Drosophila ovary, the germline-specific Orb protein mediates the translational activation of a variety of mRNAs localized in the oocyte. One of the Orb target mRNAs is orb itself, and this autoregulatory activity ensures that Orb proteins specifically accumulate in the developing oocyte. Orb is an RNA-binding protein and is a member of the cytoplasmic polyadenylation element binding (CPEB) protein family. We report here that Cup forms a complex in vivo with Orb. We also show that cup negatively regulates orb and is required to block the precocious activation of the orb positive autoregulatory loop. In cup mutant ovaries, high levels of Orb accumulate in the nurse cells, leading to what appears to be a failure in oocyte specification as a number of oocyte markers inappropriately accumulate in nurse cells. In addition, while orb mRNA is mislocalized and destabilized, a longer poly(A) tail is maintained than in wild type ovaries. Analysis of Orb phosphoisoforms reveals that loss of cup leads to the accumulation of hyperphosphorylated Orb, suggesting that an important function of cup in orb-dependent mRNA localization pathways is to impede Orb activation.Translational regulation of localized mRNAs is essential for patterning and axes determination in many organisms. In the  Drosophila germline-specific protein Orb In eukaryotic cells, cytoplasmic polyadenylation is used to activate the on-site translation of localized mRNAs. Polyadenylation is thought to depend upon two key elements in the 3′ UTR of the mRNA. The first is the AAUAAA motif, which is bound by the cleavage and polyadenylation specificity factor (CPSF) while the second is the U-rich cytoplasmic polyadenylation element (CPE) which is bound by the cytoplasmic polyadenylation element binding protein (CPEB) orb alleles indicates that the formation of the 16-cell cyst, the specification of the oocyte and the proper expression of the TGF-α signaling molecule Gurken (Grk) at the posterior of pre-vitellogenic egg chambers requires orb activity melorb, these early steps in oogenesis appear normal; however, the specification of both the anterior-posterior (AP) and dorsal ventral (DV) polarity axes in vitellogenic egg chambers is disrupted. Orb protein is thought to function in AP axis specification by binding to oskar (osk) mRNA after it is localized to the posterior pole of the oocyte and activating its translation by a mechanism involving polyadenylation grk mRNA at the dorsal-anterior corner of the oocyte. In melorb ovaries, grk mRNA is mislocalized and little or no Grk protein is produced. Like other mutations that disrupt grk signaling, melorb eggs have ventralized chorions that either have fused or lack dorsal respiratory appendages Phenotypic analysis of strong orb mRNAs to sites in the oocyte cortex and to promote their on-site translation. As is the case for osk mRNA, it is thought to act by binding to target sequences in the orb 3′ UTR and activating polyadenylation orb activity. Since orb mRNA is synthesized in the nurse cells, and must be transported through the nurse cells into the oocyte and then localized within the oocyte to the cortex, there must be mechanisms in place that prevent the precocious activation of the orb positive autoregulatory loop. Previous studies have shown that the Drosophila Fragile X mental retardation protein dFMR1 downregulates orb mRNA translation in nurse cells dfmr1 mutations on Orb protein expression, and on oogenesis in general, are relatively modest and it seems likely that other factors may play more central roles in blocking the premature activation of the orb positive autoregulatory loop.Another Orb regulatory target is its own mRNA. Orb is required to localize orb positive autoregulatory loop is fs(2)cup (cup). cup was discovered in a screen for female sterile mutations orb targets, oskOne candidate for a gene that prevents the precocious activation of the osk and orb that contain sequence motifs recognized by the Bruno protein. Cup is thought to inhibit the translation of these mRNAs by binding to eIF4E and sequestering it from interacting with eIF4G osk mRNA is prematurely upregulated in several cup hypomorphic mutant combinations and Osk protein can be detected in stage 6–7 egg chambers osk mRNA translation is also observed in a cup mutant, Δ212cup, which lacks the canonical high affinity eIF4E binding motif Δ212 protein with eIF4E is expected to be compromised, it is only a very weak Class III allele. This observation suggests that the regulatory activities of cup during oogenesis are likely to include other functions besides sequestering the eIF4E translation factor.These interactions involve different domains of the Cup protein. For example, Cup-eIF4E interactions are mediated by a canonical and a non-canonical eIF4E binding motif in the Cup N-terminus, while the C-terminal end of the Cup protein mediates interactions with Bruno cup gene is to prevent the premature activation of the orb positive autoregulatory loop. In wild type ovaries, the orb autoregulatory loop is activated in the oocyte ensuring that high levels of Orb protein accumulate in the compartment where its activity is required. In contrast, in cup mutants the autoregulatory appears to be precociously activated and high levels of Orb protein accumulate in the nurse cells. Our data suggest that Cup employs at least two different though likely overlapping mechanisms to prevent the premature activation of the orb autoregulatory loop. The first is to limit the poly(A) tail length of orb mRNAs. In cup mutants orb mRNAs have longer poly(A) tails than in wild type. The second is to limit the accumulation of hyperphosphorylated (activated) Orb protein isoforms. In wild type ovaries, there are two Orb isoform populations, hypo- and hyperphosphorylated, that differ in the extent of phosphorylation and in their activity cup ovaries there is a shift in the isoform distribution and most the Orb protein is hyperphosphorylated. In addition to these effects on orb expression and post-translational modifications, we find that cup is required for the proper localization and stability of orb mRNA.In this paper, we present evidence that one of the other functions of the in vivo. For this purpose, we tested candidate proteins that were detected in a previous mass spectrometry analysis on Orb- immunoprecipitated wild type ovary extracts Drosophila Gld2-homolog Wisp, five predicted RNA helicases, multiple RNA binding proteins, components of the siRNA machinery and proteins involved in decapping and RNA turnover like Me31B, Trailer Hitch, Not4, Enhancer of decapping and Bicaudal-C. There were also several proteins  implicated in mRNA localization in Drosophila ovaries. We also found Cup and one of its known partners, the initiation factor eIF4E To identify components of the machinery that regulates Orb activity or localization, we searched for proteins that associate with Orb To confirm the physical association between Cup and Orb, we immunoprecipitated ovarian extracts with either Cup or Orb antibodies and then analyzed the immunoprecipitates by Western blotting. Orb, but not HA antibody is able to pull-down Cup protein . The totIn the converse experiment, we found that Cup antibody co-immunoprecipitates Orb protein . This asorb and cup is functionally important, we tested for genetic interactions. orb is weakly haploinsufficient for its activity in the establishment of DV polarity in the developing egg chamber, and 5–10% of the eggs laid by females heterozygous for the null allele 343orb have ventralized chorions due to a defect in the production of the Grk ligand. It is possible to exacerbate this haploinsufficiency by introducing a dominant negative transgene HD19G. HD19G expresses an hybrid mRNA that contains β-galactosidase protein coding sequence fused to the 3′ UTR of orb mRNA orb 3′ UTR in the chimeric mRNA competes with the 3′ UTR of the endogenous orb mRNA for Orb protein binding. This interferes with orb autoregulation and downregulates Orb protein expression. When this transgene is combined with the orb null allele, 343orb, 20–30% of the eggs laid by transgene-heterozygous mutant females are ventralized. The somewhat higher frequency of DV defects in the 343/+HD19G orb background as compared to 343/+orb is useful in that it allows a more reliable detection of mutations that dominantly suppress the DV polarity defects induced by compromising orb activity. It is expected that dominant suppressors will correspond to genes that function to repress or reduce orb activity and/or grk signaling. In contrast, mutations that dominantly enhance the 343/+HD19G orb DV polarity defects are expected to correspond to genes that are required to promote orb activity and/or grk signaling.To determine if the physical association between trans-heterozygous for a recombinant 343HD19G orb chromosome and various cup mutant alleles by crossing 343/TM3SerHD19G orb females and cup/CyO males to test for genetic interactions. Using this strategy, we analyzed 5 different cup alleles, 1cup, 3cup, 6cup, 8cup, and 1355cup3cup, 6cup and 8cup are strong loss-of-function mutations and these Class I alleles exhibit a pre-vitellogenesis oogenesis arrest. 1cup is a Class II allele and ovarian development arrests around stage 9. Finally, 1355cup, which has a P-element insertion in the 5′ UTR that downregulates its expression, is a Class III allele and arrests during vitellogenesis.We generated females 343/+HD19G orb females can be suppressed by reducing cup activity. At 25°C, the frequency of DV defects in eggs from 343/+HD19G orb; −/+cup females of severe alleles is, with one exception (3cup), about half that seen for 343/+HD19G orb females. The suppression is even more pronounced at 18°C. At this temperature, close to 30% of the eggs laid by 343/+HD19G orb females are ventralized while in the cup trans-heterozygotes the frequency of egg shell polarity defects drops to less than 10%.As illustrated in Δ212cup, which lacks the canonical eIF4E interaction domain. Unlike stronger cup alleles, which arrest oogenesis and can substantially alter the pattern of orb expression (see below), oogenesis is comparatively normal in Δ212cup and defective eggs are produced 343/+HD19G orb females. Thus, the canonical eIF4E interaction domain is unlikely to be critical for genetic interactions between cup and orb at least in the DV polarity pathway.In a separate experiment (not shown) we also tested the very weak Class III allele, cup and orb. Moreover, given what is already known about the regulatory activities of the Cup protein in other contexts cup negatively regulates orb expression and/or activity. In this case, we would expect to find that Orb protein levels are increased in cup mutant ovaries. To test this prediction, we probed Western blots of ovary extracts prepared from female homozygous mutants for three different cup alleles, 8cup, 3cup and 1355cup with Orb antibodies. We anticipated that Orb levels would be elevated in the mutants if cup functions as a “general” negative regulator of orb activity (because of Orb autoregulation), and that in this case the effects on Orb accumulation should be proportional to the strength of the cup allele. However, the results were anomalous , these high levels of protein do not persist. Instead, we find that Orb levels begin to drop as the chambers age and in older chambers (based on their location in the string of chambers) there is often little or no Orb present , but instead is distributed at approximately the same level in many if not all of the germ cells in the chamber  3cup chambers, this problem is usually not observed in the Class III 1355cup mutant where the pattern of Orb accumulation appears to be comparatively normal in early stages 1–5 1355cup chambers . These findings indicate that orb mRNA is inappropriately translated in the nurse cells beginning during mid-oogenesis (stages 6–7) in 1355cup mutant ovarioles. Interestingly, precocious expression of Osk is observed around the same time in other Class III cup mutants While the oocyte appears to be mis-specified in chambers . Howeverse cells  and Orb cup mutations on orb, we examined the pattern of orb mRNA accumulation in cup ovaries. In wild type ovaries, orb mRNA localizes preferentially to the oocyte soon after the 16-cell cyst is formed 8cup and 3cup ovaries, orb message is distributed more or less uniformly in the germ cells of the egg chamber; however, many of these chambers have a single cell which has somewhat higher levels of mRNA . Besides mislocalized mRNA, there are also chambers that have little or no orb message (see arrowheads in panel C). As was seen for Orb protein, the distribution of orb mRNA in 1355cup more closely resembles wild type than either of the stronger alleles (panel