Development of clinical risk-prediction models for uterine atony following vaginal and cesarean delivery.

BACKGROUND: Uterine atony is the most common cause of postpartum hemorrhage and is associated with substantial morbidity. Prospectively identifying women at increased risk of atony may reduce the incidence of subsequent adverse events. We sought to develop and evaluate clinical risk-prediction models for uterine atony following vaginal and cesarean delivery, using prespecified risk factors identified from systematic review. METHODS: Using retrospective data from vaginal and cesarean deliveries occurring at a single institution between 2010 and 2019, antepartum and intrapartum risk-prediction models for uterine atony, defined by supplementary uterotonic administration in addition to prophylactic oxytocin infusion, were developed using logistic regression. The C-statistic quantified the ability of the model to discriminate between cases and controls. RESULTS: Data were available for 4773 atony cases and 23 933 controls. The antepartum model included 20 risk factors and exhibited moderate discriminatory ability (C-statistic 0.61, 95% confidence interval 0.60 to 0.62). The intrapartum model included 27 risk factors and showed improved discriminatory ability (C-statistic 0.68, 95% confidence interval 0.67 to 0.69). CONCLUSIONS: We identified antepartum and intrapartum risk-prediction models to quantify patients' risk of uterine atony. Models performed similarly for all delivery modes, races, and ethnic groups. Future work should further improve these models through inclusion of more comprehensive prediction data.

Quantifying the incidence of clinically significant respiratory depression in women with and without obesity class III receiving neuraxial morphine for post-cesarean analgesia: a retrospective cohort study.

BACKGROUND: Obesity is a suspected risk factor for respiratory depression following neuraxial morphine for post-cesarean analgesia, however monitoring guidelines for obese obstetric patients are based on small, limited studies. We tested the hypothesis that clinically significant respiratory depression following neuraxial morphine occurs more commonly in women with body mass index (BMI) ≥40 kg/m2 compared with BMI <40 kg/m2. METHODS: We conducted a single-center, retrospective chart review (2006-2017) of obstetric patients with clinically significant respiratory depression following neuraxial morphine, defined as: (1) opioid antagonist administration; (2) rapid response team activation (initiated in April 2010); or (3) tracheal intubation due to a respiratory event. The incidence of respiratory depression was compared between women with BMI ≥40 kg/m2 and BMI <40 kg/m2. RESULTS: In total, 11 327 women received neuraxial morphine (n=1945 BMI ≥40 kg/m2; n=9382 BMI <40 kg/m2). Women with BMI ≥40 kg/m2 had higher rates of sleep apnea, hypertensive disorders, and magnesium administration. Sixteen cases of clinically significant respiratory depression occurred within seven days postpartum. The incidence did not significantly differ between groups (odds ratio 2.2, 95% CI 0.6 to 6.9, P=0.174). Neuraxial morphine was not deemed causative in any case, however women with BMI ≥40 kg/m2 had higher rates of tracheal intubation unrelated to neuraxial morphine (2/1945 vs. 0/9382, P=0.029). CONCLUSIONS: Respiratory depression in this population is rare. A larger sample (∼75 000) is required to determine whether the incidence is higher with BMI ≥40 kg/m2. Tracheal intubation was higher among the BMI ≥40 kg/m2 cohort, likely due to more comorbidities.

A cluster quasi-randomized controlled trial of an interactive, monthly obstetric anesthesiology curriculum: impact on resident satisfaction and knowledge retention.

BACKGROUND: Increasingly, evidence supports the use of educational paradigms that focus on teacher-learner interaction and learner engagement. We redesigned our monthly obstetric anesthesia resident didactics from a lecture-based curriculum to an interactive format including problem-based learning, case discussion, question/answer sessions, and simulation. We hypothesized that the new curriculum would improve resident satisfaction with the educational experience, satisfaction with the rotation, and knowledge retention. METHODS: Fifty-three anesthesiology residents were prospectively recruited and quasi-randomized through an alternating-month pattern to attend either interactive sessions or traditional lectures. Residents completed a daily satisfaction survey about quality of teaching sessions and a comprehensive satisfaction survey at the conclusion of the rotation. Knowledge retention was assessed with a knowledge test completed on the final day. The primary outcome was daily satisfaction with the curriculum, and secondary outcomes included overall satisfaction with the curriculum, overall rotation satisfaction, and within-resident difference between pre- and post-knowledge test scores. RESULTS: No differences were observed in daily resident satisfaction after interactive sessions vs traditional lectures. Furthermore, no differences were observed between the interactive sessions and traditional lecture groups in overall satisfaction with the curriculum, overall satisfaction with the entire rotation or within-resident difference between pre- and post-knowledge test scores. CONCLUSIONS: Our study failed to demonstrate improvement in resident satisfaction or knowledge retention following implementation of an interactive curriculum on a month-long obstetric anesthesia rotation. Reasons may include misalignment of the intervention with measured study outcomes, lack of sensitivity of the survey tools, and inadequate training of faculty presenters.

