Associations between anogenital distance and measures of fertility in lactating North American Holstein cows: A validation study.

Anogenital distance (AGD) has been defined in dairy cows as the distance from the center of the anus to the base of the clitoris. Initial reports on nulliparous Holstein heifers and first- and second-parity Holstein cows have found inverse relationships between AGD and measures of fertility. Our primary objective was to determine the relationship between AGD and measures of fertility in a larger population of North American Holstein cows to validate our previous finding that AGD is inversely related to fertility. Secondary objectives were to determine the associations between AGD and parity, and milk yield. Using digital calipers, we measured AGD in 4,709 Holstein cows [mean ± standard deviation (SD); parity 2.3 ± 1.4; days in milk (DIM) 154 ± 94; 305-d mature equivalent (ME) milk yield 13,759 ± 2,188 kg] from 18 herds in Western Canada and 1 herd in the USA. Anogenital distance (mm) was normally distributed with a mean (±SD) of 132 ± 12, ranging from 95 to 177, and a median of 133. Anogenital distance was linearly but inversely associated with pregnancy to first artificial insemination (P/AI1). For every 1-mm increase in AGD, the estimated probability of P/AI1 decreased by 0.8%. The optimum AGD cut-point that predicted probability of P/AI1 with sensitivity and specificity of 45 and 55%, respectively, was 129 mm. Consequently, data were categorized into either short (≤129) or long (>129) AGD groups across parities, and associations between AGD, parity (first, second, and third+), and fertility measures were determined. Rates of P/AI1 were greater (36 vs. 30%) in short- than in long-AGD cows; short-AGD cows required fewer AI per conception (2.3 vs. 2.4) and had fewer days open (137 vs. 142), and a greater proportion of short-AGD cows (67 vs. 64%) was pregnant by 150 DIM compared with long-AGD cows. The rates of pregnancy up to 150 (hazard ratio of 0.91) and 250 DIM (hazard ratio of 0.93) were smaller in long- than in short-AGD cows. Anogenital distance had a weak positive association with both parity (r = 0.22) and 305-d ME milk yield (r = 0.04). Results indicate an inverse relationship between AGD and measures of fertility in lactating cows, validating our earlier report. We infer that although selecting cows for short AGD is expected to have an adverse effect on milk yield, the anticipated gain in fertility will outweigh the small decline in milk yield, strengthening the potential of AGD as a novel reproductive phenotype for use in future breeding programs to improve fertility.

Relationship of anogenital distance with fertility in nulliparous Holstein heifers.

Anogenital distance (AGD), defined as the distance from the center of the anus to the base of the clitoris, in lactating dairy cows of first and second parity, has been reported to be inversely related to fertility and moderately heritable. Thus, AGD may be a useful reproductive phenotype for future genetic selection to improve fertility. The objectives of this study were to (1) characterize AGD in nulliparous dairy heifers; and (2) determine if the inverse relationship between AGD and fertility, found in lactating dairy cows, is also evident in nulliparous heifers. We measured AGD in 1,692 Holstein heifers from 16 herds in Western Canada (Alberta and British Columbia) and one herd in the United States (Washington State). Data were analyzed using MEANS, UNIVARIATE, LOGISTIC, ROC, GLIMMIX, and LIFETEST procedures of SAS (SAS Institute Inc.). Mean (±standard deviation) age at AGD measurement was 13.9 ± 1.5 mo, and AGD was normally distributed with a mean of 107.3 ± 10.5 mm, ranging from 69 to 142 mm. With every 1-mm increase in AGD, the predicted probability of pregnancy was reduced by 1.9%. Receiver operating characteristic curve analysis was used to determine the optimum threshold AGD that predicted the probability of pregnancy. Based on the optimum threshold AGD, data from heifers were categorized into short (≤110 mm) and long (>110 mm) AGD groups, and associations between AGD groups and fertility measures were determined. Heifers with short AGD required fewer services per conception (1.5 vs. 1.7) than heifers with long AGD. Consequently, heifers with short AGD conceived earlier (448.4 vs. 454.3 d) and had greater pregnancy to first AI than those with long AGD (58.3 vs. 49.6%). Moreover, heifers with long AGD had reduced hazard (hazard ratio of 0.59) for pregnancy up to 450 d of life compared with those with short AGD. In summary, AGD was normally distributed and highly variable in the population. In addition, an inverse relationship between AGD and fertility measures in nulliparous heifers was evident, confirming an earlier report of a similar relationship in lactating dairy cows. These findings strengthen the potential for AGD to be used as a fertility trait and management tool in future selection programs.