H). However, even in this mutant the relative proportion of message in the nurse cells appears higher than in wild type chambers of similar stages (panel I).To further investigate the effects of cup activity is required not only to regulate the translation of orb mRNA but also for its proper localization. In addition, since many chambers  have little orb mRNA, it would appear that cup is required either for the synthesis of orb mRNA, or to stabilize its accumulation. The latter possibility is supported by recent studies using a tissue culture system by Igreja and Izaurralde cup activity is also required for the normal accumulation of orb mRNA, we used quantitative real-time PCR. Compared to a normalized wild type value of 1.0 (for pre-vitellogenic egg chambers) the level of orb mRNA in 8cup, 3cup and 1355cup mutant ovaries is 0.28, 0.32 and 0.32 respectively . These results would be consistent with the tissue culture experiments and would argue that cup is also required for orb mRNA stability.These findings indicate that the orb mRNA, one of the other known Orb regulatory targets, osk mRNA, is mislocalized and prematurely translated in cup ovaries osk mRNA is also reduced in level when cup activity is compromised. To address this question, we used quantitative RT-PCR to estimate the relative abundance of osk mRNA in the three cup mutants. osk mRNA in the cup mutants than in wild type and that the extent of the reduction roughly corresponds to the severity of the allele. For example, 8cup, the most severe mutant had approximately 22% of the osk mRNA found in wild type ovaries. Since the amount of Osk protein detected in Western blots of extracts from the three cup mutants is close to that found in wild type (pre-vitellogenic) chambers  tail length orb mRNAs in nurse cells and promote their deadenylation. This would ensure that orb mRNAs remained repressed until they are localized in the oocyte. In cup mutants, orb mRNAs would have elongated poly(A) tails and this would increase their translational efficiency. A prediction of this model is that the poly(A) tails of orb mRNAs will be elongated in cup mutant ovaries. To determine whether this is true, we measured the lengths of orb mRNA poly(A) tails in wild type, 8cup and 3cup ovaries using the ligation-mediated poly(A)-tail assay orb mRNA poly(A) tails towards longer tails in both 8cup and 3cup ovaries (1335cup using the anchored poly(A)-tail assay  tails. While this would be expected to increase their translational efficiency cup alleles. The abnormally high levels of Orb protein seen in the nurse cells suggests that the orb autoregulatory loop is also prematurely activated in the cup mutants. We've previously shown that orb activity is regulated, at least in part, by phosphorylation and that phosphorylation depends upon Casein Kinase II (CK2) ck2 activity, there is a shift in the orb phosphoisoform profile towards the hypophosphorylated isoforms. Correlated with this shift, we find that orb functioning in both autoregulation and in grk signaling is disrupted. The two phosphoisoform populations are also found in quite distinct protein complexes that likely to differ in their activities. The hypophosphorylated proteins are in slowly sedimenting complexes and are associated with the translational Bruno repressor orb autoregulatory loop would be to upregulate Orb phosphorylation.The findings described above indicate that cup affects the Orb phosphorylation status, we separated the hypo- and hyperphosphorylated Orb isoforms in wild type and cup mutant ovaries on SDS-PAGE gels. Since cup ovaries arrest early in oogenesis, we compared the Orb protein in cup ovaries with wild type ovaries containing only pre-vitellogenic stages. As shown in cup mutants. In wild type ovaries, the rapidly migrating lower isoform is always more abundant than the upper isoform  in 8cup appear to be a good deal higher than in wild type. Fourth, while Orb protein levels are elevated, orb mRNAs levels are reduced in cup mutants compared to wild type. Since a similar reduction is observed for osk mRNA, it would appear that cup activity is needed either directly or indirectly to ensure the stability of at least two localized mRNAs. These unexpected effects of cup on mRNA stability would be consistent with recent studies in tissue culture cells, which have shown that tethering Cup protein to a reporter mRNAs stabilizes the mRNA even as it represses translation orb autoregulatory loop is inappropriately activated is further supported by the finding that there is a shift in the orb mRNA poly(A) profile towards longer poly(A) tails in cup mutants.The studies reported here indicate that the cup alleles also suggests that there may be problems in the specification or maintenance of oocyte identity. This possibility is supported by the fact that two other oocyte markers, Encore and orb mRNA, are also not properly localized. However, since we do see chambers in which these oocyte markers appear to be more highly concentrated in the presumptive oocyte, it seems likely that the loss of cup activity does not disrupt the initial specification of the oocyte, but rather affects the maintenance of oocyte identity. In this context, it is interesting to note that orb activity is required for the proper specification of the oocyte, and in strong orb mutants, oocyte identity is not properly established or maintained. Perhaps the presence of excessive amounts of Orb in the nurse cells of these cup mutant chambers might also compromise oocyte identity, but by a different mechanism. Instead of failing to express key oocyte determinants as in orb mutants, Orb would promote the premature expression of these determinants in the nurse cells.The abnormal pattern of Orb protein accumulation in the two strong orb autoregulatory loop becomes precociously activated in cup mutants? One factor that likely contributes to the premature activation of the orb autoregulatory loop is a failure to deadenylate orb mRNAs. Recent studies by Igreja and Izaurralde cup has a similar activity in the female germline, then it should directly antagonize Orb, which is thought to activate translation by promoting polyadenylation. A prediction of this model is that orb poly(A) tail lengths should increase when cup activity is reduced. Indeed, this is the case; we found that the poly(A) tails of orb mRNAs in cup mutants are elongated compared to wild type.An important question is why the orb mRNA translation efficiency, a simple change in translational efficiency would not fully account for the effects of cup mutations. In particular, one might expect to observe a phenotype similar to that seen in dfmr1 mutants where Orb protein levels are increased proportionally in both the nurse cells and the oocyte. However, in cup mutants there is a disproportionate increase in Orb protein levels in the nurse cells. This difference suggests that the orb autoregulatory loop is also precociously activated in nurse cells. In previous studies, we found that like the CPEB protein in Xenopus oocytes ck2 activity is partially compromised, there is a marked shift in the phosphoisoform profile towards the hypophosphorylated isoforms. Accompanying this shift in phosphoisoform profile, orb functions in DV polarity and orb autoregulation are disrupted. The results described here indicate that a shift in the opposite direction, i.e. towards the hyperphosphorylated isoforms occurs in cup mutant ovaries. Based on our previous work, such a shift in the phosphoisoform profile would be expected to upregulate orb activity. This change in phosphorylation status, like a failure in deadenylation, would be expected to contribute to the overexpression of Orb in cup mutant egg chambers. If this change in phosphorylation status were to occur in the nurse cells, it could lead to the precocious activation of the orb autoregulatory loop in the nurse cells and a disproportionate increase in the amount of Orb protein in these cells. Similarly, it could explain why the orb target osk mRNA is prematurely translated in cup mutants While an increase in poly(A) tail length would be expected to generally increase cup is a translational repressor, a plausible idea is that it indirectly downregulates phosphorylation by repressing the synthesis of CK2, some other kinase or the factors that control kinase activity. Alternatively (or in addition), the presence of Cup protein in Orb complexes could impede hyperphosphorylation. Consistent with this expectation, we were unable to detect Cup in Western blots of extracts from the two strongest alleles, 8cup and 3cup. 1355cup is thought to encode a wild type protein and Cup-Orb complexes can be detected in this mutant (data not shown); however, as reported by Keyes and Spradling Since cup mutant ovaries, it leaves open the question of what happens in wild type ovaries. Since Cup promotes deadenylation, it should directly antagonize Orb activation of translation as long as the Cup protein remains associated with Orb containing mRNPs. Thus, a simple expectation is that Cup would dissociate from Orb mRNP complexes when Orb is hyperphosphorylated and activates translation. However, this does not seem to be the case as we find that Cup is associated with both hypo- (inactive) and hyper- (active) phosphorylated Orb (orb and cup regulatory targets including orb mRNA and like Cup it is thought to associate with newly synthesized mRNAs and repress translation while they are being transported into the oocyte While a model in which Cup inhibits phosphorylation (either directly or indirectly) would help explain why the levels of the hyperphosphorylated Orb isoforms increase in ated Orb . Also, l343/Tm3SerHD19G orb flies were described in cup alleles used in this study were 1cup, 3cup, 6cup, 8cup and 1355cup. 1355cup was recovered in a P-element mutagenesis screen 1cup, 3cup, 6cup and 8cup were EMS-induced alleles recovered by Schüpbach and Wieschaus Δ212cup lacks the cup-eIF4E interaction domain and is described by Nakamura et al., 2, 250 mM sucrose, 0.05% NP40, 0.5% Triton-X, 1 mM PMSF, 1 µg/ml protease inhibitor cocktail, 1 mM dithiothreitol, 1 mM NaF, 40 µM NaVO3, 40 µM Na3VO4 and 500 µg of RNase A). The homogenate was centrifuged and the supernatant mixed with Orb antibody-coupled protein A/G beads , and incubated at room temperature for 2 hours. The beads were washed with Co-IP buffer and the proteins that were immunoprecipitated analyzed by Western blots. The amount of ovary extract which was loaded in one control lane is approximately 10% of the amount of ovary extract that was used in the immunoprecipitation reactions loaded in one experimental lane.Ovaries of 2–3 days old were fed on yeast overnight and dissected in 1× PBS. Ovary extract was prepared by homogenizing ovaries in Co-IP buffer . Blots were blocked in 5% milk/1× TBST for 2 hours, then incubated in primary antibodies  against 6H4 and 4H8, respectively) was performed at room temperature for approximately 2 hours. Microscopy was performed on a Perkin Elmer confocal microscope.Ovaries were dissected and fixed in freshly made 4% paraformaldehyde. After rinsing, the ovaries were blocked in 10 mg/ml BSA/PBSTT. The samples were incubated in primary antibodies (1∶30 anti-Orb 6H4 and 4H8 in 1 mg/ml BSA/PBSTT) overnight at 4°C, then rinsed. Incubation in secondary antibodies . The two proteins are also not properly concentrated into the presumptive oocyte in many older mutant egg chambers. Instead, several cells have high levels of Encore and Orb (see arrowheads).(TIF)Click here for additional data file.Figure S2Poly(A) tails are elongated in 1355cup. Poly(A) tails of orb mRNA from wild type and 1355cup (as indicated) were analyzed using the anchored poly(A)-tail assay orb specific primers (F2 and F4) were derived from the orb 3′ UTR. The amplification products were analyzed on an agarose gel and visualized with ethidium bromide.(PDF)Click here for additional data file.Table S1cup negatively regulates orb and suppresses the ventralization phenotype of 343HD19Gorb. Females that were trans-heterozygous for 343HD19Gorb and five different alleles of cup mutants were generated by crossing 343/TM3SerHD19G orb females (n = 10 in each cross) with cup/CyO males. Independent crosses were set up and scored at 18°C and 25°C. The number of embryos scored at each temperature is shown in the table. While approximately 20–30% of embryos laid by 343/+HD19G orb females were ventralized, suppression of this phenotype was seen in trans-heterozygotes of all five alleles. Approximately 2.5–7.3% of embryos laid by cup mutant transheterozygotes were ventralized at 18°C. Suppression was weaker but still very obvious and statistically significant at 25°C, where 8.6–20.4% of embryos were ventralized.(DOC)Click here for additional data file.


