Search

The GTPase Cdc42 regulates polarized growth in most eukaryotes. In the bipolar yeast Schizosaccharomyces pombe, Cdc42 activation cycles periodically at sites of polarized growth. These periodic cycles are caused by alternating positive feedback and time-delayed negative feedback loops. At each polarized end, negative feedback is established when active Cdc42 recruits the Pak1 kinase to prevent further Cdc42 activation. It is unclear how Cdc42 activation returns to each end after Pak1-dependent negative feedback. We find that disrupting branched actin-mediated endocytosis disables Cdc42 reactivation at the cell ends. Using experimental and mathematical approaches, we show that endocytosis-dependent Pak1 removal from the cell ends allows the Cdc42 activator Scd1 to return to that end to enable reactivation of Cdc42. Moreover, we show that Pak1 elicits its own removal via activation of endocytosis. These findings provide a deeper insight into the self-organization of Cdc42 regulation and reveal previously unknown feedback with endocytosis in the establishment of cell polarity., (© 2024 Harrell et al.)

The conserved GTPase Cdc42 is a major regulator of polarized growth in most eukaryotes. Cdc42 periodically cycles between active and inactive states at sites of polarized growth. These periodic cycles are caused by positive feedback and time-delayed negative feedback loops. In the bipolar yeast S. pombe , both growing ends must regulate Cdc42 activity. At each cell end, Cdc42 activity recruits the Pak1 kinase which prevents further Cdc42 activation thus establishing negative feedback. It is unclear how Cdc42 activation returns to the end after Pak1-dependent negative feedback. Using genetic and chemical perturbations, we find that disrupting branched actin-mediated endocytosis disables Cdc42 reactivation at the cell ends. With our experimental data and mathematical models, we show that endocytosis-dependent Pak1 removal from the cell ends allows the Cdc42 activator Scd1 to return to that end to enable reactivation of Cdc42. Moreover, we show that Pak1 elicits its own removal via activation of endocytosis. In agreement with these observations, our model and experimental data show that in each oscillatory cycle, Cdc42 activation increases followed by an increase in Pak1 recruitment at that end. These findings provide a deeper insight into the self-organization of Cdc42 regulation and reveal previously unknown feedback with endocytosis in the establishment of cell polarity., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing or financial interests.

Fission yeast cytokinesis is driven by simultaneous septum synthesis, membrane furrowing and actomyosin ring constriction. The septum consists of a primary septum flanked by secondary septa. First, delivery of the glucan synthase Bgs1 and membrane vesicles initiate primary septum synthesis and furrowing. Next, Bgs4 is delivered for secondary septum formation. It is unclear how septum synthesis is coordinated with membrane furrowing. Cdc42 promotes delivery of Bgs1 but not Bgs4. We find that after primary septum initiation, Cdc42 inactivators Rga4 and Rga6 localize to the division site. In rga4Δrga6Δ mutants, Cdc42 activity is enhanced during late cytokinesis and cells take longer to separate. Electron micrographs of the division site in these mutants exhibit malformed septum with irregular membrane structures. These mutants have a larger division plane with enhanced Bgs1 delivery but fail to enhance accumulation of Bgs4 and several exocytic proteins. Additionally, these mutants show endocytic defects at the division site. This suggests that Cdc42 regulates primary septum formation and only certain membrane trafficking events. As cytokinesis progresses Rga4 and Rga6 localize to the division site to decrease Cdc42 activity to allow coupling of Cdc42-independent membrane trafficking events with septum formation for proper septum morphology., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To identify high-risk categories for high-order multiple pregnancy (HMP) in in vitro fertilization (IVF), establish clinic-specific HMP risk data for counseling use, and verify their utility in reducing HMP., Design: Before and after intervention study., Setting: Two IVF programs using the same embryology laboratory and IVF protocols., Patient(s): All IVF patients undergoing fresh embryo transfers., Intervention(s): Use of clinic-specific age, diagnosis, and embryo score (ES) risk data in assessing individual HMP risk during informed consent., Main Outcome Measure(s): HMP and pregnancy outcomes., Result(s): In determining clinic-specific high risk categories and developing outcomes-based HMP risk data for counseling, the good outcome rate (GR) was defined as the percentage of singleton or twin deliveries per cycle and the bad outcome rate included no pregnancy or nondelivered pregnancies (miscarriages, multifetal reduction) and HMP per cycle. During 1995 to 1999, age <35 years, calculated morphologic ES, and donor egg (DE) cycles were factors shown by logistic regression to statistically significantly affect the GR. The optimal GRs for DE <35 and >or=35 years (donor age), and non-DE cycles <35 years were achieved with two (57.7%), three (43.2%), and three (43.2%) embryos transferred, respectively. A DE <35 years with >or=3 embryos transferred had the highest risk for HMP. The GR correlated (0.91) with the ES according to the formula: GR = 3.3 + 2.0 ES, when ES range was between 4 and 26. Clinic-specific risks for HMP based on age, diagnosis, and ES were developed and considered while counseling for ET during 2004. The clinic-specific HMP risk data made for a reduction in the HMP rate of 90.9% for DE-IVF (11.8% to 1%) and 53.8% for all IVF (9.1% to 4.2%), without decreases in clinical pregnancy or delivery rates. Physicians showing the greatest decline (64%) in HMP had no reduction in pregnancy or delivery rates., Conclusion(s): The use of clinic-specific HMP risk data in counseling based on age, diagnosis, and ES provided a 53% to 64% reduction in HMP without affecting rates of pregnancy or delivery. The clinic-specific ES system correlated closely with good outcomes. A standardized ES system may provide useful information for counseling during ET informed consent.

