The role of pre- and post-anaphase microtubules in the cytokinesis phase of the cell cycle

The cytokinesis phase, or C phase, of the cell cycle results in the separation of one cell into two daughter cells after the completion of mitosis. Although it is known that microtubules are required for proper positioning of the cytokinetic furrow 1 and 2, the role of pre-anaphase microtubules in cytokinesis has not been clearly defined for three key reasons. First, inducing microtubule depolymerization or stabilization before the onset of anaphase blocks entry into anaphase and cytokinesis via the spindle checkpoint [3]. Second, microtubule organization changes rapidly at anaphase onset as the mitotic kinase, Cdc2–cyclin B, is inactivated [4]. Third, the time between the onset of anaphase and the initiation of cytokinesis is very short, making it difficult to unambiguously alter microtubule polymer levels before cytokinesis, but after inactivation of the spindle checkpoint. Here, we have taken advantage of the discovery that microinjection of antibodies to the spindle checkpoint protein Mad2 (mitotic arrest deficient) in prometaphase abrogates the spindle checkpoint, producing premature chromosome separation, segregation, and normal cytokinesis 5 and 6. To test the role of pre-anaphase microtubules in cytokinesis, microtubules were disassembled in prophase and prometaphase cells, the cells were then injected with anti-Mad2 antibodies and recorded through C phase. The results show that exit from mitosis in the absence of microtubules triggered a 50 minute period of cortical contractility that was independent of microtubules. Furthermore, upon microtubule reassembly during this contractile C-phase period, ∼30% of the cells underwent chromosome poleward movement, formed a midzone microtubule complex, and completed cytokinesis

Pharmacokinetics in vivo and pharmacodynamics ex vivo/in vitro of meropenem and cefpirome in the Yucatan micropig model: continuous infusion versus intermittent injection

ObjectiveTo investigate the pharmacodynamic disposition of two recently developed β-lactam antibiotics, meropenem and cefpirome, in the Yucatan micropig model, and to compare the bactericidal activity of these drugs against bacteria in this in vitro/ex vivo micropig model after administration by both intermittent injection and continuous infusion.MethodsCefpirome (1 g) was given to the micropig over a 12-h period by direct intravenous injection and 6-h continuous infusion (500 mg). Meropenem (250 mg) was administered either by 30-min intravenous and 8-h continuous infusion. The two drugs were assayed by HPLC. The pharmacodynamics of these drugs were evaluated by means of (1) serum killing curve against Klebsiella pneumoniae producing extended-spectrum β-lactamase, stably derepressed Enterobacter cloacae and methicillin-susceptible penicillinase-producing Staphylococcus aureus, and (2) calculations of index of surviving bacteria (ISB).ResultsThe bactericidal activity of meropenem against K. pneumoniae and E. cloacae in this in vitro/ex vivo model was excellent, with a 4 log decrease at peak concentrations. Meropenem produced a mixed concentration- and time-dependent, killing effect against E. cloacae and K. pneumoniae. The ISB value ranged from 25% to 30% for E. cloacae. With concentrations above MIC for S. aureus (1 mg/L), cefpirome has a time-dependent bactericidal activity, as shown by the ISB ranging from 20% to 80% after 4 h and between 20% and 40% after an 8-h drug exposure. For both antibiotics, the higher concentrations obtained just after intermittent injection had a rapid and strong killing effect against the strains tested, but the trough levels had no bactericidal activity. The continuous infusions produce consistent concentrations of antibiotic that can be maintained above the MIC, and the bactericidal activity of which ranges from 2 to 4 log10 decrease of inoculum.ConclusionsIn the present study the micropig has been shown to be an adequate model for the pharmacodynamic investigation of cefpirome and meropenem. In general, continuous infusion appears to optimize the pharmacodynamic profile of the two tested β-lactam antibiotics. However, against Gram-negative bacilli, the administration of a loading dose prior to continuous infusion of β-lactams would eliminate the only potential pharmacokinetic disadvantage of continuous infusion and ensure the rapid onset of antimicrobial activity

Nuf2 and Hec1 Are Required for Retention of the Checkpoint Proteins Mad1 and Mad2 to Kinetochores

Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable kinetochore-microtubule attachments and chromosome alignment in mitosis 1, 2, 3, 4, 5, 6, 7, 8 and 9. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at kinetochores that have no or few stable kinetochore microtubules [5]. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to kinetochores and that kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint [6]. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from kinetochores that have not yet formed stable kinetochore-microtubule attachments

"Me, I'm Living It": The Primary Health Care Experiences of Women who use Drugs in Vancouver’s Downtown Eastside: Summary of Findings from the VANDU Women's Clinic Action Research for Empowerment Study