This opportune case study describes visual and stepping behaviours of an 87 year old female (P8), both prior to, and following two falls. Before falling, when asked to walk along a path containing two stepping guides positioned before and after an obstacle, P8 generally visually fixated the first stepping guide until after foot contact inside it. However, after falling P8 consistently looked away from the stepping guide before completing the step into it in order to fixate the upcoming obstacle in her path. The timing of gaze redirection away from the target (in relation to foot contact inside it) correlated with absolute stepping error. No differences in eyesight, cognitive function, or balance were found between pre- and post-fall recordings. However, P8 did report large increases in fall-related anxiety and reduced balance confidence, supporting previously suggested links between anxiety/increased fear or falling and maladaptive visual/stepping behaviours. The results represent a novel insight into how psychological and related behavioural factors can change in older adults following a fall, and provide a possible partial rationalisation for why recent fallers are more likely to fall again in the following 12 months. These findings highlight novel possibilities for falls prevention and rehabilitation. Walking safely through our cluttered world requires visual identification of hazards in our path, so that we can plan and execute appropriate stepping actions safely to avoid falling. Falls in older adults (OA) have a pronounced influence on morbidity and mortality The detrimental effects of fear of falling (FOF) in OA has received much attention in the literature since 1982, when Murphy and Isaacs Previous research has shown that older adults with FOF will adopt suboptimal eye movement patterns when walking through complex terrain The current study describes a rare opportunity to compare recordings of eye movement patterns, stepping characteristics, and a range of sensory and psychological measures recorded from an 87 year old female (P8) both four weeks prior to, and following, two falls occurring within the home environment (bathroom and garden path) which each resulted in minor cuts and bruises. Designing a study such as this would be practically and ethically unfeasible as researchers cannot predict when an OA will fall for the first time. Therefore, the current results represent a particularly novel insight in to how visual/stepping characteristics can be influenced by the occurrence of a fall and associated increases in FOF.P8 was a participant in a study reported by Young and Hollands We hypothesized that anxiety or FOF directly resulting from the falls would lead to maladaptive changes in eye and stepping behaviour. Our prediction based on this hypothesis was that P8 would report increased anxiety and reduced balance confidence during the post-fall session, and would demonstrate changes in her eye and stepping behaviours characterized by earlier gaze transfer from the stepping guide resulting in reduced stepping accuracy.P8 gave written and informed consent prior to the start of each experimental session. Ethical approval for both the research protocol and the consent documentation used was granted by the local ethics committee at The University of Birmingham, and was carried out in accordance with the principals laid down by the Declaration of Helsinki.2) on a backing luminance of 10 cd/m2. A lower visual field of >60° represents an absence of any deficit in this measure The battery of sensory measures taken during both pre- and post fall sessions were as follows: Peripheral cutaneous sensation under the ball of each foot was measured using a 3-point aesthesiometer. The final score from this measure represents the minimum distance between two points at which an individual can successfully discriminate their being two, as opposed to one single point. Functional balance was assessed using the Berg Balance Scale The psychological measures taken during both pre- and post-fall sessions were as follows: Falls efficacy was measured using the Falls Efficacy Scale Cognitive functioning was examined using the Mini Mental State Examination, where a score of >26/30 indicates no major deficit in this area Within each of the two pre-fall and two post-fall sessions P8 repeated 20 trials, where she was asked to walk along a 10 m walkway and place her foot inside a stepping guide made from lightweight packaging foam, with raised edges and a boarder width of 40 mm×40 mm. Inside the stepping guide was an area of 190 mm×415 mm . The raised edges on the stepping guides were designed to encourage participants to make accurate steps into the centre of the guide by imposing a degree of postural threat. P8 was asked to place her right foot into the first stepping guide, step over the obstacle with either foot and place her left foot in the second stepping guide. In each of the stepping guides P8 was asked to place her feet “as accurately as possible into the centre of each target”. This stepping guide was followed by one stepping obstacle with a width, height, and depth of 670 mm, 210 mm and 12 mm, and a second stepping guide . The obsReflective markers were placed on each corner of the initial stepping guide, and on both pairs of P8’s shoes; on the toe and heel, and on the medial and lateral sides (mid-foot markers), placed equidistantly between the toe and heel markers. The position of each marker was sampled at 120 Hz using a Vicon MX motion capture system . Kinematic data were passed through a low-pass butterworth filter with a cut-off frequency of 5 Hz. The centre of the targeting foot and the centre of the stepping guide were calculated as the mid-point between the respective markers in both the medio-lateral (M-L) and anterior-posterior (A-P) planes. Absolute foot placement error was calculated at the absolute distance between the centre of the foot and the centre of the stepping guide see . HoweverThe time of heel contact and toe-down events were determined as the maximum vertical acceleration of the heel and toe markers, respectively, identified by zero crossing in the respective jerk profiles. Foot contact times were determined as the first instance of either heel or toe contact following each step. Stance duration was defined as the elapsed time between foot contact and toe off during the single stance phase of the targeting foot prior to the step in to the stepping guide. Swing duration was calculated as the elapsed time between toe off and foot contact in the stepping guide.−1 within the analogue signal representing the vertical component of eye-in-head angle. The time difference between the eye movement away from the stepping guide and foot contact inside it was then calculated for each trial.Gaze behaviour was assessed using a high-speed ASL-500 head mounted gaze tracker. The horizontal and vertical components of eye-in-head angle were generated by the ASL control unit and were each sampled at 120 Hz and synchronised with the motion capture data via analog inputs. The ASL controller also produced a digital video of P8’s visual scene, superimposed with a crosshair representing the area of gaze fixation. This video was sampled at 30 Hz and used to identify the frame number in which P8 transferred her gaze from the stepping guide. This frame number was multiplied by 4 in order to select the corresponding section of the analog data. The precise timing of the saccadic eye movement away from the target was calculated by identifying an absolute local velocity peak with a threshold of 100°sp<0.05). This analysis was carried out for each of the following variables: 1) the time of gaze transfer from the stepping guide relative to foot contact inside it, 2) absolute error with respect to the centre of the stepping guide, 3) M-L error, 4) A-P error, 5) stance duration prior to stepping in the stepping guide, 6) swing duration of the step in to the stepping guide. Pearson’s product moment correlation analysis was used to determine the extent of the relationship between the time of gaze transfer and absolute error.Within a normally distributed data set, 95% of all data points lie within 2 standard deviations (2SD) of the mean. Therefore, if the difference between mean values recorded during pre-  and post-fall  sessions is greater than 2SD of all trials from pre-fall sessions, we can conclude that the results reflect a statistically significant difference  = −.151 p = .351). However, there was a significant negative correlation during post-fall sessions (r(40) = −.464 p<.005)  .The current data show that during pre-fall sessions P8 demonstrated eye movement patterns and stepping behaviours indicative of older adults deemed to be at a low-risk of falling. However, following the falls P8 transferred her gaze away from the target significantly earlier with respect to foot contact, thus compromising her stepping accuracy; a behaviour typical of high-risk older adults The significant correlation between the time of gaze redirection from the stepping guide and absolute error see  supportsUntil recently it was thought that the motor plans relating to body motion during gait were fixed at foot-off, meaning that the movement of the swinging foot and centre of mass were pre-planned and ballistic in nature Chapman and Hollands With respect to the error of P8’s foot placement in the stepping guide, we cannot conclude precisely how P8 interpreted the instructions to place her feet “as accurately as possible into the centre of each target”. For example, P8 may have considered where the ‘centre’ of her foot was and attempted to place that part of her foot on to the area where she perceived the centre of the stepping guide to be. Alternatively P8 may have attempted to place her foot in to the stepping guide so that the space around her foot was equidistant from the inside edge of the stepping guide. In the instance of the latter, we still cannot be sure whether P8 was concentrating on the placement of her whole foot, or rather just the portion of her foot visible to her (i.e. the toe). Indeed, P8’s tendency to place her foot in a more anterior position during post-fall sessions  may reprIt is curious that P8 only exhibited increased variability of foot placement in A-P, and not M-L directions during post-fall sessions. This finding is especially surprising considering that, in a similar task, older adults deemed to be at a high-risk of falling will exhibit increased M-L and A-P foot placement variability, compared to their low-risk counterparts It should be considered that the test battery used was not exhaustive, and therefore we cannot unanimously state that there was no physiological change that may have influenced P8’s visual and stepping behaviours following the falls. Nevertheless, it is clear that P8 experienced a clear reduction in both balance confidence and falls-efficacy, and an increase in state anxiety following the falls, collectively indicating an increased FOF . Based oPrevious work has shown that from every 3 older adults (OA) who fall, 2 will fall again in the following 12 months With respect to P8 it is pertinent to question what came first; the maladaptive visual strategy or the fall. Whilst we cannot reliably comment on the specific factors that predisposed P8 to fall on these occasions, we do suggest that the alterations in visual sampling will have occurred in accordance with changes in FOF Importantly, the significant correlation between premature gaze transfers and increased stepping errors during post-fall sessions  suggestsIn general, the nature of falls in OA will vary widely, as will the nature of each person’s recovery. Therefore, we cannot suggest that the falls experienced by P8’s are indicative of other OA’s experiences. This is a clear limitation of the current study, and an important consideration. However, our proposals for a link between FOF, eye movement patterns and stepping accuracy are based, not only on the current results, but also on the congruent relationships established in the literature After falling, P8 exhibited significant changes in her eye movement patterns, by transferring her gaze earlier from the stepping guide with respect to foot contact. P8 also showed significant reductions in stepping accuracy during post-fall trials. A significant correlation between visual and stepping behaviours during post-fall sessions support previous claims for a relationship between the two behaviours. The only changes found in the battery of sensory and psychological tests reflected a remarkable increase in P8’s FOF. Taken together, and based on previous findings from group studies, the current results support claims for a relationship between FOF and visual behaviours in OA, leading to reduced stepping accuracy.This opportune case study provides a rare insight in to how falling can influence a range of behaviours associated with safe gait, and highlights areas that should be targeted by future falls-prevention and rehabilitation strategies.


