Pseudo contrastive labeling for predicting IVF embryo developmental potential.

In vitro fertilization is typically associated with high failure rates per transfer, leading to an acute need for the identification of embryos with high developmental potential. Current methods are tailored to specific times after fertilization, often require expert inspection, and have low predictive power. Automatic methods are challenged by ambiguous labels, clinical heterogeneity, and the inability to utilize multiple developmental points. In this work, we propose a novel method that trains a classifier conditioned on the time since fertilization. This classifier is then integrated over time and its output is used to assign soft labels to pairs of samples. The classifier obtained by training on these soft labels presents a significant improvement in accuracy, even as early as 30 h post-fertilization. By integrating the classification scores, the predictive power is further improved. Our results are superior to previously reported methods, including the commercial KIDScore-D3 system, and a group of eight senior professionals, in classifying multiple groups of favorable embryos into groups defined as less favorable based on implantation outcomes, expert decisions based on developmental trajectories, and/or genetic tests.

Is the aging human ovary still ticking?: Expression of clock-genes in luteinized granulosa cells of young and older women.

BACKGROUND: It has been shown - mostly in animal models - that circadian clock genes are expressed in granulosa cells and in corpora luteum and might be essential for the ovulatory process and steroidogenesis. OBJECTIVE: We sought to investigate which circadian clock genes exist in human granulosa cells and whether their expression and activity decrease during aging of the ovary. STUDY DESIGN: Human luteinized granulosa cells were isolated from young (age 18-33) and older (age 39-45) patients who underwent in-vitro fertilization treatment. Levels of clock genes expression were measured in these cells 36 h after human chorionic gonadotropin stimulation. METHODS: Human luteinized granulosa cells were isolated from follicular fluid during oocyte retrieval. The mRNA expression levels of the circadian genes CRY1, CRY2, PER1, PER2, CLOCK, ARNTL, ARNTL2, and NPAS2 were analyzed by quantitative polymerase chain reaction. RESULTS: We found that the circadian genes CRY1, CRY2, PER1, PER2, CLOCK, ARNTL, ARNTL2, and NPAS2, are expressed in cultured human luteinized granulosa cells. Among these genes, there was a general trend of decreased expression in cells from older women but it reached statistical significance only for PER1 and CLOCK genes (fold change of 0.27 ± 0.14; p = 0.03 and 0.29 ± 0.16; p = 0.05, respectively). CONCLUSIONS: This preliminary report indicates that molecular circadian clock genes exist in human luteinized granulosa cells. There is a decreased expression of some of these genes in older women. This decline may partially explain the decreased fertility and steroidogenesis of reproductive aging.

Heparanase procoagulant activity is elevated and predicts survival in non-small cell lung cancer patients.

BACKGROUND: Heparanase is implicated in angiogenesis and tumor progression. We had earlier demonstrated that heparanase may also affect the hemostatic system in a non-enzymatic manner. It forms a complex and enhances the activity of the blood coagulation initiator- tissue factor (TF). Although increased heparanase antigen level in the plasma and biopsies of cancer patients was previously demonstrated, in the present study we evaluated, for the first time, the heparanase procoagulant activity in the plasma of patients with lung cancer. MATERIALS AND METHODS: Sixty five patients with non-small cell lung cancer at presentation and twenty controls were recruited. Plasma was studied for TF / heparanase procoagulant activity, TF activity and heparanase procoagulant activity using chromogenic assay and heparanase antigen levels by ELISA. RESULTS: Heparanase antigen levels were higher in the study group compared to control (P=0.05). TF / heparanase activity, and even more apparent, heparanase procoagulant activity were significantly higher in the study group compared to controls (P=0.008, P<0.0001, respectively). No significant difference was observed in the TF activity between the groups. Survival of patients with heparanase procoagulant activity higher than 31 ng/ml predicted a mean survival of 9 ± 1.3 months while heparanase procoagulant activity of 31 ng/ml or lower predicted a mean survival of 24 ± 4 months (P=0.001). Heparanase procoagulant activity was higher than 31 ng/ml in the four cases of thrombosis detected during the follow-up period. CONCLUSIONS: Elevated heparanase procoagulant activity in patients with lung cancer reveals a new mechanism of coagulation system activation in malignancy. Heparanase procoagulant activity can potentially be used as a predictor for survival.

A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes.