The cell cycle of Chlamydomonas reinhardtii: the role of the commitment point.

Chlamydomonas reinhardtii cells can double their size several times during the light period before they enter the division phase. To explain the role of the commitment point (defined as the moment in the cell cycle after which cells can complete the cell cycle independently of light) and the moment of initiation of cell division we investigated whether the timing of commitment to cell division and cell division itself are dependent upon cell size or if they are under control of a timer mechanism that measures a period of constant duration. The time point at which cells attain commitment to cell division was dependent on the growth rate and coincided with the moment at which cells have approximately doubled in size. The timing of cell division was temperature-dependent and took place after a period of constant duration from the onset of the light period, irrespective of the light intensity and timing of the commitment point. We concluded that at the commitment point all the prerequisites are checked, which is required for progression through the cell cycle; the commitment point is not the moment at which cell division is initiated but it functions as a checkpoint, which ensures that cells have passed the minimum cell size required for the cell division.

Blue light delays commitment to cell division in Chlamydomonas reinhardtii.

In this study, we describe the effect of red and blue light on the timing of commitment to cell division in Chlamydomonas reinhardtii. The time point and cell size after which cells can complete their cell cycle with one division round were determined for cultures that were exposed to various red and blue light periods. We show that the commitment point of cells grown in blue light is shifted to a later time point and a larger cell size, when compared with cells grown in red light. This shift was reduced when cultures were exposed to shorter blue light periods. Furthermore, this shift occurred only when exposure to blue light started before the cells attained a particular size. We conclude that the critical cell size for cell division, which is the cell size at which commitment to cell division is attained, is dependent on spectral composition.

Low-expression genes induced by nitrogen starvation and subsequent sexual differentiation in Chlamydomonas reinhardtii, isolated by the differential display technique.

Genes that are expressed upon a shift to nitrogen-free medium, an event that initiates gametogenesis, were identified in Chlamydomonas reinhardtii by using the differential display technique. Ten different cDNAs were isolated and shown to have increased levels of their transcripts upon removal of the nitrogen source. The initial kinetics of RNA accumulation allowed an ordering of the genes with respect to the timing of their expression, with individual genes being expressed very early, early, intermediately, or late after induction. For very early genes, significantly increased transcript levels were detected within 30 min. This fast response suggests that gene expression is rapidly activated after removal of the nitrogen source. The accumulation of transcripts from the very early, early, and intermediate genes preceded the appearance of mating competence. Though transcript levels of several very early genes fluctuated during subsequent incubation in nitrogen-free medium, most of them exhibited maxima when the highest level of mating competence was attained. One of these very early genes was shown to encode a urate oxidase type-II enzyme.

Identification and localization of a thylakoid-bound carbonic anhydrase from the green algae Tetraedron minimum (Chlorophyta) and Chlamydomonas noctigama (Chlorophyta).

In order to broaden our understanding of the eukaryotic CO2-concentrating mechanism the occurrence and localization of a thylakoid-associated carbonic anhydrase (EC 4.2.1.1) were studied in the green algae Tetraedron minimum and Chlamydomonas noctigama. Both algae induce a CO2-concentrating mechanism when grown under limiting CO2 conditions. Using mass-spectrometric measurements of 18O exchange from doubly labelled CO2, the presence of a thylakoid-associated carbonic anhydrase was confirmed for both species. From purified thylakoid membranes, photosystem I (PSI), photosystem II (PSII) and the light-harvesting complex of the photosynthetic apparatus were isolated by mild detergent gel. The protein fractions were identified by 77 K fluorescence spectroscopy and immunological studies. A polypeptide was found to immunoreact with an antibody raised against thylakoid carbonic anhydrase (CAH3) from Chlamydomonas reinhardtii. It was found that this polypeptide was mainly associated with PSII, although a certain proportion was also connected to light harvesting complex II. This was confirmed by activity measurements of carbonic anhydrase in isolated bands extracted from the mild detergent gel. The thylakoid carbonic anhydrase isolated from T. minimum had an isoelectric point between 5.4 and 4.8. Together the results are consistent with the hypothesis that thylakoid carbonic anhydrase resides within the lumen where it is associated with the PSII complex.

Low external pH induces HOG1-dependent changes in the organization of the Saccharomyces cerevisiae cell wall.