Cardiac Magnetic Resonance (CMR) imaging is increasingly recognized as the gold standard for evaluating left ventricular function (LVF). Measurement of LVF is typically done manually by tracing endocardial and epicardial borders on a stack of short axis (SA) cine images of the heart and is based on the modified Simpson assumption that the left ventricle approximates a spherical shape. A drawback of this method is that it is laborious and time-consuming. The recent introduction of computer-based inline VF software that allows for automatic assessment raised the prospect for significant reductions in CMR post processing times. Initial results have found that fully automatic analysis is unreliable as compared to manual calculations. However, usually only 1 or 2 contours are inaccurate using the automatic software suggesting that, if these are corrected manually, a semi-automatic approach may be helpful. This study sought to compare fully automatic inline VF tracking to this semi-automatic method using the manual technique as gold standard.This was a retrospective cohort study of subjects scanned on a 1.5 T MRI scanner  from January 1, 2012 to July 7, 2012. 8-12 SA steady state free precession slices from the base to apex of the heart were acquired for each subject and automatic inline calculations of LVF were performed. 77% of patients received gadolinium contrast prior to SA cine acquisition to shorten overall procedure time.The SA cine images with automatic contours were then transferred to an independent workstation  and the contours were corrected using standard software , if needed, by 2 independent observers . This constituted the semi-automatic analysis Figure . Origina100 subjects were included, with 38% female and 62% male and a mean age of 54.13 ± 14.94. Fully automated inline tracking failed for 9 cases. For the 91 successful cases, an overview of the results is presented in Table There was consistent underestimation of EF with automatic LVF analysis compared to semi-automatic and manual approaches, but semi-automatic and manual measurements were strongly correlated. There was significant reduction in post processing time with semi-automatic as compared to manual analysis. Semi-automatic analysis of LVF is accurate compared to the manual method and may improve workflow by reducing post processing time.Not funded.


Most egg yolk precursors are synthesized by the liver, secreted into the blood and transferred into oocytes, to provide nutrients and bioactive molecules for the avian embryo. Three hundred and sixteen distinct proteins have been identified in egg yolk. These include 37 proteases and antiproteases, which are likely to play a role in the formation of the yolk (vitellogenesis), as regulators of protein metabolism. We used a transcriptomic approach to define the protease and antiprotease genes specifically expressed in the hen liver in relation to vitellogenesis by comparing sexually mature and pre-laying chickens showing different steroid milieu.Using a 20 K chicken oligoarray, a total of 582 genes were shown to be over-expressed in the liver of sexually mature hens (1.2 to 67 fold-differences). Eight of the top ten over-expressed genes are known components of the egg yolk or perivitelline membrane. This list of 582 genes contains 12 proteases and 3 antiproteases. We found that “uncharacterized protein LOC419301/similar to porin” (GeneID:419301), an antiprotease and “cathepsin E-A-like/similar to nothepsin” (GeneID:417848), a protease, were the only over-expressed candidates  that are present in the egg yolk. Additionally, we showed the 4-fold over-expression of “ovochymase-2/similar to oviductin” (GeneID:769290), a vitelline membrane-specific protease.Our approach revealed that three proteases and antiproteases are likely to participate in the formation of the yolk. The role of the other 12 proteases and antiproteases which are over-expressed in our model remains unclear. At least 1/3 of proteases and antiproteases identified in egg yolk and vitelline membrane proteomes are expressed similarly in the liver regardless of the maturity of hens, and have been initially identified as regulators of haemostasis and inflammatory events. The lack of effect of sex steroids on these genes expressed in the liver but the products of which are found in the yolk suggests that these may be passively incorporated into the yolk rather than actively produced for that purpose. These results raise the question of the biological significance of egg yolk proteases and antiproteases, and more generally of all minor proteins that have been identified in egg yolk. Egg yolk constitutes a major source of proteins, minerals, vitamins and lipids for the developing embryo. The major egg yolk proteins, with the exception of immunoglobulins, are synthesized by the liver of laying hens. Protein synthesis and lipogenesis are highly stimulated in the liver at sexual maturity in hens (15 to 20 fold) to support the incorporation of 0.75 kg proteins and 1.5 kg of triglycerides into the yolk over a year of commercial production. The production of estrogen and at a lower magnitude testosterone by the theca of the growing follicle increases 2 to 3 weeks before the production of the first egg and contributes to the development of reproductive organs and the synthesis of egg constituents . Once seThe transfer of the major egg yolk proteins such as Very Low Density Lipoproteins (VLDL) containing essentially apovitellenin and apolipoprotein-B, but also vitellogenins and some other plasma proteins from the blood such as serum albumin to the interstitial fluid of the thecae is possible due to the presence of broad discontinuities in the capillary endothelium. The fluid in the theca spaces is then “filtered” by the basal lamina, which selectively permits the passage of particles depending on their size and their molecular charge . These pEgg yolk precursors are further processed within the oocyte by the action of proteases, the activities of which are regulated by protease inhibitors (antiproteases) or autolysis, to produce the final yolk constituents. It is assumed that cathepsin D, an aspartate protease, plays a major role in the generation of mature egg yolk constituents ,13. ReceThe present study provides an original overview of hepatic proteases and antiproteases that may be involved in the formation of the egg yolk and the perivitelline membrane, in fertilization or that could play a significant role during embryonic development as key components of the egg yolk. It also emphasizes the complementarities of both proteomic and transcriptomic strategies to better define the relevance of proteins which are associated with a specific milieu, and sheds lights on new approaches that can be implemented to answer questions related to their predicted biological significance.P < 0.01) are over-expressed in the livers of laying hens compared with livers of pre-laying pullets, with ratios ranging from 1.2 to 67. When analyzing the 582 over-expressed genes, we found that 70% of them were known, 18% have a homolog in other species and 12% are unknown , the activity of which is associated with phospholipid biosynthesis. This protein has been identified in chicken bursal lymphocytes . By simiOur data also revealed the presence of two predicted proteins, “PREDICTED: dual 3',5'-cyclic-AMP and -GMP phosphodiesterase 11A”  and “PREDICTED: nebulin-related-anchoring protein ”, which are highly expressed in the liver at sexual maturity of hens  and whose function remains to be explored. However, by similarity to mammals, “PREDICTED: dual 3',5'-cyclic-AMP and -GMP phosphodiesterase 11A” would have a role in signal transduction (Table P < 0.05). Three genes, PREDICTED: A disintegrin and metalloproteinase with thrombospondin motifs 5 , PREDICTED: metalloendopeptidase OMA1, mitochondrial  and “Signal peptide peptidase-like 2A precursor”  were not significantly over-expressed in the liver of laying hens, when compared to the liver of pre-laying pullets by q-RT-PCR. Two of these three genes  encode proteins, which are predicted to be integral to the membrane [The analyses of the over-expressed genes related to proteases and antiproteases using Gene Ontology, Interpro and Uniprot databases revealed 18 genes Table , which rmembrane . The mammembrane . HoweverGallus gallus has not yet been explored, but as a predicted aspartic protease it might assist cathepsin D in the processing of egg yolk precursors. “Uncharacterized protein LOC419301/similar to porin” is a predicted antiprotease containing one kunitz domain and nine whey-acidic protein domains. The presence of these multiple domains with potential inhibitory activities towards serine proteases suggests that this “uncharacterized protein LOC419301/similar to porin” could constitute a potent regulator of proteolytic activities within the egg yolk. This protein is related to “WAP four-disulfide core domain proteins”. Members of this family include elafin and secretory leucocyte protease inhibitor, which are involved in various aspects of mucosal immunity [To better appreciate which of these proteases and antiproteases are components of the egg, we compared this list of 15 candidates with proteases and antiproteases that were identified in the egg yolk by proteomics ,15. Thirimmunity . An altePart of the perivitelline membrane is formed in the follicle and depends on the expression of proteins by theca and granulosa cells but proteins from the liver are also important . In thisTo summarize, in the list of the 15 proteases and antiproteases over-expressed by the liver of laying hens by both oligoarray and q-RT-PCR, only three of them were actually known components of the egg yolk  or the vitelline membrane . Further analyses were therefore needed to better appreciate the respective role of the other 12 proteases and antiproteases that are overexpressed in the liver of mature hens, in the process of egg yolk or vitelline membrane formation and also to explore the biological significance of all the other candidates present in the egg yolk including those not specifically expressed by the liver in relation to egg yolk formation.Gallus gallus, has not been yet validated. However, when considering ovalbumin, a protein which does not possess a classical signal peptide and which is unambiguously secreted into the egg, it is predicted to be secreted through an unconventional secretory pathway using SecretomeP. These results suggest that SecretomeP can be informative to predict non-classical secreted proteins in Gallus gallus. Using this approach applied to the list of 15 genes, four have a signal peptide and six would be secreted through an unconventional pathway . In fact, it has been shown that ubiquitin specific peptidases are involved in the maintenance of genome integrity  and in tFrom the comparative analyses between the present transcriptomic approach and the published proteomic data, we emphasise the fact that the majority of egg yolk proteases and antiproteases are not differentially expressed in the liver of mature hens and immature pullets. Thirty-seven proteases and antiproteases were identified in egg yolk ,15. Most(a)) and Ovalbumin-related protein X and Ovostatin . These results suggest that these egg yolk components might be expressed locally by other tissues than the liver, i.e., theca or granulosa cells.Additionally, eight of these thirty-seven proteases and antiproteases identified by proteomics were not found