic reconstruction of the family Bovidae. The study revealed that nucleolar organizing regions (NORs) are phylogenetically informative. It was shown that at least one, or sometimes two of these characters punctuate divergences that include nodes that are the most basal in the tree, to those that are the most recent. In this context, the shared presence of three NORs in saola and species of Syncerus and Bubalus strongly suggests the saola's placement within the subtribe Bubalina. This contrasts with Robertsonian rearrangements which are informative only at the generic level. These findings suggest that NORs are an important and frequently overlooked source of additional phylogenetic information within the Bovidae that may also have applicability at higher taxonomic levels, possibly even for Pecora.

In utero characterisation of fetal growth by ultrasound scanning in the rabbit.

Fetal development is an important factor influencing the susceptibility of adults to metabolic diseases. In order to study the influence of fetal growth on further development in animal models like the rabbit, methods of measurement of fetal and placental size and viability must be established and validated. In this study, 42 New Zealand does bred naturally (N=12) or transferred with in vivo produced embryos (2, 4 or 6 embryos/doe) have been scanned every 2-3 days with a 7.5 MHz transabdominal probe from Day 7 post-coitum until term to measure fetal and placental growth. Vesicle, placental, fetal length and head size have thus been determined according to number of fetuses and time. In late gestation, the fetuses that were transferred in limited numbers to the uterus of does were significantly larger than their natural breeding counterparts probably due to reduced litter size.

Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos.

Full-term development has now been achieved in several mammalian species by transfer of somatic nuclei into enucleated oocytes [1, 2]. Although a high proportion of such reconstructed embryos can evolve until the blastocyst stage, only a few percent develop into live offspring, which often exhibit developmental abnormalities [3, 4]. Regulatory epigenetic markers such as DNA methylation are imposed on embryonic cells as normal development proceeds, creating differentiated cell states. Cloned embryos require the erasure of their somatic epigenetic markers so as to regain a totipotent state [5]. Here we report on differences in the dynamics of chromosome methylation between cloned and normal bovine embryos before implantation. We show that cloned embryos fail to reproduce distinguishable parental-chromosome methylation patterns after fusion and maintain their somatic pattern during subsequent stages, mainly by a highly reduced efficiency of the passive demethylation process. Surprisingly, chromosomes appear constantly undermethylated on euchromatin in morulae and blastocysts, while centromeric heterochromatin remains more methylated than that of normal embryos. We propose that the abnormal time-dependent methylation events spanning the preimplantation development of clones may significantly interfere with the epigenetic reprogramming, contributing to the high incidence of physiological anomalies occurring later during pregnancy or after clone birth.

Scaffold attachment regions stimulate HSP70.1 expression in mouse preimplantation embryos but not in differentiated tissues.

Eukaryotic interphase chromatin is thought to be organized into topologically discrete, independent domains acting as units upon which differential patterns of gene expression are established. Sequences which attach chromatin to in vitro preparations of a nucleoprotein matrix (scaffold attachment regions [SARs]) may act as domain boundaries, but their role remains poorly defined compared with those of other elements such as locus control regions. We have produced mice homozygous for a transgene which is transcribed as early as the activation of the embryonic genome at the two-cell stage and which is expressed ubiquitously in a number of differentiated tissues. Transgenic lines were generated in the presence or absence of flanking SAR sequences, creating an original model which enabled us to examine the effects of these elements at different developmental stages. In the preimplantation mouse embryo, flanking SARs stimulated transgene expression in a copy-dependent manner. In contrast, in the differentiated tissues of newborn and adult mice, no significant SAR-dependent increase in transgene expression was found, correlation with copy number was lost, and position effects were observed. These results suggest a limited capacity of SARs to act as insulating elements but are consistent with a proposed model of SAR-mediated chromatin opening and closing.

Evidence for the involvement of mouse heat shock factor 1 in the atypical expression of the HSP70.1 heat shock gene during mouse zygotic genome activation.