Low environmental pH strongly affected the organization of the Saccharomyces cerevisiae cell wall, resulting in rapidly induced resistance to beta1,3-glucanase. At a molecular level, we found that a considerable amount of Cwp1p became anchored through a novel type of linkage for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins, namely an alkali-labile linkage to beta1,3-glucan. This novel type of modification for Cwp1p did not require the presence of a GPI-derived structure connecting the protein with beta1,6-glucan. In addition, we found high levels of Cwp1p, which was double-anchored through both the novel alkali-sensitive bond to beta1,3-glucan and the alkali-resistant GPI-derived linkage to beta1,6-glucan. Further cell wall analyses demonstrated that Pir2p/Hsp150 and possibly other Pir cell wall proteins, which were already known to be linked to the beta1,3-glucan framework by an alkali-sensitive linkage, were also more efficiently retained in the cell wall at pH 3.5 than at pH 5.5. Consequently, the alkali-sensitive type of linkage of cell wall proteins to beta1,3-glucan was induced by low pH. The low pH-induced alterations in yeast cell wall architecture were demonstrated to be dependent on a functional HOG1 gene, but not on the Slt2p-mediated MAP kinase pathway. Consistent with this observation, DNA microarray studies revealed transcriptional induction of many known high-osmolarity glycerol (HOG) pathway-dependent genes, including four cell wall-related genes, namely CWP1, HOR7, SPI1 and YGP1.

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants.

In Candida albicans wild-type cells, the beta1, 6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via beta1,6-glucan to beta1,3-glucan. The remaining GPI-CWPs are linked through beta1,6-glucan to chitin. The beta1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to beta1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Delta and pmt1Delta mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through beta1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a beta1, 6-glucan-deficient hemizygous kre6Delta mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.

Polar glycerolipids of Chlamydomonas moewusii.

The fatty acid and polar lipid compositions of the unicellular green alga Chlamydomonas moewusii were characterized. Since this organism is an important plant model for phospholipid-based signal transduction, interest was focused on the lipids phosphatidic acid, phosphatidylinositolphosphate and phosphatidylinositolbisphosphate. A phosphatidylinositol:phosphatidylinositolphosphate: phosphatidylinositolbisphosphate ratio of 100:1.7:1.3 was found. The polyphosphoinositides accounted for 0.8 mol% of the total phospholipids and their fatty acid compositions were similar to that of phosphatidylinositol except for the enrichment of linolenic acid in phosphatidylinositol phosphate. Phosphatidic acid accounted for 0.67 mol% of the phospholipids. Major structural glycerolipids were monogalactosyldiacylglycerol (35 mol%), digalactosyldiacylglycerol (15 mol%), sulfoquinovosyldiacylglycerol (10 mol%), diacylglyceryltrimethylhomoserine (16 mol%), phosphatidylglycerol (9 mol%), phosphatidylethanolamine (8 mol%) and phosphatidylinositol (6 mol%). Relative changes in the total fatty acid compositions found during growth on nutrient-limited medium reflected mainly alterations in the compositions of the chloroplast lipids phosphatidylglycerol and monogalactosyldiacylglycerol. [32P]Pi-incorporation studies revealed that it took 6 days before the amount of label in the major phospholipids was proportional to their abundance.

Chlamydomonas contains calcium stores that are mobilized when phospholipase C is activated.

Mastoparan induces Ca(2+)-dependent deflagellation of the unicellular green alga Chlamydomonas moewusii Gerloff, as well as the activation of phospholipase C and the production of inositol 1,4, 5-trisphosphate (InsP(3); T. Munnik et al., 1998, Planta 207: 133-145). Even in the absence of extracellular Ca(2+), mastoparan still induces deflagellation (L.M. Quarmby and H.C. Hartzell, 1994, J Cell Biol 124: 807-815; J.A.J. van Himbergen et al., 1999, J Exp Bot, in press) suggesting that InsP(3) mediates Ca(2+) release from intracellular stores. To test this hypothesis, cells were pre-loaded with (45)Ca(2+) and their plasma membranes permeabilized by digitonin. Subsequent treatment of the cells with mastoparan (3.5 microM) induced release of intracellular (45)Ca(2+). Mastoparan also activated phospholipase C in permeabilized cells, as demonstrated by the breakdown of (32)P-phosphatidylinositol 4,5-bisphosphate and the production of diacylglycerol. The mastoparan analogues mas7 and mas17 were also effective and their efficacy was correlated with their biological activity. X-ray microanalysis showed that electron-dense bodies (EDBs) are a major Ca(2+) store in C. moewusii. Analysis of digitonin-permeabilized cells showed that EDBs lost calcium at digitonin concentrations that released radioactivity from (45)Ca(2+)-labelled cells, suggesting that (45)Ca(2+) monitored the content of EDBs. X-ray microanaysis of living cells treated with mastoparan also revealed that calcium was released from EDBs.