to be expressed by the liver regardless of sexual maturity: Similar to transmembrane protease serine 13, Similar to transmembrane protease serine 9, Similar to ubiquitin specific protease 42, Ovalbumin related protein Y, Similar to alpha-1 antitrypsin, Similar to ovomucoid . Regarding the five remaining candidates that are expressed by the liver, two of them are found to be not differentially expressed in our model  and under the supervision of two authorized scientists S. Réhault-Godbert and J. Gautron . Animals were housed and fed according to the recommendations defined by the Institut National de la Recherche Agronomique (INRA). Eight ten-week old pre-laying pullets and eight thirty-eight-week old laying hens  were euthanized to collect the livers that were quickly frozen in liquid nitrogen and stored at -80°C until further use.Total RNA was extracted from frozen tissue samples using a commercial kit RNA-L  according to the manufacturer's procedure, and simultaneously treated with DNase. RNA concentrations were measured at 260 nm and the integrity of RNA was evaluated on agarose 1% and with an Agilent 2100 Bioanalyzer . RNA samples were kept frozen at -80°C until further use.Only RNA samples with a 28S/18S ratio > 1.3 were considered for labeling and hybridization. Thirty micrograms of total RNA were used for labeling the cDNA with the Superscript® Plus Indirect cDNA Labelling System . A balanced block design was used for hybridization where half of the sample were labeled with Alexa® 555 fluorescent dye and the other half with Alexa® 647  using the Superscript® Plus Indirect cDNA Labelling System . After synthesis and purification, the efficiency of the cDNA sample labeling as well as the quantity of labeled cDNA was determined using a Nanodrop ND 1000  by measuring absorbance at 260, 320, 554, 650 and 750 nm. Samples were then hybridized on a micro-array ggallus_ARK-Genomics_20K (GEO: GPL6049) produced by Centre de Ressources Biologiques GADIE . The features corresponding to the oligo-array are as follows. This 20.7 K chicken oligo array contains 20,676 features that include 20,460 oligos (about 70 mers) and 216 control spots of the Operon Production Tracking Oligo opHsV04NC000001, corresponding to 12595 different genes . A dynaFive micrograms of total RNA samples that were used for microarrays experiments were reverse-transcribed using the superscript II kit  and random hexamers . Classical PCR was performed using primers for proteases and antiproteases , referred to as the Crossing point (Cp) on light cycler and defined as the cycle at which fluorescence rises above a defined base line, was determined for each sample and control cDNA. A calibration curve was calculated using the Cp values of the control cDNA samples and relative amount of unknown samples were deduced from this curve. To account for variations in mRNA extraction and reverse transcription reaction between samples, probe mRNA levels were corrected relative to Tata Binding Protein (TBP). Levels of Tata box binding mRNA were determined using Sybr Green reaction and the following primers 5’GCGTTTTGCTGCTGTTATTATGAG3’ (forward) and 5’TCCTTGCTGCCAGTCTGGAC3’ (backward).The profiling of differentially expressed genes in liver between mature and immature hens was determined using Anapuce R package . Spot inP < 0.05) in gene expression between livers of laying hens and livers of pre-laying pullets.The log of the ratio sample/TBP RNA was used for statistical analysis using StatView version 5, software . One-way analysis of variance was performed to detect differences Click here for file


Data on the clinical outcome after spinal fusion in the elderly patient are rare. To our knowledge there has been no clinical outcome assessment for instrumented spinal fusion in elderly patients comparing posterolateral fusion with intervertebral fusion. Aim of the current study was to evaluate the clinical outcome of elderly patients who underwent a spinal fusion procedure for degenerative spinal stenosis with instability. Main hypothesis was to test whether it is necessary to force an intervertebral fusion for a better clinical outcome in spinal fusion surgery of the elderly or not.Two subgroups - posterolateral fusion versus intervertebral fusion (cage vs. non-cage) were compared with regard to functional outcome, fusion rates and complications after a mean follow up of 3.8 years. Questionnaires were completed by the patients before surgery and at final follow-up. Changes in mean VAS and ODI scores (decrease from the baseline VAS and ODI scores) were compared.The mean final follow up for all subjects was 3.8 years. Of the 114 patients, 2 patients were deceased at the time of the follow-up, 5 patients didn't want to participate and 107 patients completed the questionnaires. This resulted in an overall follow-up rate of 93%. At final follow-up, the patients demonstrated significant improvement in the VAS and ODI- compared with the preoperative scores in both groups. But overall there were no significant differences between both groups regarding the outcome assessment using the ODI and VAS.The results of this study shows that elderly patients aged over 75 benefit from instrumented lumbar fusion. The study suggests that there is no need to force an intervertebral fusion because elderly patients do not seem to benefit from this procedure. As the population ages, the number of spinal fusions performed in elderly patients is continuously increasing. There is a historic conflict, however, concerning the safety and efficacy of spinal surgery in the elderly -3.Instrumented spinal fusion in elderly patients has been problematized due to the risk of screw loosening and comorbidity. But data on clinical outcomes after spinal fusion in the elderly are rare. Limitations of most studies include small study populations, evaluation of perioperative complication rate, and radiographic assessment.To our knowledge, previous studies have not assessed clinical outcomes for instrumented spinal fusion in elderly patients comparing posterolateral fusion with intervertebral fusion (Cage versus non-cage).Therefore, the aim of the current study was to evaluate the clinical outcomes of elderly patients who underwent a spinal fusion procedure for degenerative spinal stenosis with degenerative scoliosis. Two subgroups - posterolateral fusion versus intervertebral fusion (cage vs. non-cage) were compared with regard to functional outcome, fusion rate, and complications after a mean follow-up of 3.8 years. Main hypothesis was to test wether it is necessary to force an intervertebral fusion for a better clinical outcome in spinal fusion surgery of the elderly or not.Charts and records of 114 patients who underwent spinal fusion surgery between January 2005 and June 2008 were reviewed. Inclusion criteria were age over 65, degenerative spinal stenosis with instability, presence of a completed questionnaire and surgery done by the author (S.E.). Follow up was performed by the author (S.E.) and co-author (R.A.).® and autologous bone obtained from the decompression procedure. Laminectomy, partial resection of the facet and a foraminotomy was performed in all patients because of severe central  and foraminal stenosis. The decision for the number of levels to fuse was based on functional myelography  as shown in Figure All patients underwent fusion with pedicle screws and rod instrumentation  without or with intervertebral cages  introduced by posterior lumbar interbody fusion. The bone graft for posterolateral fusion according to Wiltse was a mix of EndobonePostoperative management included early mobilization with bracing for 12 weeks on the first postoperative day.The patient population was divided into 2 subgroups according to the type of fusion they underwent. Group 1 received instrumented posterior lumbar interbody fusion (porous titanium cage without bone grafts). This group included 54 patients . Group 2 received a posterolateral lumbar fusion and included 60 patients . The average number of fused levels was 1.5 ± 0.6 in group 1 (Range 1-4) and 1.8 ± 0.9 in group 2 (Range 1-5).The review of anesthesia records for the patients showed that 35 patients in group 1 were of ASA (American Society of Anesthesiologists) class II and that 19 were of ASA class III. Nine (9) patients of group 2 were of ASA class I, 31 of ASA class II and 20 were of ASA class III.See Table All patients who were still alive in July 2010 were called for assessment for the study by the postgraduate (R.A.). They were given the questionnaires including the Oswestry Disability Index (ODI) and a visual analogue scale (VAS) score to assess their functional outcome and quality of life at final follow up. Changes in mean VAS and ODI scores (decrease from the baseline VAS and ODI scores) were compared.Fusion was assessed at final follow up on plain anteroposterior and lateral radiographs using the criteria suggested by Christensen et al. In additional, surgical time, need for red cell transfusions, and need for re-operation were documented.Ethical board approval of the University of Münster, Germany for the current study was given by the ethical board. [AZ 2010-218-f-s].For an effect size of 0.5 in functional outcome with α = 0.05 and β = 0.80, calculations revealed that 53 patients would be needed in each group. The changes in the ODI and VAS were evaluated with use of the Wilcoxon-Mann-Whitney test. Chi quadrat testing was done for differentiation in fusion rate and complication.The average intraoperative blood loss was recorded as a mean of 760 mL in group 1 (Range: 510 - 1100 mL) and 470 mL in group 2 (Range: 320 - 580 mL). Average time of surgery was 2 h 23 min for group 1 and 1 h 28 min for group 2. The need for transfusions (red cell units) in group 1 was on average 1.88 (Range: 0-5) and in group 2 on average 0.35 (Range: 0-2).The mean final follow up for all subjects was 3.8 years. Of the 114 patients, 2 patients were deceased at the time of the follow-up, 5 patients didn't want to participate and 107 patients completed the questionnaires. This resulted in an overall follow-up rate of 93%.The mean hospital stay was 12.6 days in Group 1 and 13.1 days in Group 2. The complication rate was almost the same in both groups. Revision surgeries were needed in group 1 with 2 repeat decompressions, 1 screw misplacement and 1 adjacent level disease. In group 2 there was a revision surgery needed in 5 cases due to pseudarthrosis mainly of L5/S1, 1 repeat decompression and 1 adjacent level disease.At final follow-up, the patients in both groups demonstrated significant improvements in VAS and ODI-scores as compared to preoperative scores.Group 1: The mean VAS score was 8.4 before surgery, and 5.0 at final follow up, showing 42% improvement. The mean ODI score was 53.05 ± 11.47 before surgery, and 38.59 ± 14.5., showing 29% improvement.Group 2: The mean VAS score was 8.8 before surgery, and 5.2 at final follow up, showing 45% improvement. The mean ODI score was 53.43 ± 10.12 before surgery, and 39.01 ± 14.02, showing 27% improvement.Overall, there were no significant differences between the two groups regarding outcome assessment using ODI and VAS. A sub-group analysis of patients who had complications wasn't done.Fusion rate was determined by plain radiographs evaluated by the author (S.E.) and an independent radiologist. The fusion rate was similar in both groups with 41 out of 54 patients in group 1 showing successful fusion and 46 of the 60 