The mouse HSP70.1 gene, which codes for a heat shock protein (hsp70), is highly transcribed at the onset of zygotic genome activation (ZGA). This expression, which occurs in the absence of stress, is then repressed. It has been claimed that this gene does not exhibit a stress response until the blastocyst stage. The promoter of HSP70.1 contains four heat shock element (HSE) boxes which are the binding sites of heat shock transcription factors (HSF). We have been studying the presence and localization of the mouse HSFs, mHSF1 and mHSF2, at different stages of embryo development. We show that mHSF1 is already present at the one-cell stage and concentrated in the nucleus. Moreover, by mutagenizing HSE sequences and performing competition experiments (in transgenic embryos with the HSP70.1 promoter inserted before a reporter gene), we show that, in contrast with previous findings, HSE boxes are involved in this spontaneous activation. Therefore, we suggest that HSF1 and HSE are important in this transient expression at the two-cell stage and that the absence of typical inducibility at this early stage of development results mainly from the high level of spontaneous transcription of this gene during the ZGA.

Nuclear reprogramming and pluripotency of embryonic cells: Application to the isolation of embryonic stem cells in farm animals.

Despite their biological and biotechnological interest, pluripotent embryonic stem cell lines (ES cells) have been isolated from cultured embryos only in a very limited number of mammalian species. Here we review the main molecular mechanisms that have been shown in mouse or primates to regulate the maintenance of pluripotency in vitro. We describe the main signaling pathways that participate in the self-renewal of ES cells and provide an outlook on the epigenetic associated mechanisms. We also propose a practical approach to stem cell differentiation that examines the relationships between the genotype of embryos and their culture conditions and consider nuclear reprogramming as a valuable approach in ES cell derivation in farm animals.

Dairy cattle exploratory and social behaviors: is there an effect of cloning?

While an increasing number of animals are produced by means of somatic cloning, behavioral studies on cloned animals are still rare. The aim of this study was to investigate whether the somatic cloning procedure has an influence on locomotion, exploratory, vocal and social behaviors of heifers. Ten heifers were used in the present study. Five of them were cloned heifers derived from somatic cells of three different Prim'Holstein cows and five others were same-age control heifers produced by artificial insemination. In addition to observations of social behaviors in the stable group, each animal was placed individually for a short time in an unfamiliar environment. Our results failed to show any statistical differences between clones and their controls both in frequencies of agonistic and non-agonistic behaviors. However, cloned heifers showed significantly more non-agonistic and less agonistic behaviors towards other cloned partners than towards control ones. This result also stood for control heifers. As far as their Hierarchical Index was concerned, three cloned heifers were highest ranking and two others lowest ranking. In this herd, social dominance appeared to be linked to body weight and age rather than to a cloning effect. In an unfamiliar environment, cloned and control subjects exhibited the same level of locomotion and vocalization. However, cloned heifers showed more exploratory behaviors than did control ones. This difference could be due to environmental factors during the postnatal period rather than to cloning.

Assessing the quality of products from cloned cattle: an integrative approach.

Scientific expertise was developed during a 3-year study to evaluate a large number of bovine female clones (n=37; from 4 to 36 months of age) and their products through a multidisciplinary approach and compare them to non-cloned breed, age and sex-matched contemporary control animals (n=38) maintained under the same conditions at the same experimental farm of INRA. In clone and control groups, most parameters measured for health and development of the animals as well as evaluation of milk and meat products were within the normal range for the breed. The strict comparison between cloned animals and controls allowed us to detect slight significant differences between the two groups. Cloned heifers reached puberty significantly later (+62 days) and at higher body weight (+56kg) than controls. There were slight differences in antigen-specific induced proliferation of lymphocytes after vaccination with ovalbumin before 10 months of age, but responses were normal responses in older animals. There were differences in the fatty acid (FA) composition of milk and muscle arising from two families of clones, suggesting a possible deviation in lipid metabolism as assessed by higher Delta-9 desaturase activity indices in both milk and muscle from clones compared to controls. Nutritional evaluation of milk and meat using the rat model did not reveal any difference between products derived from clones versus controls.

[Reliability and reproducibility of introcular pressure (IOP) measurement with the Icare® Home rebound tonometer (model TA022) and comparison with Goldmann applanation tonometer in glaucoma patients]. / Fiabilité et reproductibilité des mesures de la pression intraoculaire par le tonomètre Icare® Home (modèle TA022) et comparaison avec les mesures au tonomètre à aplanation de Goldmann chez des patients glaucomateux.

