Computational Tools for Quantifying Concordance in Single-Cell Fate.

Cells are dynamic biological systems that interact with each other and their surrounding environment. Understanding how cell extrinsic and intrinsic factors control cell fate is fundamental to many biological experiments. However, due to transcriptional heterogeneity or microenvironmental fluctuations, cell fates appear to be random. Individual cells within well-defined subpopulations vary with respect to their proliferative potential, survival, and lineage potency. Therefore, methods to quantify fate outcomes for heterogeneous populations that consider both the stochastic and deterministic features of single-cell dynamics are required to develop accurate models of cell growth and differentiation. To study random versus deterministic cell behavior, one requires a probabilistic modelling approach to estimate cumulative incidence functions relating the probability of a cell's fate to its lifetime and to model the deterministic effect of cell environment and inheritance, i.e., nature versus nurture. We have applied competing risks statistics, a branch of survival statistics, to quantify cell fate concordance from cell lifetime data. Competing risks modelling of cell fate concordance provides an unbiased, robust statistical modelling approach to model cell growth and differentiation by estimating the effect of cell extrinsic and heritable factors on the cause-specific cumulative incidence function.

Analysis of cardiac stem cell self-renewal dynamics in serum-free medium by single cell lineage tracking.

Cardiac colony forming unit-fibroblasts (cCFU-F) are a population of stromal cells residing within the SCA1+/PDGFRα+/CD31- fraction of adult mouse hearts, and which have functional characteristics akin to bone marrow mesenchymal stem cells. We hypothesise that they participate in cardiac homeostasis and repair through their actions as lineage progenitors and paracrine signaling hubs. However, cCFU-F are rare and there are no specific markers for these cells, making them challenging to study. cCFU can self-renew in vitro, although the common use of serum has made it difficult to identify cytokines that maintain lineage identity and self-renewal ability. Cell heterogeneity is an additional confounder as cCFU-F cultures are metastable. Here, we address these limitations by identifying serum-free medium (SFM) for growth, and by using cCFU-F isolated from PdgfraGFP/+ mice to record fate outcomes, morphology and PDGFRα expression for hundreds of single cells over time. We show that SFM supplemented with basic fibroblast growth factor, transforming growth factor-ß and platelet-derived growth factor, enhanced cCFU-F colony formation and long-term self-renewal, while maintaining cCFU-F potency. cCFU-F cultured in SFM maintained a higher proportion of PDGFRα+ cells, a marker of self-renewing cCFU-F, by increasing Pdgfra-GFP+ divisions and reducing the probability of spontaneous myofibroblast differentiation.

Quantifying intrinsic and extrinsic control of single-cell fates in cancer and stem/progenitor cell pedigrees with competing risks analysis.

The molecular control of cell fate and behaviour is a central theme in biology. Inherent heterogeneity within cell populations requires that control of cell fate is studied at the single-cell level. Time-lapse imaging and single-cell tracking are powerful technologies for acquiring cell lifetime data, allowing quantification of how cell-intrinsic and extrinsic factors control single-cell fates over time. However, cell lifetime data contain complex features. Competing cell fates, censoring, and the possible inter-dependence of competing fates, currently present challenges to modelling cell lifetime data. Thus far such features are largely ignored, resulting in loss of data and introducing a source of bias. Here we show that competing risks and concordance statistics, previously applied to clinical data and the study of genetic influences on life events in twins, respectively, can be used to quantify intrinsic and extrinsic control of single-cell fates. Using these statistics we demonstrate that 1) breast cancer cell fate after chemotherapy is dependent on p53 genotype; 2) granulocyte macrophage progenitors and their differentiated progeny have concordant fates; and 3) cytokines promote self-renewal of cardiac mesenchymal stem cells by symmetric divisions. Therefore, competing risks and concordance statistics provide a robust and unbiased approach for evaluating hypotheses at the single-cell level.

Effect of sire fertility and timing of artificial insemination in a Presynch + Ovsynch protocol on first-service pregnancy rates.