subjects in group 2 showing successful fusion.2 = 0,0086; posterolateral fusion: X2 = 0,5914).Chi-quadrat testing reveals no statistically significant correlation (at a significance level of 5%) between fusion rate and complication rate comparing the different surgical techniques  population is about 4% and is predicted to rise by 2050 to 12-13% [In the past, and sometimes even today, advanced age was considered a contraindication for elective spinal surgery, so older patients were treated conservatively. However, conservative treatment can lead to increasing physical pain and limitations in quality of life ,7.The surgical procedures vary from laminotomy or wide central laminectomy alone to an anterior release with posterior decompression and fusion with instrumentation. Because of advanced age, medical co-morbidities lower bone density, and more spinal stiffness the surgical risks and complications are higher. The main objective of the surgical selection is to achieve the greatest benefit with the least complex intervention.Reviewing the literature, there are many studies dealing with the clinical outcome after spinal fusion surgery of the elderly -13.But the literature reveals different opinions about fusion surgery in the elderly, especially how to treat degenerative spinal stenosis with concomitant instability. The question of whether it is necessary to do an intervertebral fusion by implanting a cage or a posterolateral fusion  is sufficient remains controversial.The literature shows that complication rates after surgery of the lumbar spine in elderly patients vary from 8 to 80%, with further variation in the rate of minor complications that do not lead to prolongation of hospital stay which usually account for more than half of the complications ,14-18.However, data on the clinical outcome after spinal fusion in the elderly patient are rare. Existing studies have either small numbers of patients, are focused on the perioperative complication rate ,15,17, oTherefore the aim of the current study was to evaluate clinical outcomes of patients over 65 who underwent a spinal fusion procedure for degenerative spinal stenosis with concomitant instability. Two subgroups - posterolateral fusion versus intervertebral fusion (cage vs. non-cage) were compared with regard to functional outcome, fusion rate and major complications after a mean follow up of 3.8 years.Looking at the most recent literature the existing studies show that the assessment of success after spinal fusion surgery in the geriatric patient has been focused on the perioperative complication rate, with little attention directed toward improvement in function, quality of life, patient satisfaction, or improvement in perceptions of pain and the need for medication.Okuda et al were the first to assess a large number of elderly patients on the basis of health status questionnaires after posterior lumbar interbody fusion, but they compared patients with a mean age of 74 to patients with a mean age of 59. They found no differences in the clinical and functional outcomes between the two groups and stated that posterior lumbar interbody fusion is a safe and accurate procedure for geriatric patients .Andersen et al reported that superior outcomes after lumbar spinal fusion in elderly patients can be achieved using instrumentation, but the aim of their study was to compare instrumented and non-instrumented lumbar spinal fusions performed using fresh frozen allograft in patients older than 60 years with regard to functional outcome and fusion rates. The outcome was better for patients in which a solid fusion was obtained. However, instrumentation was associated with a larger number of additional surgeries, which resulted in a lesser degree of improvement .A study by Glassmann et al support the efficacy of lumbar decompression and fusion in selected patients over 65 years of age. In their retrospective evaluation they had an improvement in SF-36 and ODI at a 2 year follow up which is in line with our study results .Another study by Becker et al investigated the clinical outcome in elderly patients who underwent spinal fusion with pedicle screws and rod instrumentation with or without intervertebral cages introduced by posterior lumbar interbody fusion. They were able to demonstrate that e elderly patients benefit from spinal fusion but they do not distinguish between the two methods .To our knowledge, there is no other study that compares the clinical and functional outcomes after instrumented spinal surgery in the elderly with regard to intervertebral fusion or posterolateral fusion.Using the ODI and VAS, we found no differences between the two subgroups  of elderly patients. Overall a significant improvement in life quality was found at final follow up regardless the used fusion technique or whether a revision procedure was done in the meantime. A subgroup analysis of the patients who were in need of a revision surgery was not done. But looking at the ODI and VAS they don't have obvious lower scores.With regard to intraoperative blood loss, need for transfusion of red cell units and surgical time, there was a significant difference between the two subgroups that showed superiority for the instrumented posterolateral fusion group.The present study has some limitations. The duration of the follow-up was relatively short. The long-term results of these surgical procedures are needed. The second issue is that there is little information available regarding fusion rates in elderly patients. The fusion rate in the present study was evaluated indirectly as conventional x-ray was used for follow-up. Owing to ethical issues concerning high radiation, CT scans were not performed. But whether osseous fusion rate correlates with the clinical outcome remains a controversial issue. It was demonstrated by Pfeiffer et al. that there is only a weak correlation between intervertebral fusion and the clinical outcome . AdditioThe third issue is the sagittal balance. Even if several investigators have stressed the importance of maintaining sagittal balance to avoid lumbar "flat back," accelerated adjacent segment degeneration, pain, and inferior functional outcome only limited evidence exists on how sagittal alignment affects clinical outcome ,24. But Explanation for this phenomenon is that in addition to sagittal balance, clinical outcomes of instrumented lumbar fusion in patients with degenerative lumbar spine disease are influenced by a variety of pathophysiologic factors, including residual compression of the neural tissues, recurrence of spinal canal stenosis, irreversible changes to the nerve root, or cauda equina.The results of this study show that elderly patients over 75 benefit from instrumented lumbar fusion. The study further suggests that there is no need to advocate for an intervertebral fusion because elderly patients do not seem to benefit from this procedure. With regard to clinical and functional outcomes, an instrumented posterolateral fusion is sufficient. Additionally, the intraoperative blood loss, need for transfusions and surgical time are clearly reduced for elderly patients receiving posterolateral fusion.There was no involvement of a manufacturer in this study.- In the past five years have you received reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future? Is such an organization financing this manuscript (including the article-processing charge)? If so, please specify.No.- Do you hold any stocks or shares in an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future? If so, please specify.No.- Do you hold or are you currently applying for any patents relating to the content of the manuscript? Have you received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript? If so, please specify.No.- Do you have any other financial competing interests? If so, please specify.No.- Are there any non-financial competing interests  to declare in relation to this manuscript? If so, please specify.No.SE carried out the surgery, follow up and drafted the manuscript. RA has done the assessment of the patients. AW helped revising the manuscript and with statistics. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/189/prepub


In this report, we describe the isolation of a highly related virus from an adult patient who suffered acute respiratory illness in Malaysia. Although there was no direct evidence of bat origin, epidemiological study indicated the potential exposure of the patient to bats before the onset of disease. The current study further demonstrates that spillover events of different strains of related orthoreoviruses from bats to humans are occurring on a regular basis, which calls for more intensive and systematic surveillances to fully assess the true public health impact of these newly discovered bat-borne zoonotic reoviruses.Bats are increasingly being recognized as important reservoir hosts for a large number of viruses, some of them can be highly virulent when they infect human and livestock animals. Among the new bat zoonotic viruses discovered in recent years, several reoviruses ( Up-to-date, approximately 1400 pathogen species are recognized to infect humans. Although fewer than 200 of these are viruses, on average, more than two new species of viruses infecting humans are reported worldwide every year and novel viruses, especially RNA viruses, are more likely to cause future emerging infections Acute respiratory infections constitute the most common and widespread human infectious diseases, especially amongst patients seen at the outpatient clinics. Despite the application of various diagnostic tests, the aetiological agents of a substantial proportion of acute respiratory illnesses are still not known. Novel viruses with the ability to cause respiratory infections and spread via the respiratory route, such as the SARS coronavirus and pandemic influenza A (H1N1), are of particular public health interest because of their pandemic potential and their ability of rapid spread via aerosol or droplet. The recent isolations of several novel zoonotic orthoreoviruses from humans with acute respiratory illness and the demonstration of their human-to-human transmission highlights the potential of emergence of a pandemic strain via mutation and/or re-assortment of their segmented double-stranded RNA genomes Reoviridae are a large and diverse group of non-enveloped viruses with segmented double-stranded RNA genomes Orthoreovirus contain 10 genome segments and have been isolated from a broad range of mammals, birds and reptiles. Viruses under this genus are divided into two phenotypic groups, fusogenic and non-fusogenic, based on the ability of the virus to cause cell-cell fusion and formation of giant syncytial cells Viruses belonging to the family Pteropine orthoreovirus (PRV), replacing the existing species, Nelson Bay orthoreovirus. The new name has a better fit with other existing species names in the genus such as Avian orthoreovirus (ARV) and Mammalian orthoreovirus (MRV).Since 2006, two fusogenic orthoreoviruses, Melaka virus and Kampar virus, have been independently isolated in Malaysia from respiratory samples of patients with acute respiratory illness though no death has been reported th isolate from human patients within a four-year period in the absence of a targeted surveillance program. It can be predicted that more human cases will be detected if systematic surveillance is put in place for nations where fruit bats are present in significant