Objective: To evaluate pregnancy rates (PRs) in women undergoing artificial insemination with sperm alternately prepared by one of five techniques: sperm washing, Percoll gradient centrifugation, swim-up, swim-down, or refrigeration/heparin treatment., Design: Each treatment group alternated in a different order through the five sperm preparations. Pregnancy rates were compared for each sperm preparation., Setting: Two infertility centers, one located in an academic institution and the other a regional hospital., Patient(s): Three hundred sixty-three women undergoing 898 artificial inseminations with husband semen with a progressive motile sperm count of >20 million sperm per mL were randomly placed in the five treatment groups., Main Outcome Measure(s): Pregnancy rates., Result(s): The overall ongoing PR per insemination was 9.7% (87/898), including 6.12% for natural cycles (n = 196), 12.8% for clomiphene citrate-stimulated cycles (n = 101), and 10.3% for gonadotropin-stimulated cycles (n = 601). The highest ongoing PRs for sperm preparations followed the swim-up technique (13.2%, 26/197) and the Percoll gradient centrifugation technique (12.7%, 26/204)., Conclusion(s): These data suggest that the swim-up and Percoll gradient preparations result in higher PRs than the wash, swim-down, and refrigeration/heparin techniques.

A simple technique for high-resolution imaging of distant objects is described and experimentally demonstrated. The technique, referred to as Fourier telescopy, is a variant of Fourier microscopy, which additionally uses phase closure for correction of intervening aberrations. It is an active-illumination technique that is scalable to angular resolutions of 1 nrad and to illuminators of extremely low power. A laboratory experiment demonstrates reconstruction of images of two simple objects with an angular resolution of 83 µrad.

This article discusses the use of balanced incomplete block design for process evaluation and presents a case study of its use. This technique produces a weighted ranking of program elements, showing the relative importance of each element and allowing comparison of process and content elements. The article presents a case study in which the technique was used to evaluate the Chronic Disease Self-Management Program. Participants and lay course leaders were asked to rank 13 course elements for their helpfulness. The most valued element, sharing or unstructured interactions among participants, was not an explicitly planned part of the intervention. Some of the elements least valued (nutrition, use of community resources, and medication use) are elements most emphasized by the health care system and by patient education. We found that balanced incomplete block design was easy to administer and tally. The results could be readily applied to program redesign and to needs assessment.

Joint ventures between service and academia are designed to enhance the quality of client services, enrich faculty teaching experiences and skills, and strengthen communication channels. The joint venture described in this article is an example of how public health nursing services and academia can be united through faculty participation in administration. Included in the discussion are the impetus for the project, the contract negotiations, the positive outcomes and disadvantages of the venture, and questions that should be raised when a similar venture is considered.

The rattail fish, Coryphaenoides armatus, lives at ocean depths of 3000 m. As an adaptation for pumping oxygen into the swim bladder against the extreme pressures at the ocean bottom, the hemoglobin from this fish at low pH exhibits an extraordinarily low affinity for ligands. In this study, continuous wave and time-resolved Raman techniques are used to probe the binding site in this hemoglobin. The findings show an association between the low-affinity material and a highly strained heme-proximal histidine linkage. The transient Raman studies reveal differences in the protein structural dynamics at pH 6 and 8. The emerging picture derived from both this and earlier studies is that in vertebrate hemoglobins the heme-proximal histidine linkage represents a key channel through which species- and solution-dependent variations in the globin are communicated both statically and dynamically to the heme to produce an extensive range of ligand binding properties. Also presented is a new model that relates both intensity and frequency of the resonance Raman band involving the iron-proximal histidine stretching mode to specific protein controlled structural degrees of freedom. There emerges from this model a mechanism whereby modifications in the proximal heme pocket can further reduce the affinity of an already highly strained T state structure of hemoglobin.