Sire fertility may influence pregnancy rate (PR) by differences in sperm survival in the female reproductive system and time required for capacitation and transport of sperm to site of fertilization. A predicted fertility index, Estimated Relative Conception Rate, was used to select 3 high-fertility artificial insemination (AI) sires (+3) and 3 average AI sires (-1). Ovulation can be predicted to occur at approximately 28 h following GnRH administration when used in an Ovsynch protocol. The objective of this study was to determine if AI at 2 times, 0 or 24 h after GnRH administration, in a Presynch + Ovsynch protocol resulted in different first-service PR when average or high-fertility sires were used. Lactating Holstein cows (n = 1,457) from 2 dairy herds located in the Piedmont region of North Carolina were utilized for 12 mo. Timing of AI did not affect first AI PR and no interaction of sire-fertility group and timing of AI was detected. First AI PR did not differ between sire-fertility groups (23.2 vs. 29.4%) for average and high-fertility groups, respectively. First-lactation cows were 53% more likely to conceive than older cows, and cows bred during April through June were 66% less likely to become pregnant compared with cows bred from October through January. No interactions were detected among parity, season, sire-fertility group, or time of AI. Using only 3 sires per group based on Estimated Relative Conception Rate estimates resulted in large variability of sire conception within groups, although group averages differed by 6 points.

Effect of presence of clinical and subclinical endometritis at the initiation of Presynch-Ovsynch program on the first service pregnancy in dairy cows.

The present study examined the effect of presence of clinical or subclinical endometritis at the initiation of Presynch-Ovsynch estrous synchronization program on the first service pregnancy rate in dairy cows. Lactating Holstein cows (N=275) were given a thorough reproductive examination at 32-38 days in milk, 3 days prior to the scheduled start of Presynch-Ovsynch program. Based on the reproductive exam findings the cows were diagnosed and classified into three groups as clinical endometritis, subclinical endometritis and normal. All cows received two set-up injections of 25mg PGF(2alpha) (Lutalyse((R)), Pfizer Animal Health, New York, NY, USA) i.m., 14 days apart starting at 35-42 days in milk (DIM). All cows received 75microg of GnRH (Cystorelin, Merial, Iselin, NJ, USA) i.m. 14 days after the second pre-synchronization injection of PGF(2alpha), followed by a third injection of 25mg PGF(2alpha) i.m. 7 days later. Cows received a second injection of 75microg of GnRH i.m. 54h after the third PGF(2alpha), and received timed artificial insemination at the time of the second GnRH injection or 24h later. Multivariate logistic regression was used to analyze the odds of pregnancy at the first service. Variables included in the model were endometritis status (clinical endometritis, subclinical endometritis and normal), farm (two), presence of corpus luteum (CL, yes or no), timing of second GnRH in relation to AI (0 or 24h), sire fertility (bulls with greater compared with lesser estimated relative conception rates), parity (primiparous and multiparous) and their interactions. Of all variable included in the model, cows with corpus letuem (OR=1.83 versus OR=1.00; P=0.05) 3 days prior to the scheduled start of Presynch-Ovsynch program and primiparous cows (OR=1.00 versus OR=0.55; P=0.04) had increased odds of becoming pregnant at the first service. No differences were found in the odds of first service pregnancy among clinical, subclinical endometritis and normal cows (P>0.1). In summary, presence of clinical or subclinical endometritis at the initiation of Presynch-Ovsynch estrous synchronization program does not harm the first service pregnancy rate in dairy cows.

Fertility following fixed-time AI or insemination at observed estrus in Ovsynch and Heatsynch programs in lactating dairy cows.