A genetic map of melon enriched for fruit traits was constructed, using a recombinant inbred (RI) population developed from a cross between representatives of the two subspecies of Cucumis melo L.: PI 414723 (subspecies agrestis) and 'Dulce' (subspecies melo). Phenotyping of 99 RI lines was conducted over three seasons in two locations in Israel and the US. The map includes 668 DNA markers (386 SSRs, 76 SNPs, six INDELs and 200 AFLPs), of which 160 were newly developed from fruit ESTs. These ESTs include candidate genes encoding for enzymes of sugar and carotenoid metabolic pathways that were cloned from melon cDNA or identified through mining of the International Cucurbit Genomics Initiative database (http://www.icugi.org/). The map covers 1,222 cM with an average of 2.672 cM between markers. In addition, a skeleton physical map was initiated and 29 melon BACs harboring fruit ESTs were localized to the 12 linkage groups of the map. Altogether, 44 fruit QTLs were identified: 25 confirming QTLs described using other populations and 19 newly described QTLs. The map includes QTLs for fruit sugar content, particularly sucrose, the major sugar affecting sweetness in melon fruit. Six QTLs interacting in an additive manner account for nearly all the difference in sugar content between the two genotypes. Three QTLs for fruit flesh color and carotenoid content were identified. Interestingly, no clear colocalization of QTLs for either sugar or carotenoid content was observed with over 40 genes encoding for enzymes involved in their metabolism. The RI population described here provides a useful resource for further genomics and metabolomics studies in melon, as well as useful markers for breeding for fruit quality.

The internal pressure and stress environment of the scoliotic intervertebral disc--a review.

The aetiology, in terms of both initiation and progression, of the deformity in idiopathic scoliosis is at present unclear. Even in neuromuscular cases, the mechanisms underlying progression are not fully elucidated. It is thought, however, that asymmetrical loading is involved in the progression of the disease, with evidence mainly from animal studies and modelling. There is, however, very little direct information as to the origin or mechanism of action of these forces in the scoliotic spine. This review describes the concept of intervertebral disc pressure or stress and examines possible measurement techniques. The biological and mechanical consequences of abnormalities in these parameters are described. Future possible studies and their clinical significance are also briefly discussed. Techniques of pressure measurement have culminated in the development of 'pressure profilometry', which provides stress profiles across the disc in mutually perpendicular axes. A hydrated intervertebral disc exhibits mainly hydrostatic behaviour. However, in pathological states such as degeneration and scoliosis, non-hydrostatic behaviour predominates and annular peaks of stress occur. Recent studies have shown that, in scoliosis, high hydrostatic pressures are seen with asymmetrical stresses from concave to convex sides. These abnormalities could influence both disc and endplate cellular activity directly, causing asymmetrical growth and matrix changes. In addition, disc cells could be influenced via nutritional changes consequent to end-plate calcification. Evidence suggests that the stress environment of the scoliotic disc is abnormal, probably generated by high and asymmetrical loading of non-muscular origin. If present in the scoliotic spine during daily activities, this could generate a positive feedback of cellular changes, resulting in curve progression. Future advances in understanding may rely on the development of computer models owing to the difficulties of in-vivo invasive measurements.

Spatially variable retardation plate for beam brightness enhancement in a high-power laser.

We present a simple and robust method for brightness enhancement, efficiently transforming a radially polarized LG (0,1)(*) mode into a nearly Gaussian beam of much higher quality. We use for this a spatially variable retardation plate and a spatial filter. The analysis shows that the transformation yields an increase in brightness by a factor of 3.4. In the experiment, we transformed a high-power Nd:YAG radially polarized (0,1)(*) LG beam with power of 70 W and M(2)=2.6 into a nearly Gaussian beam with M(2)=1.36. This resulted in brightness enhancement by a factor of 2.6.

Efficient extracavity generation of radially and azimuthally polarized beams.

We demonstrate an efficient transformation of a linearly polarized Gaussian beam to a radially or an azimuthally polarized doughnut (0,1)* Laguerre-Gaussian beam of high purity. We use a spatially variable retardation plate, composed of eight sectors of a lambda/2 retardation plate, to transform a linear polarization distribution to radial/azimuthal distribution. We transformed an Nd:YAG Gaussian beam with M(2)=1.3 to a radially and azimuthally polarized (0,1)* Laguerre-Gaussian beams with M(2)=2.5 and degree of radial/azimuthal polarization of 96-98%.