Does a first-case on-time-start initiative achieve its goal by starting the entire process earlier or by tightening the distribution of start times?

BACKGROUND: We explored whether a previously successful initiative to improve first-case on-time starts succeeded because (i) preoperative steps started earlier (but the process did not necessarily improve) or (ii) the process was brought into better control. METHODS: We analysed 35 months of data comprising 28 882 first cases to calculate the difference of the time a patient entered the operating room (OR) vs the scheduled entry time. Median and inter-quartile range were used to evaluate changes in distribution parameters. A statistical process-control methodology was used to compare the differences in performance between the pre- and post-intervention phases. RESULTS: Post-intervention first cases entered the OR on average within 4 min [95% confidence interval (CI): 4-5 min] of the scheduled start time, as opposed to within 8 min (95% CI: 8-8 min) in the pre-intervention period. The median delay decreased from 5 min (95% CI: 5-5 min) to 2 min (95% CI: 2-2 min). The inter-quartile range of the difference between the scheduled start time and the first case in room time decreased from 13 min (95% CI: 13-13 min) to 10 min (95% CI: 9-10 min). CONCLUSIONS: The reduction in inter-quartile range demonstrates that improvement in on-time starts resulted from the process being in better control. The start time of preoperative preparatory activities did not move earlier, which means that OR and preoperative staff do not need to arrive at work earlier. Improvements resulting from the process being in control were sustained.

Enniatin B-induced cell death and inflammatory responses in RAW 267.4 murine macrophages.

The mycotoxin enniatin B (EnnB) is predominantly produced by species of the Fusarium genera, and often found in grain. The cytotoxic effect of EnnB has been suggested to be related to its ability to form ionophores in cell membranes. The present study examines the effects of EnnB on cell death, differentiation, proliferation and pro-inflammatory responses in the murine monocyte-macrophage cell line RAW 264.7. Exposure to EnnB for 24 h caused an accumulation of cells in the G0/G1-phase with a corresponding decrease in cyclin D1. This cell cycle-arrest was possibly also linked to the reduced cellular ability to capture and internalize receptors as illustrated by the lipid marker ganglioside GM1. EnnB also increased the number of apoptotic, early apoptotic and necrotic cells, as well as cells with elongated spindle-like morphology. The Neutral Red assay indicated that EnnB induced lysosomal damage; supported by transmission electron microscopy (TEM) showing accumulation of lipids inside the lysosomes forming lamellar structures/myelin bodies. Enhanced levels of activated caspase-1 were observed after EnnB exposure and the caspase-1 specific inhibitor ZYVAD-FMK reduced EnnB-induced apoptosis. Moreover, EnnB increased the release of interleukin-1 beta (IL-1ß) in cells primed with lipopolysaccharide (LPS), and this response was reduced by both ZYVAD-FMK and the cathepsin B inhibitor CA-074Me. In conclusion, EnnB was found to induce cell cycle arrest, cell death and inflammation. Caspase-1 appeared to be involved in the apoptosis and release of IL-1ß and possibly activation of the inflammasome through lysosomal damage and leakage of cathepsin B.

Postoperative residual curarization from intermediate-acting neuromuscular blocking agents delays recovery room discharge.

BACKGROUND: Postoperative residual curarization (PORC) [train-of-four ratio (T4/T1) <0.9] is associated with increased morbidity and may delay postoperative recovery room (PACU) discharge. We tested the hypothesis that postoperative T4/T1 <0.9 increases PACU length of stay. METHODS: At admission to the PACU, neuromuscular transmission was assessed by acceleromyography (stimulation current: 30 mA) in 246 consecutive patients. The potential consequences of PORC-induced increases in PACU length of stay on PACU throughput were estimated by application of a validated queuing model taking into account the rate of PACU admissions and mean length of stay in the joint system of the PACU plus patients recovering in operation theatre waiting for PACU beds. RESULTS: PACU length of stay was significantly longer in patients with T4/T1 <0.9 (323 min), compared with patients with adequate recovery of neuromuscular transmission (243 min). Age (P=0.021) and diagnosis of T4/T1 <0.9 (P=0.027), but not the type of neuromuscular blocking agent, were independently associated with PACU length of stay. The incidence of T4/T1 <0.9 was higher in patients receiving vecuronium. Delayed discharge significantly increases the chances of patients having to wait to enter the PACU. The presence of PORC is estimated to be associated with significant delays in recovery room admission. CONCLUSIONS: PORC is associated with a delayed PACU discharge. The magnitude of the effect is clinically significant. In our system, PORC increases the chances of patients having to wait to enter the PACU.