The objective of this study was to compare the conception rate for fixed-timed artificial insemination (FTAI) and observed heat artificial insemination (HAI) prior to the scheduled FTAI in Ovsynch and Heatsynch synchronization protocols. In Experiment 1, lactating dairy cows (n=535) received two set-up injections of 25mg prostaglandin F(2alpha) (PGF(2alpha)) i.m., 14 days apart starting at 36+/-3 days in milk (DIM). Cows were blocked by parity and were randomly allocated to either Ovsynch or Heatsynch groups. All cows received 100 microg of GnRH i.m. 14 days after the second set-up injection of PGF(2alpha), followed by a third injection of 25mg PGF(2alpha) i.m., 7 days later. In the Ovsynch group, HAI cows (n=29) were bred on standing estrus after the third PGF(2alpha) before the scheduled second GnRH, whereas FTAI cows (n=218) that were not observed in estrus, received a second injection of 100 microg of GnRH i.m., 48 h after the third PGF(2alpha) and received TAI 8 h after the second GnRH. In the Heatsynch group, all cows (n=288) received 0.5 mg of estradiol cypionate (ECP) 24 h after third PGF(2alpha) and HAI cows (n=172) were bred on standing estrus and FTAI cows (n=116) that were not observed in estrus, received TAI 72 h after the third PGF(2alpha). In Experiment 2, repeat breeder cows (n=186) were randomly assigned to either Ovsynch or Heatsynch groups. The FTAI and HAI cows were inseminated similar to Experiment 1. All cows were observed for estrus three times daily. The associations with the conception rate were modeled with logistic regression separately for Experiments 1 and 2. Of all the variables included in the model in Experiment 1, type of AI (HAI versus FTAI, P=0.0003) and parity (primiparous versus multiparous, P=0.05) influenced the first service conception rate. Over-all conception rate and first service conception rate for HAI cows were higher compared to FTAI cows (33.8% versus 21.3%, and 35.3% versus 21.0%; P=0.001). In the Heatsynch group, cows that received HAI had significantly higher over-all conception rate and first service conception rate compared to FTAI (35.2% versus 17.3% and 36.0% versus 15.5%; P=0.0001). The conception rates in repeat breeder cows for HAI and FTAI (30.1% versus 22.3%) were not different (P>0.1). In conclusion, it was recommended to include AI at observed estrus and fixed-time AI for cows not observed in estrus in order to improve the conception rate in synchronization protocols.

Recent declines in PAH, PCB, and toxaphene levels in the northern Great Lakes as determined from high resolution sediment cores.

Sediment cores were collected from two sites in Grand Traverse Bay, Lake Michigan in May 1998, dated using 210Pb geochronology, and analyzed for polychlorinated biphenyl (PCB) congeners, polycyclic aromatic hydrocarbons (PAHs), and toxaphene. The extraordinarily high sediment focusing and accumulation rates in these cores relative to other Great Lakes sediments allowed quantification of high-resolution temporal trends in the burial of hydrophobic organic contaminants. The focus-corrected accumulation rate of total PCBs (sum of 105 congeners) in 1998 was 0.50 ng/cm2-year at both sites. Toxaphene and total PAH (t-PAH; sum of 33 compounds) surficial accumulations varied at each site and ranged from 0.08 to 0.41 ng/cm2-year for toxaphene and 25 to 52 ng/cm2-yr for t-PAHs at the two sites. The maximum t-PAH accumulation rate was in sediment dated from 1942, and PAH accumulation decreased from 1942 to 1980 with a first-order rate of decline 0.017 yr(-1). Both toxaphene and t-PCB accumulations peaked in sediment deposited in 1972, afterwhich their accumulations decreased with nearly identical rates of decline (0.027 yr(-1) and 0.028 yr(-1), respectively).

Prescribing rates for psychotropic medication amongst east London general practices: low rates where Asian populations are greatest.