numbers.Epidemiological investigation by screening of sera collected from human volunteers on the Pulau Island in Malaysia revealed that 14 of 109 (13%) were positive for Pular virus, indicating that this group of viruses may be able to switch host and infect humans more frequently than other bat-borne viruses, such as the Nipah virus The investigation conducted in this study was approved by the ethics committee of the Malaysian National Public Health Laboratory. All patients (subjects) in this manuscript have given written informed consent (as outlined in the PLoS consent form) to publication of their case details. No identification of the subjects is to be revealed in any publication.A nation-wide influenza surveillance program in Malaysia was initiated in 2004 under the national influenza pandemic preparedness plan. As a requirement, attending doctors from two respective designated government polyclinics per state were instructed to collect throat swab specimens from patients with influenza-like illnesses. Simultaneously, clinical and epidemiological information of the patients were recorded in a standardized questionnaire and sent together with the clinical specimens to the national public health laboratory for influenza virus isolation and identification.2 and examined daily for evidence of any cytopathic effect (CPE). For those samples showing CPE, a 0.5 ml-aliquot of the culture supernatant containing suspended infected cells was carefully harvested and placed in a microfuge tube. The infected cells were pelleted and washed twice with sterile phosphate buffered saline (PBS) by centrifugation at 1000 g for 10 minutes. After the last wash, the infected cells were resuspended in 200 µl of sterile PBS and 10 µl of the suspension cells were carefully layered onto each well of a 12-well Teflon coated slide . The slide was allowed to dry over a warm plate (42°C) placed in a BSC-II cabinet. The slide was fixed for 10 minutes in cold acetone as soon as it was dried. Initial identification of virus was made by indirect immunofluorescence test using a panel of commercial monoclonal antibodies for identification of respiratory viruses commonly known to infect humans .For virus isolation, throat swab samples in virus transport medium (VTM) were gently vortexed after the addition of crystalline penicillin (500 I.U.), streptomycin (200 µg) and amphoteracin (50 µg). After incubation for an hour at room temperature, the solution was inoculated in duplicates  onto monolayer of MDCK , Vero  and Hep-2  cells freshly cultured in JM cell culture tubes 2 tissue culture flask was washed twice with 5 ml of sterile PBS and treated with 2 ml of 0.25% trypsin containing 0.1% EDTA . The MDCK cells were then suspended in 20 ml of DMEM culture medium containing 10% fetal calf serum. A 5-ml aliquot of the cell suspension was transferred into each of 4 new 25-cm2 flasks and incubated at 37°C in an ambient of 5% CO2. Once the cells reached confluent growth, the flasks were treated as above. The suspended cells in each flask were inoculated with 107 TCID50 of each of the four viruses included in this study. At 24 hours post-inoculation, the infected cells (with full CPE) in the suspension were harvested and washed 5 times with sterile PBS by centrifugation at 1000 g for 10 minutes in each wash. A 25-cm2 flask of suspended mock-infected MDCK cells was processed exactly the same way as a negative control. After the last wash, the mock-infected cells were distributed and added into the respective infected cells in the ratio of 1∶4. The volume of the mixed cell suspension was adjusted with sterile PBS to a cell concentration of approximately 1000 cells per 10 µl and subsequently 10 µl of the final cell suspension was transferred into each well of the Teflon coated slides. The slides were allowed to air dry in a BSL-2 cabinet, irradiated by UV-light and fixed in cold acetone for later use.Virus infected cells fixed in wells of Teflon coated slides were used for serological investigation. Briefly, a fully confluent monolayer of MDCK cells in a 25-cmHuman sera were tested for the presence of virus-specific IgM and IgG antibodies by indirect immunofluorecence assay using the fixed infected cells prepared above. Convalescent human serum samples were serially diluted from 1∶20 to 1∶2560 with sterile PBS. Twenty microlitres of the diluted sera was used for immuno staining as previously described 50 of each of the four viruses using a previously published method Cross neutralization activity of convalescent human sera was tested using a micro-neutralization test against 100 TCIDExtraction and purification of dsRNA, and genome segment analysis were carried our as previously described by our group A 46-year old policeman developed a sudden onset of high fever with a sore throat and prostrating myalgia on 6 March 2010. The fever was associated with severe anteriorly throbbing headache and mild photophobia, but without blurring of vision. There was no neck stiffness, abnormal muscle movement, seizure or loss of consciousness. The myalgia was described as generalized and associated with generalized body weakness, lethargy, malaise and loss of appetite. His sore-throat was described as severe in nature which was aggravated by swallowing of both solid and liquid food. The sore-throat was associated with hoarseness of voice and nasal blockage. There was no associated nasal discharge, cough or breathing difficulty. On the following day, he developed moderate abdominal pain over the epigastrium, but not associated with vomiting or diarrhoea. There was no history of skin rashes or a tendency to bleed. His illness was not relieved with self-medication of anti-pyretics.He received medical treatment at a government health clinic on 8 March 2010. At the outpatient clinic, he was noted to be febrile (an axillary temperature of 39.8°C), ill-looking with mild erythematous flushing over his face and chest that blanched on pressure. He was noted to have mild conjunctivitis, but there was no jaundice or petechiae spots noted. His tonsils were enlarged and injected, but there was no white exudates noted over the oral pharynx. He was not in any respiratory distress and his lungs were clear with good bilateral air-entry on auscultation. Other systemic examinations were essentially normal and there was no significant enlarged lymph node noted. A provisional diagnosis of influenza-like illness was made. A throat swab was collected in viral transport medium and sent to the laboratory for virology investigation. He was given erythromycin and a higher dose of anti-pyretic. On follow-up on 12 March 2010, he still felt weak and lethargic with mild myalgia and poor appetite though his fever had resolved on 11 March 2010.The patient had no past significant severe illness that needed hospitalization, neither was he on regular medication for any chronic illness. There was no history of recent travel overseas or into caves or jungles. There was no history of direct contact or handling of any domestic or wild animal or contact with neighbours or friends having similar illness. None of his other family members, living in the same household, developed similar illness within the month following his illness.nd child) and two daughters (5th and 6th child) on 20 April 2010 and the status of their anti-PRV7S antibodies is shown in nd son had IgM and IgG antibodies to PRV7S though none of them had ever experienced any febrile illness within a month of the onset of the patient's clinical symptoms.The patient and his family live in a double storey-linked house in Sikamat, a small town within the state of Negeri Sembilan, in the central western region of peninsular Malaysia. There is no fruit tree in the immediate surroundings of the house. However, for the past two years, the patient and his wife have regularly returned to stay over the weekend in their village house and were at the house on the weekends before the onset of his disease. The village house is situated in a 2-hectare orchard about 20 kilometres away from his main residence and is surrounded with various types of tropical fruit trees. Fruit bats are known to fly around in his orchard at night and at times were known to fly into the house, but none were found roosting inside the house. He has not directly handled any bats or killed any of them. He and his wife did not consume any fruit partially eaten by animal or insect or those drop on the ground. The patient has 6 children, two sons and four daughters. His two older sons, aged 22 and 20, occasionally assist him in the orchard during the weekend, but did not stay overnight in the village house. Samples of venous blood were collected from his wife, one son . Further cross-reactivity analyses indicated that convalescent sera from patients infected with three different PRV strains all had high level of cross reactivity , Table 3Orthoreovirus.Profiling of the genome segments for PRV7S on SDS-polyacrylamide gel indicated that its electropherotype is similar to those of other Malaysian PRVs, and is most closely related to that of PRV3M . AnalysiPhylogenetic trees based on all four S-class genome segments further confirmed that PRT7S is a member of the PRV species group .Pteropine orthoreovirus (PRV) species group. Considering that the detection of such events was “accidental” in the sense that no targeted surveillance was conducted, it can be predicted that the real frequency of spillover will be higher.The isolation of yet another orthoreovirus from a patient suffering acute respiratory disease in Malaysia confirms the trend of regular human infection by members of the To date, there are four confirmed outbreaks of human respiratory illness caused by this group of viruses, three in Malaysia and one in Bali/Hong Kong Pteropus poliocephalus in Australia), PRV2P  and PRV6XR (isolated from Rousettus leschenaultia in China), PRV1NB was clustered closer to PRV6XR than to PRV2P despite the fact that PRV1NB and PRV2P were isolated from bats in the same genus, Pteropus, while PRV6XR was from the genus Rousettus. The viral genetic diversity is also reflected by the four viruses isolated from human patients. Although they are closely related, they represented four independent spillover events associated with four distinctive viruses.While there is the possibility of PRV infection in other wildlife animal(s), it is clear that bats are at least one of the natural reservoir hosts based on the fact that closely related PRVs have been isolated from three different bat species in three well separated geographic locations in Australia, Malaysia and China It is important to emphasize that among all three outbreaks investigated in Malaysia so far, each was associated with multiple human infections. Although one cannot exclude the possibility that this could be a result of independent bat-to-human transmission, epidemiological tracing suggested they were more likely the result of human-to-human transmission In summary, recent studies demonstrated that there are a large number of genetically diverse PRVs circulating among different bats in geographically separated locations. Many, if not all of them, have the potential to infect human and cause respiratory disease outbreaks. These viruses can be transmitted between humans and the resulting clinical presentations vary with different individuals, from symptomless to severe respiratory illness or diarrhoea