Variations in the v2 region of the Raman spectra of cryogenically trapped photoproducts of different liganded myoglobins as a function of ligand (CO, O2, and n-butyl isocyanide) and species (whale, tuna, elephant) are reported. These variations are attributed to differences in the population of "open" (ligand accessible) and "closed" (ligand inaccessible) conformations of the distal heme pocket. Based on these findings and those derived from other spectroscopies including x-ray crystallography, NMR, IR spectra, and ESR, a working model is presented which accounts for how the conformation of the distal heme pocket, the geometry of the bound ligand, the identity of the ligand, and the dynamics of the dissociated ligand are all interconnected.

Inhomogeneous broadening of the 760-nanometer photoproduct band of carboxymyoglobin at cryogenic temperatures has been demonstrated with a dynamic hole burning technique. Line-shape changes and frequency shifts in this spectral band are generated by ligand recombination and are shown not to be the result of structural relaxation below 60 K. The observation of dynamic hole burning exposes the relation between the structural disorder responsible for the inhomogeneous broadening and the well-known distributed ligand rebinding kinetics. The findings provide direct evidence for the functional relevance of conformational substrates in myoglobin rebinding. In addition, a general protocol for evaluating the relative contributions of structural relaxation and hole burning to the spectral changes accompanying rebinding in hemeproteins is presented.

A high incidence of luteal phase defect (LPD) has been reported using subcutaneous pulsatile gonadotropin-releasing hormone for induction of ovulation. We reviewed all patients treated with the combination of subcutaneous pulsatile gonadotropin-releasing hormone during the follicular phase and human chorionic gonadotropin during the luteal phase (GnRH-hCG) who underwent endometrial biopsy during a treatment cycle. All of these patients had biopsy-proven LPD which persisted despite traditional therapy with progesterone vaginal suppositories and/or clomiphene citrate. The mean number of biopsies out of phase per patient prior to GnRH-hCG treatment was 2.8 +/- 0.2 (+/- SEM). When treated with GnRH-hCG, 15/16 patients (94%) showed a normal endometrial biopsy. The probability of this result occurring by chance alone allowing for a 50% treatment independent correction rate is less than .001. These results show that the combination of subcutaneous pulsatile gonadotropin-releasing hormone and luteal-phase human chorionic gonadotropin can result in normal endometrial maturation in a high percentage of cycles when administered as described. It appears to be an effective alternative to traditional treatment modalities for luteal phase defect should one be needed.

The geminate recombination of CO with Hb following dissociation by a 10-ns laser pulse has been studied as a function of pH (9.2 and 7.0 without inositol hexaphosphate and 6.0 with inositol hexaphosphate) and temperature (5-35 degrees C). The hemoglobins studied included adult, Rothschild, rabbit, opossum, and carp. Despite significant differences in their structural and functional properties, the first four of these hemoglobins show similar trends in the yields, rates, and activation energies of the geminate recombination. The nature of the "cage recombination" in hemoglobin is discussed in the light of such findings. Neither a slow diffusion model nor a model based upon a specific non-heme binding site accounts for the observations.

We studied the effect of the opiate antagonist naloxone on the peripheral GH response to the alpha 2-receptor agonist clonidine in eight normally cycling women during the mid-luteal phase. In a randomized, double-blind, cross-over design, each subject received clonidine and naloxone on one occasion and clonidine and placebo on the other. In seven of eight subjects, an attenuation of the GH response was associated with naloxone administration. The maximal GH increment above baseline (delta GHMAX) of 7.8 +/- 2.0 micrograms/L (mean +/- SEM) with placebo was higher than the delta GHMAX of 4.2 +/- 0.9 micrograms/L with naloxone (p = 0.05). Likewise, the area above baseline under the GH level-time curve following clonidine (delta GHAREA) was higher with placebo compared to naloxone (477 +/- 175 micrograms/L x min vs. 228 +/- 62 micrograms/L x min), although this difference was not quite statistically significant (p = 0.09). As expected, with placebo the increase in GH following clonidine was statistically significant by repeated measures analysis of variance (p = 0.001). The smaller increase in GH levels when naloxone was given was not significant. Both delta GHMAX and delta GHAREA values were significantly positively correlated with estradiol levels when placebo was given, but not when naloxone was given. GHRH was not detectable following clonidine administration under either the placebo or the naloxone conditions. Our data support the hypothesis that estrogen enhances the response of GH to provocative stimuli in women, at least in part by increasing endogenous opioid tone in the hypothalamus.