OBJECTIVES: The aim of this study was to examine the contribution of Asian ethnicity to the variation in rates of practice prescribing for antidepressant and anxiolytic medication, taking into account other population and practice organizational factors. METHODS: A practice-based cross-sectional survey was carried out of the prescribing of antidepressants and anxiolytics (daily defined dosages) in 164 general practices. The study was set in East London and the City Health Authority, which includes the multiethnic inner London boroughs of Hackney, Tower Hamlets, Newham and the City of London. The main outcome measures were the annual prescribing rates for each group of drugs, calculated as the total annual daily defined dosages divided by the practice population, and the ratio of antidepressant/ anxiolytic annual prescribing rates. RESULTS: Prescribing rates for antidepressants showed a 25-fold variation between practices; this was greater for anxiolytics. The median annual prescribing rate for all antidepressants combined was 4.13 (interquartile range 2.50-5.88). For all anxiolytics and hypnotics combined the median annual prescribing rate was 3.55 (interquartile range 1.71-6.36). Univariate analysis showed that Asian ethnicity alone accounted for 28% of the variation in antidepressant prescribing and 20.5% of the variation in the anxiolytic prescribing. A backwards multiple regression model using 10 explanatory practice and population variables accounted for 47.7% of the variance in antidepressant prescribing and 34% of the variance in the anxiolytic prescribing. CONCLUSION: In practices where the proportion of Asian patients is high, both antidepressant and anxiolytic prescribing is low. This is important for understanding interpractice prescribing variation and for setting levels of drug budgets. This study confirms that the low rates of non-psychotic disorders presented by Asian populations is not a selective feature of access to secondary care, but is evident in the prescribing behaviour of GPs. Uncertainty remains as to how much this is due to a lower prevalence rate, "culture-bound syndromes" or practical difficulties in diagnosis and management within the general practice setting.

Pink triangles: antecedents and consequences of perceived workplace discrimination against gay and lesbian employees.

A model of perceived sexual orientation discrimination was tested in a national sample of 534 gay and lesbian employees. The effects of legislation, organizational policies and practices, and work group composition on perceived sexual orientation discrimination were examined, as well as the attitudinal and organizational outcomes associated with discrimination. Gay employees were more likely to report discrimination when employed in groups that were primarily heterosexual and in organizations that lacked supportive policies and were not covered by protective legislation. Disclosure of sexual orientation at work was related to discrimination and antecedent variables. Perceived discrimination was associated with negative work attitudes and fewer promotions. Organizational policies and practices had the strongest impact on perceived discrimination and were directly related to outcomes.

Odor recognition and identification: effect of labels over time.

The effect of labels on recognition and identification of odors over time was assessed. 30 men and 30 women were presented 20 odors; half of the participants were also told a name for the odor as a label. Five min. and 60 min. later, all participants were given 20 odors (10 from the original set, 10 new) and asked whether each odor was new or old (odor recognition). The group given labels was also asked to recall the label provided (odor identification). Analysis indicated a significant effect of time on recognition. Significantly more odors were recognized at 5 min. than 60 min. The effect of label was also significant, with recognition being better for the Label condition than the No-Label condition. As for odor identification, women identified more labels than did men. Overall, odor recognition was better with labels soon after exposure, and the women were better at remembering the labels than the men.

Do GPs prescribe antidepressants differently for South Asian patients?

BACKGROUND: In spite of evidence from controlled trials and published guidelines, general practitioners prescribe antidepressants in lower doses and for shorter courses than are recommended [2]. However, these studies have not examined the effect of ethnicity on antidepressant prescribing by general practitioners. OBJECTIVES: To compare the antidepressant treatment of South Asian patients with White patients. METHODS: Patients, between 16 and 65 years prescribed an antidepressant between November 1993-1995, were selected from an east London training practice by searching the practice computer system EMIS. From a total of 438 patients identified, 40 cases were selected on the basis of their surname [3] as South Asian, and 50 cases formed the White comparison group. Data was collected retrospectively from the computer and paper records and analysed using Stata. The main outcome measures were presenting symptoms, maximum dose of antidepressant prescribed, duration of treatment and continuity of care. RESULTS: Women formed 2/3 of each group, the mean age in both groups being similar. Psychological symptoms were noted in the majority of both groups, but South Asians presented more physical complaints than the White group (67.5% compared to 22%, Chi squared=18.86, P=0.00001). The South Asian group were significantly more likely to be prescribed amitriptyline at doses of 75 mg or less than the White group (Fisher exact 2 tailed test, P=0.008), had significantly shorter median durations of antidepressant treatment (60 days, compared with 160 days for the White group, Mann Whitney test P=0.005). No differences were found between the groups in their continuity of care. CONCLUSION: The results suggest that successful drug treatment of depressed South Asian patients may be less likely than in White patients.