Ewes were assigned randomly to receive 37.5 IU of porcine follicle stimulating hormone (FSH-P) daily for the 3 days preceding implant removal (Day 0). Synchronization of estrus was carried out using a 1.5 mg norgestomet (Crestar) ear implant for 12 days. Ewes in estrus were mated two to three times with rams of proven fertility. At the time of first mating each ewe was administered 1000 IU of human chorionic gonadotrophin (hCG) to induce ovulation. Surgical embryo recovery was performed on Days 4 and 6 after onset of estrus (Day 0) and recovered embryos were subjected to comparative metabolism studies with in vitro derived embryos at the same stage of development. The number of corpora lutea (CL), unovulated follicles and overall ovarian activity were recorded for each ewe during the breeding and non-breeding seasons. While the pattern of oxidation was similar among in vitro and in vivo derived embryos, a low pyruvate to lactate ratio was the preferred substrate of embryos derived in vitro. A high level of production of CO2 and lactate resulted from a stress response to the suboptimal culture environment. The first marked increase in the metabolism of glucose by ovine embryos was detected in compact morula stage, but there was no significant increase in the oxidation of glucose after the morula stage. Two different concentrations of glucose were compared, but this did not affect metabolism. However, the rate of incorporation and metabolism of glucose tended to be higher at the 0.56 mmol/L glucose dosage.The synchronization and ovulatory responses of Sangsari cross bred ewes and metabolism of energy substrates in 8-cell stage embryos to hatched blastocysts stage produced  In addition, pyruvate (0.33 mmol/L) and lactate (3.3 mmol/L) together were beneficial to early embryonic development in sheep[in vitro and in vivo derived embryos from the 8-cell to hatched blastocyst stages Enhancing the understanding of metabolic activity of pre implantation embryos with respect to improving the yield and quality of embryos was also an objective of this study.Synchronization of estrus followed by induction of ovulation are basic techniques in all embryo transfer programs. Superovulation ensures recovery of multiple embryos during one estrous cycle, thus allowing us to maximally exploit the female germ pool. Reports of synchronization of estrus and superovulation of ewes have been previously discussed,2. The pruvate 0.3 mmol/L in sheep inferredA total of 23 ewes (Sangsari cross bred) between 2 and 5 years of age that were subjected to standard feeding and management protocols were used. Ewes were isolated from rams except at the time of mating. After deworming, ewes were tagged for identification and assigned randomly to either be bred during the normal breeding season  or out of season . All animal experiments were approved by the Zabol University and Semnan Province Agricultural Jihad sheep breeding farm Animal Ethics Committee in accordance with the care and use of animals for scientific purposes follow internationally recognized guidelines, approved by the Iranian higher education and research ministry.− 19- nor− preg-4-en-3,20 dione)  that remained in place for 12 days. Prior to removal of the progestagen ear implant, each ewe was treated twice daily at 12 h intervals with FSH-P  with 16 IU, 12 IU and 9.5 IU on Day -2, Day -1 and Day 0. Day 0 was the day the Crestar implant was removed. Beginning at 8 h after implant removal, ewes were observed for estrus three times each day, both visually and in the presence of teaser rams. When detected in estrus, ewes were mated two to three times with fertile rams. At the time of the first mating, each ewe was administered 1000 IU hCG  or Day 6 (n = 6 for breeding season and n = 6 for non-breeding season) using Dulbecco’s Phosphate Buffer Saline  as the flushing medium. On Day 4 each oviduct and uterine horn was flushed, whereas, only the uterine horns were flushed on Day 6 post mating. The ovarian response of each ewe was recorded with respect to the number of corpora lutea, unovulated follicles and overall ovarian activity as described previously[−  and lactate production from glucose was also determined. Anabolic metabolism was estimated by determining the amount of substrate carbon incorporated by the embryos. A specially washed and heat sterilized 20 mL scintillation vial served as the incubation apparatus. Embryos were washed through two changes (2 mL) of energy-substrate-free PBS and placed in a 50 μL drop of radioactive medium for a final wash before being transferred into a sterile, 600 μL micro centrifuge tube  containing 50 μL radioactive incubation medium. This tube was placed in the scintillation vial with the lid of the tube acting as a support. Simultaneously, 0.5 mL of 2.5 mol/L sodium hydroxide was added to the bottom of the vial, which was then closed and incubated at 39°C for 4 h. At the end of the incubation period, the tube with embryos was taken out and the radioactivity trapped as CO2 was determined after the addition of scintillation cocktail. In all experiments, at least two sham incubations were run concurrently to correct for background counts. The metabolic turnover was estimated from the disintegrations per minute detected in the sample and the specific activity of the parent substrate, and converted to picomoles/embryo/h on the basis that each glucose molecule gives rise to 6 molecules of CO2[ glucose-, along in vitro, then subjected to comparative metabolism studies with in vivo derived embryos at the same stage of development for assessment of energy differences and requirement for further development up to hatched blastocyst stage. The in vitro development of in vivo and in vitro derived embryos was assessed at 4 h intervals. The metabolism of 8- to 16- cell stage embryos up to the hatched blastocyst stage was investigated using either glucose, pyruvate or lactate as the sole energy substrate. Surgically recovered embryos at the 8-cell to morula and blastocyst stages and derived in vivo were subjected to comparative metabolism of energy substrate with embryos at the same stage derived in vitro.The collected oocytes first were fertilized and developed up to the 8-cell stage Recovered embryos were classified according to International Embryo Transfer Society (IETS) guidelines, and was1) Eight-cell embryos were used to assess metabolism before the so- called block to development stages prior to activation of the embryonic genome.2) Morula stage embryos obtained on Day 6 of culture were used to elucidate changes in metabolism after the block to development and before compaction of blastomeres and the period of rapid growth to the blastocyst stage.in vivo derived early-stage blastocysts were cultured in vitro for 24 or 48 h to obtain mid-stage and expanded blastocysts, respectively.3) Blastocysts obtained on Days 7 to 9 post-mating were classified as early- and mid-stage blastocysts and expanded blastocysts before being used for metabolic studies. The overall diameter of blastocysts, including the zona pellucida, was measured with an ocular micrometer at two axes perpendicular to each other and expressed as the mean of the two measurements. These measurements were made before metabolic studies were performed to find relationships between size and metabolic activity. The In vivo derived early blastocysts were grown in vitro for 24 h to obtain hatched blastocysts.4) Hatching or hatched blastocysts were obtained on Days 8 to 10 of culture and subjected to metabolic studies to assess changes during the process of hatching and post-hatching. The data obtained from all experiments were subjected to appropriate statistical analyses as described previously. In the Irrespective of season of the year, the majority of ewes exhibited estrus 24 h following implant removal and in the non-breeding season only 4 ewes exhibited estrus at 48 h after implant removal Table. The meaIt has been reported that ear implants containing 3 mg progesterone offer a reliable means for synchronization of estrus in ewes, whereasFlushing the uterine horns and oviducts on Day 4 post mating during the breeding and nonbreeding seasons yielded embryos with developmental stages varying between 8-cells to morula, with, two embryos classified as degenerated, but the remaining embryos had normal morphology and were considered as transferable to a recipient ewe. Flushing only uterine horns on Day 6 during the breeding and non-breeding seasons yielded embryos varying from the 16-cell to hatched blastocyst stages and all had normal morphology and were considered as transferable to a recipient ewe.An overall analysis of embryos quality, irrespective of season, revealed that morulae and blastocysts were recovered only from ewes flushed on Day 6 post-estrus as expected. The embryos were classified according to International Embryo Transfer Society (IETS) guidelines, washed in vitro metabolism of energy substrates by 8-cell stage embryos derived in vivo was compared to that for the same stage embryos derived in vitro. The pattern of oxidation was similar between the two types of embryos, but oxidation and CO2 production and lactate production was greater for in vitro as compared to in vivo derived 8-cell stage embryos (c versus d = (p < 0.05). In general the two concentrations of glucose did not differ in their metabolic fate, although the rate of glucose incorporation tended to be higher in the presence of 0.56 mmol/L glucose. However, production of lactate and uptake of glucose carbon were higher in the presence of 0.56 mmol/L glucose. No lactate production was detected apart from the higher oxidation of lactate by the in vivo derived embryos of both stages (Tablein vivo collected morulae and early blastocysts produced lower rates of CO2 (p < 0.05) than their counterparts derived in vitro. Similarly, the mid-stage blastocysts cultured for 24 h produced more CO2 as compared to values for freshly collected and cultured mid-stage blastocysts. In general, lactate production by the freshly obtained embryos was about one half that for in vitro derived embryos. However, long pre-exposure to culture conditions narrowed the differences between the two sources at the hatched blastocyst stage (72 h of IVC) and were not significant. Incorporation of glucose carbon was independent of the source of embryos, but there was a tendency for a lower uptake by IVM/IVF embryos from mid stage to hatched blastocyst stages (TableThe es Table. The in = p < 0.0. In genein vitro and in vivo derived embryos was the lack of detectable lactate production until the 12- cell stage. When lactate was detected its production increased continuously through the blastocyst stage. In vivo derived embryos produced one-half the quantity of lactate as in vitro derived embryos. Lactate production by embryos recovered by flushing the oviduct and uterus was also about one-half that for in vitro derived embryos. However, this difference decreased as time in culture increased.The concentration of pyruvate was the same as used previously for mouse embryos. The resSeason and time of onset of estrus did not affect the ultimate number of transferable embryos in ewes. However, results of this study indicate: that ewes exhibit a lower ovulatory response compared with results reported for crossbred ewes in; a previous study. In the Dr Ahmad Azizimoghadam declare that they have no competing interests.Dr AA carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. The author read and approved the final manuscript.