The geminate recombination of CO with carboxy hemoglobin (Hb4(CO)3) following a ten nanosecond laser pulse and the overall combination of the fourth CO with Hb4(CO)3 has been studied as a function of pH in the presence and absence of inositol hexaphosphate. The results indicate that the kinetics of both reactions are independent of pH and phosphate concentration. The results are discussed in terms of a two-step mechanism: a pre-equilibrium step followed by heme--ligand bond formation. The latter is also known as the geminate recombination reaction (Hb + CO in equilibrium Hb X CO in equilibrium HbCO).

The low-temperature recombination of CO with myoglobin was studied using Fourier transform infrared spectroscopy. The bound state of carbon monoxide myoglobin has two distinct conformers observed at 1926 and 1945 cm-1 with an intensity ratio of 1 to 8. The recombination of these bands after complete photolysis at 10 K followed by a temperature jump shows distinct kinetics for the two bands. Although both bands apparently follow the nonexponential kinetics originally described by Frauenfelder and co-workers (Austin, R., Beeson, K., Eisenstein, L., Frauenfelder, H., and Gunsalus, I. (1975) Biochemistry 14, 5355-5373), the 1926 cm-1 band does not appear appreciably below 70 K. In fact, after 20 min of recombination at 70 K the 1945 cm-1 band is fully recovered, while no detectable amount of the 1926 cm-1 band is present. This is the first association of a spectroscopic marker of protein substructure with reaction kinetics.

A 15-year-old girl presented with a large abdominopelvic mass that proved to be a retroperitoneal right ovarian cystadenofibroma. The patient's left ovary contained a small adenofibroma and was also retroperitoneal but otherwise grossly normal, as were the left tube and uterus. This is the first published report in which a patient's only ovaries appeared to have been located retroperitoneally.

Background and Objective: Best-worst scaling is a theory-driven method that can be used to prioritize objects in health. We sought to characterize all studies of best-worst scaling to prioritize objects in health, to assess trends of using best-worst scaling in prioritization over time, and to assess the relationship between a legacy measure of quality (PREFS) and a novel assessment of subjective quality and policy relevance., Methods: A systematic review identified studies published through to the end of 2021 that applied best-worst scaling to study priorities in health (PROSPERO CRD42020209745), updating a prior review published in 2016. The PubMed, EBSCOhost, Embase, Scopus, APA PsychInfo, Web of Science, and Google Scholar databases were used and were supplemented by a hand search. Data describing the application, development, design, administration/analysis, quality, and policy relevance were summarized and we tested for trends by comparing articles before and after 1 January, 2017. Multivariate statistics were then used to assess the relationships between PREFS, subjective quality, policy relevance, and other possible indicators., Results: From a total of 2826 unique papers identified, 165 best-worst scaling studies were included in this review. Applications of best-worst scaling to study priorities in health have continued to grow (p < 0.01) and are now used in all regions of the world, most often to study the priorities of patients/consumers (67%). Several key trends can be observed over time: increased use of pretesting (p < 0.05); increased use of online administration (p < 0.01), and decreased use of paper self-administered surveys (p = 0.02); increased use of heterogeneity analysis (p = 0.02); an increase in having a clearly stated purpose (p < 0.01); and a decrease in comparing respondents to non-respondents (p = 0.01). The average sample size has more than doubled, from 228 to 472 respondents, but formal sample size justifications remain low (5.3%) and unchanged over time (p = 0.68). While the average PREFS score remained unchanged at 3.1/5, both subjective quality and policy relevance trended up, but changes were not statistically significant (p = 0.06 and p = 0.13). Most of the variation in subjective quality was driven by PREFS (R 2 = 0.42), but it was also positively assosciated with policy relevance, heterogeneity analysis, and using a balanced incomplete block design, and was negatively associated with not using developmental methods and an increasing sample size., Conclusions: Using best-worst scaling to prioritize objects is now commonly used around the world to assess the priorities of patients and other stakeholders in health. Best practices are clearly emerging for best-worst scaling. Although legacy measures (PREFS) to measure study quality are reasonable, there may need to be new tools to assess both study quality and policy relevance., (© 2022. The Author(s).)

Purpose: Does IDEF mapping help monitor the technical process of IUI and explore the potential improvements which might contribute to increased pregnancy and live birth rates?, Method: Retrospective analysis of 1729 homologous IUI cycles of couples attending a fertility clinic in a university hospital setting. Standardized conventional semen parameters were analyzed and the semen samples prepared via discontinuous density gradient centrifugation., Results: There was no significant association between sperm concentration, motility and morphology (analysis phase), and pregnancy outcome. Only female and male ages were significantly associated with the pregnancy outcome. There was a significant difference in the odds on clinical pregnancies and live births when analysis was ≤ 21 min initiated, and < 107 min between sample production and IUI, adjusted for male and female age., Conclusions: Adjusting for the couple's age, we could show that time intervals between semen production and analysis and IUI when kept low significantly influenced clinical pregnancies and live births.