Muscle rehabilitation in haemophilia.

Musculoskeletal dysfunction is a common manifestation of haemophilia. This dysfunction may be associated with imbalances between muscle groups. Evidence emerging from the literature suggests that the rehabilitation of this dysfunction is very relevant for the patient with musculoskeletal problems. Treatment of muscle imbalance may be linked with a reduction in recurrence of symptoms. Further research is needed to establish the relevance of this area in patients with haemophilia but the clinical evidence supports the developing work in this field.

Neuropsychological deficits in probands from multiply-affected schizophrenic families.

This study was designed to determine if schizophrenics from families with more than one psychotic relative show more severe neuropsychological deficits than schizophrenics with only one psychotic relative, non-familial schizophrenics, and a group of matched normal controls. Eighty-one schizophrenic-spectrum patients were divided into three groups on the basis of the presence of psychotic disorder among first- and second-degree relatives. The three groups of schizophrenics and the normal controls were compared for differences on a brief neuropsychological testing battery. The four groups showed significant multivariate differences. Patients from multiply-affected families showed significantly greater neuropsychological dysfunction on measures of abstract concept formation, visuomotor-coordination, and attention than patients from families that had only one psychotic relative. Schizophrenics from low-density families showed more severe deficits in fine motor-control than non-familial schizophrenics. These data suggest that abnormalities in those frontal systems that are likely to mediate fine motor control and abstract concept formation may be related to the degree of familial loading for psychotic disorder.

Maintenance treatment of the anemia of myelodysplastic syndromes with recombinant human granulocyte colony-stimulating factor and erythropoietin: evidence for in vivo synergy.

Patients with myelodysplastic syndromes (MDS) have refractory cytopenias leading to transfusion requirements and infectious complications. In vitro marrow culture data have indicated that granulocyte colony stimulating factor (G-CSF) synergizes with erythropoietin (EPO) for the production of erythroid precursors. In an effort to treat the anemia and neutropenia in this disorder, MDS patients were treated with a combination of recombinant human EPO and recombinant human G-CSF. Fifty-five patients were enrolled in the study of which 53 (96%) had a neutrophil response. Forty-four patients were evaluable for an erythroid response of which 21 (48%) responded. An erythroid response was significantly more likely in those patients with relatively low serum EPO levels, higher absolute basal reticulocyte counts and normal cytogenetics at study entry. Seventeen (81%) of the patients who responded to combined G-CSF plus EPO therapy continued to respond during an 8-week maintenance phase. G-CSF was then discontinued and all patients' neutrophil responses were diminished, whereas 8 continued to have an erythroid response to EPO alone. In 7 of the remaining 9 patients, resumption of G-CSF was required for recurrent erythroid responses. The median duration of erythroid responses to these cytokines was 11 months, with 6 patients having relatively prolonged and durable responses for 15 to 36 months. Our results also indicate that approximately one half of responding patients require both G-CSF and EPO to maintain an effective erythroid response, suggesting that synergy between G-CSF and EPO exists in vivo for the production of red blood cells in MDS.

A preliminary study of the neuropsychological heterogeneity of familial schizophrenia.

This exploratory study describes the heterogeneity of the neuropsychological deficits that characterize familial schizophrenia. Forty-six familial schizophrenics showed significantly more variability in abstraction and problem-solving and motor control than 39 non-familial schizophrenics. Cluster analyses of these two neuropsychological parameters indicate that while the non-familial schizophrenics fall into one homogeneous cluster, the familial schizophrenics fall into three relatively distinct clusters which differ significantly in their morbid risk for schizophrenic-spectrum disorder. These preliminary data suggest that frontal lobe deficits are associated with an increased familial risk for schizophrenia.