Low numbers of regulatory T cells in common variable immunodeficiency: association with chronic inflammation in vivo.

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by defective immunoglobulin production and high frequency of bacterial infections, autoimmunity and manifestations of chronic inflammation. Abnormalities of CD4+CD25high forkhead box P3 (FoxP3)+ regulatory T cells (Treg) have been associated with autoimmune and inflammatory disorders, and we hypothesized that CVID might be characterized by Treg abnormalities. CD3+ cells from patients and controls were analysed for the expression of FoxP3 mRNA by real time reverse transcription-polymerase chain reaction (RT-PCR). Peripheral blood mononuclear cells from CVID patients and controls were stained for Treg markers, analysed by flow cytometry and compared to clinical characteristics. The main findings were: (i) CVID patients had significantly decreased expression of FoxP3 mRNA and decreased proportions of CD4+CD25highFoxP3+ cells compared to controls; (ii) CVID patients with splenomegaly had even lower proportions of Treg compared to other patients and controls; (iii) serum levels of the inflammatory marker neopterin were correlated negatively with the proportions of Treg within the CVID population, while there was no significant association with bronchiectasis. We have demonstrated decreased proportions of Treg in CVID patients, particularly in those with signs of chronic inflammation. Decreased proportions of TReg are suggested to be pathogenetically important in autoimmunity, and our results suggest that TReg may have a similar role in CVID.

A survey of physician attitudes and practices concerning cost-effectiveness in patient care.

OBJECTIVE: To identify physicians' views regarding cost-containment and cost-effectiveness and their attitudes and experience using cost-effectiveness in clinical decision making. DESIGN: A close-ended 30-item written survey. SUBJECTS: 1,000 randomly selected physicians whose practices currently encompass direct patient care and who work in the California counties of Sacramento, Yolo, Placer, Nevada, and El Dorado. OUTCOME MEASURES: Physician attitudes about the role of cost and cost-effectiveness in treatment decisions, perceived barriers to cost-effective medical practice, and response of physicians and patients if there are conflicts about treatment that physicians consider either not indicated or not cost-effective. RESULTS: Most physicians regard cost-effectiveness as an appropriate component of clinical decisions and think that only the treating physician and patient should decide what is cost-worthy. However, physicians are divided on whether they have a duty to offer medical interventions with remote chances of benefit regardless of cost, and they vary considerably in their interactions with patients when cost-effectiveness is an issue. CONCLUSION: Although physicians in the Sacramento region accept cost-effectiveness as important and appropriate in clinical practice, there is little uniformity in how cost-effectiveness decisions are implemented.

The effect of educational gifts from pharmaceutical firms on medical students' recall of company names or products.

PURPOSE: To assess the influence of pharmaceutical advertising (in the form of books) directed at medical students and also to examine students' attitudes toward pharmaceutical representatives after interacting with them. METHOD: Two groups of fourth-year medical students were surveyed: 166 residency applicants to the Department of Anesthesia and Critical Care between 1991 and 1993, who were questioned during their personal interviews with the department chair, and 39 fourth-year students from the University of Chicago Pritzker School of Medicine in 1994-95, who were surveyed by telephone. The students were asked if they had ever received a book from a pharmaceutical representative and, if so, to name the book. Then they were asked to name the book-giving company or a product associated with the company. Responses were compared using chi-square analysis. RESULTS: In all, 90% of the students had received one or more books and accurately recalled titles for 89% of them. However, only 25% of the named books were accurately associated with a pharmaceutical company or product. The Pritzker students, asked to recall interactions with pharmaceutical representatives, reported being skeptical of representatives who ignored them because they were students, but they rated as helpful and informative those who conversed with them or gave them gifts. CONCLUSION: Although gifts to medical students do not necessarily engender company or product recall, attention paid to medical students by pharmaceutical representatives engenders goodwill toward the representatives and their messages.

Context dependence of mutational effects in a protein: the crystal structures of the V35I, I47V and V35I/I47V gene V protein core mutants.