PURPOSE: The Icare® Home tonometer is a new rebound tonometer, developed for intraocular pressure (IOP) self-monitoring. The main objective of our study was to evaluate the reliability and reproducibility of measurements taken with the Icare® Home tonometer in glaucoma patients compared to the Goldmann applanation tonometer. A secondary objective was to investigate factors that could influence the reproducibility of these measurements. MATERIALS AND METHODS: Fifty-two glaucoma patients were included in this prospective, non-randomized, monocentric study. IOP measurements were performed on the right eye and then on the left eye in the following order (3 measurements of IOP for each method): air tonometer (T-Air), Icare® Home tonometer by the patient (RT-P), Icare® Home tonometer by an ophthalmologist (RT-O), Goldmann applanation tonometer (GAT). RESULTS: Forty-four patients (85%) managed to take their IOP on both eyes with the Icare® Home tonometer. Mean IOPs were 14.35±3.93mmHg (T-Air), 13.43±4.65mmHg (RT-P), 14.13±4.29mmHg (RT-O), 14.74±3.84mmHg (GAT). The intraclass correlation indices (ICC) on the 3 repeated IOP measurements were 0.924, 0.872, 0.947 and 0.957, respectively. Bland-Altman analysis found a mean difference (bias) between GAT and RT-P, between GAT and RT-O, and between RT-O and RT-P, respectively, of 1.31, 0.61 and 0.70mmHg, with a 95% confidence interval of -3.34 to 5.96, -3.91 to 5.14 and -3.44 to 4.84mmHg, respectively. The reproducibility of the measurements taken with the Icare® Home tonometer did not vary according to corneal thickness or age of the patients. CONCLUSION: The Icare® Home tonometer provides reliable and reproducible IOP values in glaucoma patients, although it appears to slightly underestimate the IOP measurements compared to the Goldmann applanation tonometer.

[Selective laser trabeculoplasty: Effect of number of preoperative topical glaucoma medications on pressure lowering and success rate]. / Trabéculoplastie sélective au laser : effet du nombre de traitements antiglaucomateux topiques préopératoires sur la baisse pressionnelle et le taux de succès.

PURPOSE: To evaluate the lowering of intraocular pressure (IOP) one year after SLT and to assess if differences are related to number of pre-SLT topical treatments in ocular hypertension (OHT) and primary open angle glaucoma (POAG) patients. METHODS: Retrospective review of 106 eyes of 13 OHT and 93 POAG patients treated by SLT for insufficient IOP control, allergy, discomfort or non-compliance to glaucoma medications, excluding patients with less than 1 year of follow-up after SLT. IOP was measured by applanation before and at 1, 6 and 12 months after SLT. RESULTS: Hundred and six eyes untreated (n=13), or treated with one (n=25), two (n=40) or three or more (n=28) glaucoma medications were included. Mean IOP decreased from 19.4±3.6mmHg preoperatively to 15.7±3.1mmHg at 12 months, which corresponds to an average decrease of 18.8%. At 1 year, 62.2% (n=66) were responders (IOP reduction≥3mmHg): 92.3% without medications (n=12), 68% with one (n=17), 57.5% with two (n=23) and 50% with three or more medications (n=14). Their average IOP decreased from 20.7±3.4 to 15.2±2.9mmHg (26.6%), respectively from 20.8±2.6 to 15.8±3.2 (25%) without medications, 20.6±3.2 to 14.9±3.7 (27.3%) with one, 20.8±4.1 to 15.5±3.3 (25.1%) with two and 20.7±3.2 to 14.4±2.4mmHg (29.7%) with three medications. CONCLUSIONS: The number of responders seems to be greater in OHT and POAG patients without or with few glaucoma medications, but the IOP reduction seems to be similar regardless of the number of glaucoma medications.

[Post-traumatic endophthalmitis]. / Les endophtalmies post-traumatiques.

Post-traumatic endophthalmitis is a rare but serious complication of open globe injury, representing a major turning point for the patient's visual prognosis. Risk factors for this complication are lens capsule rupture, an intraocular foreign body, type of eye trauma and especially a delay in initial management of the trauma. Although Staphylococcus epidermidis is the most common organism, as in postoperative acute endophthalmitis, other microorganisms are more frequently represented and the multi-microbial involvement is common. The diagnosis can be difficult in the presence of inflammatory signs of trauma. Aside from rapid globe repair, neither preventive nor curative treatment have been well delineated. The class of antibiotics, the dosage, route of administration, as well as surgical treatment by vitrectomy remain topics of discussion.