Localization of synthesis of beta1,6-glucan in Saccharomyces cerevisiae.

Beta1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, beta1,3-glucan, and chitin. It has been postulated that the synthesis of beta1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1. This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules. We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of beta1,6-glucan. Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases. Next, using immunogold labeling, we tried to visualize intracellular beta1,6-glucan in cryofixed sec1-1 cells which had accumulated secretory vesicles at the restrictive temperature. No intracellular labeling was observed, but the cell surface was heavily labeled. Consistent with this, we could detect substantial amounts of beta1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles. Taken together, our data indicate that the synthesis of beta1, 6-glucan takes place largely at the cell surface. An alternative function for Kre6 and Skn1 is discussed.

Cell wall dynamics in yeast.

The yeast Saccharomyces cerevisiae is the first fungus for which the structure of the cell wall is known at the molecular level. It is a dynamic and highly regulated structure. This is vividly illustrated when the cell wall is damaged and a salvage pathway becomes active, resulting in compensatory changes in the wall.

The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and beta 1,6-glucan-deficient mutants.

The cell wall of yeast contains a major structural unit, consisting of a cell wall protein (CWP) attached via a glycosylphosphatidylinositol (GPI)-derived structure to beta 1,6-glucan, which is linked in turn to beta 1, 3-glucan. When isolated cells walls were digested with beta 1,6-glucanase, 16% of all CWPs remained insoluble, suggesting an alternative linkage between CWPs and structural cell wall components that does not involve beta 1,6-glucan. The beta 1,6-glucanase-resistant protein fraction contained the recently identified GPI-lacking, O-glycosylated Pir-CWPs, including Pir2p/Hsp150. Evidence is presented that Pir2p/Hsp150 is attached to beta 1,3-glucan through an alkali-sensitive linkage, without beta 1,6-glucan as an interconnecting moiety. In beta 1,6-glucan-deficient mutants, the beta 1,6-glucanase-resistant protein fraction increased from 16% to over 80%. This was accompanied by increased incorporation of Pir2p/Hsp150. It is argued that this is part of a more general compensatory mechanism in response to cell wall weakening caused by low levels of beta 1,6-glucan.

The Saccharomyces cerevisiae CWH8 gene is required for full levels of dolichol-linked oligosaccharides in the endoplasmic reticulum and for efficient N-glycosylation.

The Saccharomyces cerevisiae mutant cwh8 was previously found to have an anomalous cell wall. Here we show that the cwh8 mutant has an N -glycosylation defect. We found that cwh8 cells were resistant to vanadate and sensitive to hygromycin B, and produced glycoforms of invertase and carboxypeptidase Y with a reduced number of N -chains. We have cloned the CWH8 gene. We found that it was nonessential and encoded a putative transmembrane protein of 239 amino acids. Comparison of the in vitro oligosaccharyl transferase activities of membrane preparations from wild type or cwh8 Delta cells revealed no differences in enzyme kinetic properties indicating that the oligosaccharyl transferase complex of mutant cells was not affected. cwh8 Delta cells also produced normal dolichols and dolichol-linked oligosaccharide intermediates including the full-length form Glc3Man9GlcNAc2. The level of dolichol-linked oligosaccharides in cwh8 Delta cells was, however, reduced to about 20% of the wild type. We propose that inefficient N -glycosylation of secretory proteins in cwh8 Delta cells is caused by an insufficient supply of dolichol-linked oligosaccharide substrate.

The contribution of cell wall proteins to the organization of the yeast cell wall.

Our knowledge of the yeast cell wall has increased rapidly in the past few years, allowing for the first time a description of its structure in molecular terms. Two types of cell wall proteins (CWPs) have been identified that are covalently linked to beta-glucan, namely GPI-CWPs and Pir-CWPs. Both define a characteristic supramolecular complex or structural unit. The GPI building block has the core structure GPI-CWP-->beta1,6-glucan-->beta1,3-glucan, which may become extended with one or more chitin chains. The Pir building block is less well characterized, but preliminary evidence points to the structure, Pir-CWP-->beta1,3-glucan, which probably also may become extended with one or more chitin chains. The molecular architecture of the cell wall is not fixed. The cell can make considerable adjustments to the composition and structure of its wall, for example, during the cell cycle or in response to environmental conditions such as nutrient and oxygen availability, temperature, and pH. When the cell wall is defective, dramatic changes can occur in its molecular architecture, pointing to the existence of cell wall repair mechanisms that compensate for cell damage. Finally, evidence is emerging that at least to a considerable extent the cell wall of Saccharomyces cerevisiae is representative for the cell wall of the Ascomycetes.