The basis for the context dependence of the effects of core mutations on protein stability was investigated by comparing the structures of three gene V protein mutants with that of the wild-type protein. We previously examined a "swapped" mutant in which core residues Val35 and Ile47 were simply reversed so that the mutant had no hydrophobicity change from the native protein. The swapped mutant was destabilized by 3 kcal/mol per gene V protein dimer relative to the wild-type protein, demonstrating that factors other than hydrophobicity must make substantial contributions to the effects of mutations on the stability of the protein. Here we have determined the structure of this swapped mutant (V35I/I47V) as well as those of the two constituent mutants (V35I and I47V). We find that the structures of the mutant proteins are very similar to that of the wild-type protein except for the necessary addition or deletion of methylene groups and for slight positional shifts of atoms around each mutated residue. The structure of the double mutant is a composite of the structures of the two single mutants. In the mutant structures, the V35I mutation fills a cavity that exists in the wild-type protein and the I47V mutation creates a new cavity. The structures of the mutants indicate further that the reason the V35I and I47V mutations do not have opposite effects on stability is that the cavity in the wild-type protein filled by the V35I mutation is not optimally shaped for accommodating the additional methylene group of the isoleucine. These results support the concepts that the details of core packing have substantial influence on the effects of core mutations on protein stability and that these packing effects are major determinants of the context dependence of core mutation effects on stability.

Loss of alveolar macrophages during anesthesia and operation in humans.

Pulmonary macrophages play an important role in the host defense against infection, and the importance of this role is probably enhanced when the upper airway defenses are circumvented by endotracheal intubation. Studies in animals suggest that exposure to volatile anesthetics compromises the viability and function of alveolar macrophages. We studied the effect of surgery and anesthesia on the alveolar macrophages of 41 human subjects undergoing lower abdominal procedures of varying lengths during nitrous oxide-isoflurane anesthesia. Alveolar macrophages were harvested from bronchoalveolar lavage fluid obtained before incision and compared to those recovered just before emergence from anesthesia. Macrophages were analyzed for aggregation and viability, assessed by the ability of viable cells to exclude trypan blue dye. Operations lasting 2 h or less led to little aggregation and had little effect on viability. However, there was a strong correlation between loss of macrophages and the duration of surgery and anesthesia. Aggregation increased and viability decreased as a function of procedure length. Studies are needed to determine whether prolonged surgery contributes to the incidence of postoperative pulmonary complications by disturbing the function and survival of alveolar macrophages in humans.

Relationship between in vivo activity and in vitro measures of function and stability of a protein.

The in vivo activities of mutant proteins are readily measured and can potentially be used to estimate changes in in vitro properties such as stability or function, but this connection has not been rigorously established. Gene V protein is a small protein produced by bacteriophage f1 that binds to single-stranded DNA and to RNA and for which fitness can be assayed both in vivo and in vitro. We have assembled a large number of temperature-sensitive mutants of the gene V protein of bacteriophage f1 and measured their ability to support phage growth and replication in vivo. We have also purified many of these mutant gene V proteins and measured their stabilities and ssDNA binding affinities in vitro. Mutations at surface residues frequently yielded temperature-sensitive mutants, but remarkably, no overall correlation between in vivo activity and in vitro measures of either stability or function was found for this group. Mutations at buried residues often lead to the temperature-sensitive phenotype. At buried sites temperature sensitivity was strongly correlated with in vitro stability changes, but not with in vitro ssDNA binding affinity. The implication of these observations for protein engineering efforts is that phenotypes conferred by amino acid substitutions at buried sites can be used to identify mutants whose stabilities fall into ranges of interest, while phenotypes of mutants with surface substitutions may be much less readily interpreted, even in the case of a single-stranded-DNA-binding protein.

In vivo characterization of mutants of the bacteriophage f1 gene V protein isolated by saturation mutagenesis.

The gene V protein of bacteriophage f1 binds to single-stranded nucleic acids and is essential for propagation of phage f1. We tested the function of gene V protein mutants with single amino acid substitutions in two ways: by the ability of the mutant proteins to support phage growth, and by the ability of the mutant proteins, when expressed at high levels, to inhibit the growth of Escherichia coli. The results of the tests were used to identify sites in the protein that are relatively tolerant or intolerant to substitution, where tolerant sites are defined as those where most substitutions do not affect the function of the protein. The two assays generally yielded similar results for the tolerance of sites to substitution. Many sites that are less than 10% exposed to the solvent are relatively intolerant of substitution, with even very conservative substitutions leading to loss of function in some cases such as Ile to Leu at residue 6. Some buried sites such as Ile47 are more tolerant, with even a substitution of Ile to Thr leading to a functional protein based on the ability of the proteins to inhibit the growth of E. coli. Some surface sites in the protein (> 10% exposure to solvent) that are thought to be near the location of bound oligonucleotides, such as Arg16, Val19, Ser20, Arg21, Tyr26, Lys46 and Arg80, are sensitive to substitution. Other side-chains thought to be close to bound oligonucleotides, including Leu28, can be replaced with a number of amino acids with little loss of function based on either assay. Most non-Gly/Pro surface residues thought to be distant from the locations of bound oligonucleotides are relatively tolerant of substitution, except for two small residues (Ala11 and Thr14), two aromatic residues (Tyr34 and Tyr56), two residues that are only partially exposed to solvent (Asn29 and Val70), and three residues that have been proposed to be at the dimer-dimer interface formed when gene V protein binds to nucleic acids (Glu40, Tyr41 and Arg82).