[Disc-fovea angle adjustment for peripallary retinal nerve fiber layer analysis by a spectral domain optical coherence tomography. Preliminary study]. / Analyse des fibres nerveuses rétiniennes péripapillaires ajustée selon l'angle papillo-maculaire en tomographie par cohérence optique en domaine spectral. Étude préliminaire.

INTRODUCTION: Circumpapillary retinal nerve fiber layer (cpRNFL) analysis by spectral domain optical coherence tomography (SD-OCT) has become essential for the assessment of glaucoma patients. The foveal projection is conventionally below the disc plane, creating an angle with the horizontal meridian, the disc-fovea angle. The purpose of this study is to evaluate the role of adjustment of cpRNFL analysis based on this angle. MATERIALS AND METHODS: This study concerns 40 control eyes and 55 eyes affected with and followed for primary open angle glaucoma (POAG). After precise localization of the optic disc center and the axis connecting it to the center of the fovea, a circular peripapillary scan is performed with Spectralis OCT (Heidelberg Engineering, Germany). The mean thickness in each of six papillary sectors and the global mean thickness of the cpRNFL were evaluated. The ROC (receiver operating characteristic) analysis evaluated the diagnostic capabilities of the various sectors before and after adjustment of the analysis for the disc-fovea angle. RESULTS: The disc-fovea angle was not different between the two groups (-7.0 ± 1.2° for controls vs. -6.6 ± 1.2° for POAG, P=0.70). There is a significant variance of this angle in both groups (the angle varies in the control group between -22.5° to +1.8° and in the POAG group between -18° to +2.4°). The global mean and inferior temporal (IT) thickness of the cpRNFL show the best diagnostic performance. Adjustment for disc-fovea angle does not increase the diagnostic accuracy of the various sectors analyzed. Although there is an increase in the area under the curve for the IT sector after adjustment, it is not statistically significant (0.910 ± 0.056 vs. 0.936 ± 0.045, P=0.06). CONCLUSIONS: There is a significant variation in disc-fovea angle. In this study, accounting for it does not significantly improve the diagnostic capabilities of cpRNFL in patients with POAG.

SSH adequacy to preimplantation mammalian development: scarce specific transcripts cloning despite irregular normalisation.

BACKGROUND: SSH has emerged as a widely used technology to identify genes that are differentially regulated between two biological situations. Because it includes a normalisation step, it is used for preference to clone low abundance differentially expressed transcripts. It does not require previous sequence knowledge and may start from PCR amplified cDNAs. It is thus particularly well suited to biological situations where specific genes are expressed and tiny amounts of RNA are available. This is the case during early mammalian embryo development. In this field, few differentially expressed genes have been characterized from SSH libraries, but an overall assessment of the quality of SSH libraries is still required. Because we are interested in the more systematic establishment of SSH libraries from early embryos, we have developed a simple and reliable strategy based on reporter transcript follow-up to check SSH library quality and repeatability when starting with small amounts of RNA. RESULTS: Four independent subtracted libraries were constructed. They aimed to analyze key events in the preimplantation development of rabbit and bovine embryos. The performance of the SSH procedure was assessed through the large-scale screening of thousands of clones from each library for exogenous reporter transcripts mimicking either tester specific or tester/driver common transcripts. Our results show that abundant transcripts escape normalisation which is only efficient for rare and moderately abundant transcripts. Sequencing 1600 clones from one of the libraries confirmed and extended our results to endogenous transcripts and demonstrated that some very abundant transcripts common to tester and driver escaped subtraction. Nonetheless, the four libraries were greatly enriched in clones encoding for very rare (0.0005% of mRNAs) tester-specific transcripts. CONCLUSION: The close agreement between our hybridization and sequencing results shows that the addition and follow-up of exogenous reporter transcripts provides an easy and reliable means to check SSH performance. Despite some cases of irregular normalisation and subtraction failure, we have shown that SSH repeatedly enriches the libraries in very rare, tester-specific transcripts, and can thus be considered as a powerful tool to investigate situations where small amounts of biological material are available, such as during early mammalian development.