Maternal morbidity following routine second trimester genetic amniocentesis.

Two cases of septic complications of routine second trimester amniocentesis are presented. The first case is a 37-year-old gravida suffering from ulcerative colitis who was admitted for amniocentesis in the 18th week of her third pregnancy. An uncomplicated transabdominal amniocentesis was performed using a sterile technique and ultrasound guidance. Twenty-eight hours later the patient had a septic abortion and sepsis. The second case is a 34-year-old gravida in the 24th week of her pregnancy who was admitted with amnionitis 10 h after an uncomplicated amniocentesis, and subsequently had a septic abortion. A high index of suspicion and rapid intervention were crucial in both cases.

Bilateral periprosthetic stress fractures in a juvenile chronic arthritis patient secondary to bilateral genu valgum.

Lateral insufficiency fractures following total hip replacement have been reported with the femoral stems positioned in varus, together with osteopenia of the lateral femoral cortex. Any abnormal alignment of the lower limbs, such as genu valgum, will alter the load distribution across the femoral cortices, and repetitive loading during walking will predispose the bones to stress fractures at any stress riser point, such as the tip of a femoral component. Bilateral femoral stress fractures post total hip replacements have not been previously described. We present a 55-year-old woman, diagnosed with juvenile idiopathic arthritis, who had undergone bilateral total hip replacements and bilateral knee replacements. The knees 15 years postoperatively were in valgus and the left knee was extremely stiff, flexing to just 5. The patient presented with bilateral thigh pain, with plain radiographs confirming bilateral periprosthetic fractures of the femur at the tip of well-fixed femoral components. There was no history of injury and her hips were functioning well up to this time. The patient required revision of both hips to long-stem uncemented components, bypassing the fractures, and revision of both knees to stemmed semi-constrained implants, thereby correcting the alignment of both lower limbs. Both fractures healed and the patient is currently pain-free and mobile with walking aids. Surgeons must remain aware that when implants are in situ, abnormal alignments will lead to abnormal forces, and stress fractures are likely to occur at any stress riser around the implant. Avoiding malalignment will avoid this complication.

UV disinfection of wastewater effluents for unrestricted irrigation.

Wastewater reuse in arid regions is important for the production of a water resource to be utilised for non-potable purposes and to prevent the environmental transmission of disease-causing agents. This study was conducted to evaluate the effect of water quality on the comparative disinfection efficiency of viruses, bacteria and spores by UV irradiation. Furthermore, the microbial quality of effluent produced by coagulation, high rate filtration (HRF) and either UV irradiation or chlorination was determined. Using low pressure collimated beam, a UV dose of 80 mWs/cm2 was needed to achieve a 3-log10 inactivation of either rotavirus SA-11 or coliphage MS2, whereas over 5-log10 inactivation of E. coli was reached with a dose of only 20 mWs/cm2. B. subtilis inactivation was found to be linear up to a dose of 40 mWs/cm2 and then a tailing up to a UV dose of 120 mWs/cm2 was observed. It is worth noting that effluent turbidity of < 5 NTU did not influence the inactivation efficiency of UV irradiation. Operation of a pilot plant to treat secondary effluent by coagulation, HRF and UV disinfection at a UV dose of 80 mWs/cm2 resulted in the production of high quality effluent in compliance with the Israel standards for unrestricted irrigation (< 10 CFU/100 mL faecal coliform and turbidity of < 5 NTU). Sulphite reducing clostridia (SRC) were found to be more resistant than coliphages and F coliform for UV irradiation. The results of this study indicated that UV disinfection is suitable for the production of effluents for unrestricted irrigation of food crops.

Dominant-negative synthesis suppression of voltage-gated calcium channel Cav2.2 induced by truncated constructs.