Engineering multiple properties of a protein by combinatorial mutagenesis.

A method for simultaneously engineering multiple properties of a protein, based on the observed additivity of effects of individual mutations, is presented. We show that, for the gene V protein of bacteriophage f1, effects of double mutations on both protein stability and DNA binding affinity are approximately equal to the sums of the effects of the constituent single mutations. This additivity of effects implies that it is possible to deliberately construct mutant proteins optimized for multiple properties by combination of appropriate single mutations chosen from a characterized library.

Genetic fusion of subunits of a dimeric protein substantially enhances its stability and rate of folding.

The gene V protein of bacteriophage f1 is a single-stranded DNA and RNA-binding protein composed of two identical subunits. We have constructed single-chain variants of the protein using short peptide linkers of five or six amino acids to connect the carboxyl terminus of one monomer to the amino terminus of the second monomer. The resulting subunit-fusion gene V proteins were found to bind single-stranded DNA nearly as tightly as the wild-type protein. Denaturation measurements show that the subunit-fusion gene V proteins are 5 kcal/mol (1 kcal = 4.18 kJ) more stable than the wild-type protein at a protein concentration of 10 microM. The rate of unfolding of the protein is essentially unaffected by the fusion of monomeric subunits, whereas the rate of folding is greatly enhanced. Our results suggest a simple way of obtaining a substantial thermodynamic stabilization for some oligomeric proteins.

Energetics of repacking a protein interior.

To test whether interactions in the hydrophobic core of a protein can be adequately modeled based on the properties of a liquid hydrocarbon, we measured the unfolding free energies of the wild-type bacteriophage f1 gene V protein and 29 mutants with apolar substitutions at positions 35 and 47. Stability changes arising from identical mutations at these two buried sites are quite different, suggesting that one site is more rigid than the other. Reversals of residues at positions 35 and 47 confirm that their environments are distinct. Mutants containing weakly polar residues at these two sites suggest that the protein interior is more polar than a liquid hydrocarbon. Interactions between residues at the two sites appear to be minimal. These observations are compatible with a view of protein interiors that incorporates properties of liquid hydrocarbons but also includes polar interactions and a site-dependent "packing energy" associated with changes in internal structure.

Repacking protein interiors.

Several goals of protein engineering may be achieved through redesign and repacking of protein interiors. The effects of interior apolar substitutions on protein stability depend strongly on the site of the substitution. One reason for this is that protein interiors have properties both of apolar liquids and of crystalline solids. Substitutions at interior sites affect the stability of a protein by changing the hydrophobicity, but each site in a protein has a characteristic energy associated with introducing packing changes, and the net stability depends on both of these factors.

Influence of interior packing and hydrophobicity on the stability of a protein.

Protein interiors contain many tightly packed apolar atoms in a nearly crystalline state. Both shielding of apolar atoms from solvent and efficient interior packing arrangements affect protein stability, but their relative importance is unclear. To separate these effects, the stabilities of wild-type and mutant gene V proteins from bacteriophage fl were studied by measuring resistance to denaturation. The effects of subtle interior packing changes, both separate from and combined with changes in buried side chain hydrophobicity, were measured. For the interior apolar-to-apolar substitutions studied, the two effects were of the same magnitude and alteration of packing without accompanying hydrophobicity changes substantially destabilized the protein.

Fibroadenomas with stromal cellularity. A clinicopathologic study of 21 patients.

Data on clinical and pathologic features of tumors from 21 patients who had breast tumors compatible with juvenile (cellular) fibroadenomas (JCF) were reviewed. All patients were black females ranging in age at time of presentation from 10 to 39 years, with a median age of 15 years. Follow-up (median period, six years) was obtained for 20 patients. Patients were categorized into two groups; 13 had solitary lesions and eight had multiple and successive lesions. No histologic differences were observed between JCF occurring as solitary or multiple tumors or between lesions occurring in patients younger and older than age 20 years. The JCF was shown to be a fairly distinct clinicopathologic entity that characteristically occurred in adolescents, but which was occasionally seen in young adults. It had a benign course, characterized by synchronous and metachronous multicentricity, rather than local recurrence.