Voltage-gated calcium channel alpha1 subunits consist of four domains (I-IV), each with six transmembrane segments. A number of truncated isoforms have been identified to occur as a result of alternative splicing or mutation. We have examined the functional consequences for expression of full-length Ca(v)2.2 (alpha1B) of its coexpression with truncated constructs of Ca(v)2.2. Domains I-II or domains III-IV, when expressed individually, together with the accessory subunits beta1b and alpha2delta-1, did not form functional channels. When they were coexpressed, low-density whole-cell currents and functional channels with properties similar to wild-type channels were observed. However, when domain I-II, domain III-IV, or domain I alone were coexpressed with full-length Ca(v)2.2, they markedly suppressed its functional expression, although at the single channel level, when channels were recorded, there were no differences in their biophysical properties. Furthermore, when it was coexpressed with either domain I-II or domain I, the fluorescence of green fluorescent protein (GFP)-Ca(v)2.2 and expression of Ca(v)2.2 protein was almost abolished. Suppression does not involve sequestration of the Ca(v)beta subunit, because loss of GFP-Ca(v)2.2 expression also occurred in the absence of beta subunit, and the effect of domain I-II or domain I could not be mimicked by the cytoplasmic I-II loop of Ca(v)2.2. It requires transmembrane segments, because the isolated Ca(v)2.2 N terminus did not have any effect. Our results indicate that the mechanism of suppression of Ca(v)2.2 by truncated constructs containing domain I involves inhibition of channel synthesis, which may represent a role of endogenously expressed truncated Ca(v) isoforms.

Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells.

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and ataxia. The ducky gene was mapped previously to distal mouse chromosome 9. High-resolution genetic and physical mapping has resulted in the identification of the Cacna2d2 gene encoding the alpha2delta2 voltage-dependent calcium channel subunit. Mutations in Cacna2d2 were found to underlie the ducky phenotype in the original ducky (du) strain and in a newly identified strain (du(2J)). Both mutations are predicted to result in loss of the full-length alpha2delta2 protein. Functional analysis shows that the alpha2delta2 subunit increases the maximum conductance of the alpha1A/beta4 channel combination when coexpressed in vitro in Xenopus oocytes. The Ca(2+) channel current in acutely dissociated du/du cerebellar Purkinje cells was reduced, with no change in single-channel conductance. In contrast, no effect on Ca(2+) channel current was seen in cerebellar granule cells, results consistent with the high level of expression of the Cacna2d2 gene in Purkinje, but not granule, neurons. Our observations document the first mammalian alpha2delta mutation and complete the association of each of the major classes of voltage-dependent Ca(2+) channel subunits with a phenotype of ataxia and epilepsy in the mouse.

Quality of life before and after laparoscopic Heller myotomy for achalasia.

BACKGROUND: Because the surgical treatment of achalasia is directed at the palliation of chronic symptoms, it is important to assess how surgery affects patients' health-related quality of life (HRQL). METHODS: We evaluated upper gastrointestinal symptoms, satisfaction, and HRQL in 19 patients with achalasia before and after undergoing a laparoscopic Heller myotomy and partial fundoplication. HRQL was assessed using the Medical Outcomes Study 36-item short form health survey (SF-36). RESULTS: The mean age of the patients was 40 years (range 16 to 74), and 58% were men. After a median follow-up of 21 months (range 2 to 35), 12 of 16 patients were satisfied with the results of their surgery. Liquid and solid dysphagia scores were improved after surgery, and the prevalence of heartburn symptoms did not change. Although all the health concepts measured by the SF-36 instrument showed some improvement, statistically significant increases (on a 0 to 100 scale) were detected in physical functioning (11.1, P = 0.02), role-physical (25.0, P = 0.05), bodily pain (12.2, P = 0.01), vitality (13.7, P = 0.02), and social functioning (18.4, P = 0.02). CONCLUSIONS: Most aspects of HRQL improve after a laparoscopic Heller myotomy and partial fundoplication for achalasia.

Enterocyte apoptosis and proliferation are increased in microvillous inclusion disease (familial microvillous atrophy).

Microvillous inclusion disease (MID) is characterized by diffuse villous atrophy without inflammatory changes. While increased apoptosis has been related to mucosal flattening in celiac disease, the role of apoptosis in the pathogenesis of MID is unknown. The aim of this study was to assess the rates of apoptosis and cell proliferation in MID and to compare them with those of normal controls and celiac disease. Small intestinal biopsies from 5 infants with MID, 10 children with normal villous architecture, and 10 children with untreated celiac disease were stained with the terminal uridine deoxynucleotidyl nick end labeling (TUNEL) method to assess apoptotic activity, and with Ki-67 immunohistochemistry to assess cellular proliferation. TUNEL and Ki-67 positive enterocytes were counted in a minimum of 20 well oriented half crypts per section. The percentage of apoptotic cells per crypt (apoptotic index) in normal, MID, and celiac biopsies was 0.03 +/- 0.01%, 0.08 +/- 0.08%, and 0.16 +/- 0.3%, respectively. Significant differences were found between normal and MID, and between normal and celiac cases. The percentage of Ki-67 positive cells per crypt (proliferation index) in normal, MID, and celiac cases was 14 +/- 2.5%, 28 +/- 9.2%, and 56 +/- 14%. Significant differences were found between the 3 groups. In conclusion, (1) enterocyte apoptosis and proliferation are increased in MID; (2) apoptosis appears to be an important factor of cell loss and may be, at least in part, responsible for villous atrophy in MID; and (3) crypts in MID are hyperplastic and not hypoplastic. HUM PATHOL 31:1404-1410.

Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma.

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.

Ion channels in presynaptic nerve terminals and control of transmitter release.

The primary function of the presynaptic nerve terminal is to release transmitter quanta and thus activate the postsynaptic target cell. In almost every step leading to the release of transmitter quanta, there is a substantial involvement of ion channels. In this review, the multitude of ion channels in the presynaptic terminal are surveyed. There are at least 12 different major categories of ion channels representing several tens of different ion channel types; the number of different ion channel molecules at presynaptic nerve terminals is many hundreds. We describe the different ion channel molecules at the surface membrane and inside the nerve terminal in the context of their possible role in the process of transmitter release. Frequently, a number of different ion channel molecules, with the same basic function, are present at the same nerve terminal. This is especially evident in the cases of calcium channels and potassium channels. This abundance of ion channels allows for a physiological and pharmacological fine tuning of the process of transmitter release and thus of synaptic transmission.

Structure of chordae tendineae in the left ventricle of the human heart.

The bicuspid (mitral) valve complex of the human heart consists of functional units which include the valve leaflets, chordae tendineae and the papillary muscles. The mechanical properties of these functional units depend to a large extent on the link between the muscle and the valve. This link is usually arranged in a branching network of avascular tendinous chordae composed of collagen and elastic fibres, which transmit contractions of the papillary muscle to the valve leaflets. In order to perform their function efficiently, the chordae have to possess a high degree of elasticity, as well as considerable strength and endurance. Human chordae tendineae originating from the left ventricles were obtained from 7 embalmed cadavers and 6 postmortem subjects of various ages. Samples washed in saline were fixed or postfixed in 9 % formol saline. Observations were made by illuminating the chordae along their axes. The reflected images originating from the superficial collagenous layers of the relaxed chordae showed a striped pattern 11 microm in width. Scanning electron and light microscopy of the chordae confirmed an undulating pattern of collagen fibrils arranged in bundles of planar waves in register and around the entire circumference of the chorda. The dimensions of the waves correlated with those of the striped reflected pattern. The observed undulating arrangement of the collagen fibrils appears to produce an inherent built-in elasticity which is likely to be of considerable advantage for a tissue which is under continuous repetitive stress. The chordae were covered by endocardium composed of a superficial layer of smooth squamous endothelial cells and an underlying dense layer of elastic fibres. It is suggested that the relaxed striped chordae, consisting of undulating collagen fibrils, straighten when the chordae become stretched by papillary muscle contraction, thereby mitigating the peak stress developed during muscle contraction. On relaxation the elastic tissue tends to return the collagen to its wavy configuration. It is also suggested that the regular wavy pattern of collagen seen in young individuals gradually changes with age by elongation of the wave pattern which eventually becomes randomised. In addition, with increasing age, substantial cushions of connective tissue appear below endocardium while the dense collagenous core has a reduced cross-sectional area which may lead to stretching and eventual rupture of the chordae.

Known calcium channel alpha1 subunits can form low threshold small conductance channels with similarities to native T-type channels.

Native T-type voltage-dependent calcium channels are low voltage-activated and have a small single channel conductance of 5-8 pS, which distinguishes them from any known cloned calcium channels whose conductances are 12-25 pS. Here, we show that when alpha1B, alpha1E, or alpha1C are expressed in COS7 cells, which contain no endogenous calcium channel subunits or calcium channels, they each exhibit a 4-7 pS channel as well as a large conductance channel. At low depolarizations, or when the alpha1 subunit is expressed in the absence of auxiliary alpha2-delta or beta subunits, the small conductance channels are seen alone, and their biophysical properties, including voltage dependence and kinetics of activation and inactivation, are very similar to native T-type calcium channels.