First paragraph: Preventing and reducing the harmful consequences of drug use has been identified as a key priority in Canada at all levels of government. Initiatives to reduce barriers to care for marginalized women who use drugs are currently underway. However, despite the significant national and international attention paid to the health and social conditions of women in Vancouver's Downtown Eastside (DTES), women who use drugs in this community report persistent health inequities and barriers to accessing a wide range of services, including primary health care, harm reduction services, mental health care, and addictions treatment. These inequities and barriers to care result from a complex interplay of social, political and economic factors. In order to provide effective, empowering, compassionate, and respectful care for women who use drugs it is crucial to understand how these factors influence health and well-being

Taxol-Stabilized Microtubules Can Position the Cytokinetic Furrow in Mammalian Cells

How microtubules act to position the plane of cell division during cytokinesis is a topic of much debate. Recently, we showed that a subpopulation of stable microtubules extends past chromosomes and interacts with the cell cortex at the site of furrowing, suggesting that these stabilized microtubules may stimulate contractility. To test the hypothesis that stable microtubules can position furrows, we used taxol to rapidly suppress microtubule dynamics during various stages of mitosis in PtK1 cells. Cells with stabilized prometaphase or metaphase microtubule arrays were able to initiate furrowing when induced into anaphase by inhibition of the spindle checkpoint. In these cells, few microtubules contacted the cortex. Furrows formed later than usual, were often aberrant, and did not progress to completion. Images showed that furrowing correlated with the presence of one or a few stable spindle microtubule plus ends at the cortex. Actin, myosin II, and anillin were all concentrated in these furrows, demonstrating that components of the contractile ring can be localized by stable microtubules. Inner centromere protein (INCENP) was not found in these ingressions, confirming that INCENP is dispensable for furrow positioning. Taxol-stabilization of the numerous microtubule-cortex interactions after anaphase onset delayed furrow initiation but did not perturb furrow positioning. We conclude that taxol-stabilized microtubules can act to position the furrow and that loss of microtubule dynamics delays the timing of furrow onset and prevents completion. We discuss our findings relative to models for cleavage stimulation

Anaphase Onset does not Require the Microtubule-Dependent Depletion of Kinetochore and Centromere-Binding Proteins

Spindle checkpoint proteins, such as Mad2 and BubR1, and the motors dynein/dynactin and CENP-E usually leave kinetochores prior to anaphase onset by microtubule-dependent mechanisms. Likewise, \u27chromosome passenger proteins\u27 including INCENP are depleted from the centromeres after anaphase onset and then move to the midzone complex, an event that is essential for cytokinesis. Here we test whether the cell cycle changes that occur at anaphase onset require or contribute to the depletion of kinetochore and centromere proteins independent of microtubules. This required the development of a novel non-antibody method to induce precocious anaphase onset in vivo by using a bacterially expressed fragment of the spindle checkpoint protein Mad1 capable of activating the APC/C, called GST-Mad1F10. By injecting PtK1 cells in nocodazole with GST-Mad1F10 and processing the cells for immunofluorescence microscopy after anaphase sister chromatid separation in nocodazole we found that Mad2, BubR1, cytoplasmic dynein, CENP-E and the 3F3/2 phosphoepitope remain on kinetochores. Thus depletion of these proteins (or phosphoepitope) at kinetochores is not required for anaphase onset and anaphase onset does not produce their depletion independent of microtubules. In contrast, both microtubules and anaphase onset are required for depletion of the \u27chromosome passenger\u27 protein INCENP from centromeres, as INCENP does not leave the chromosomes prior to anaphase onset in the presence or absence of microtubules, but does leave the centromeres after anaphase onset in the presence of microtubules

Global trends and patterns of commercial milk-based formula sales: is an unprecedented infant and young child feeding transition underway?

Objective: The marketing of infant/child milk-based formulas (MF) contributes to suboptimal breast-feeding and adversely affects child and maternal health outcomes globally. However, little is known about recent changes in MF markets. The present study describes contemporary trends and patterns of MF sales at the global, regional and country levels. Design: Descriptive statistics of trends and patterns in MF sales volume per infant/child for the years 2008–2013 and projections to 2018, using industry-sourced data. Setting: Eighty countries categorized by country income bracket, for developing countries by region, and in countries with the largest infant/child populations. Subjects: MF categories included total (for ages 0–36 months), infant (0–6 months), follow-up (7–12 months), toddler (13–36 months) and special (0–6 months). Results: In 2008–2013 world total MF sales grew by 40·8% from 5·5 to 7·8 kg per infant/child/year, a figure predicted to increase to 10·8 kg by 2018. Growth was most rapid in East Asia particularly in China, Indonesia, Thailand and Vietnam and was led by the infant and follow-up formula categories. Sales volume per infant/child was positively associated with country income level although with wide variability between countries. Conclusions: A global infant and young child feeding (IYCF) transition towards diets higher in MF is underway and is expected to continue apace. The observed increase in MF sales raises serious concern for global child and maternal health, particularly in East Asia, and calls into question the efficacy of current regulatory regimes designed to protect and promote optimal IYCF. The observed changes have not been captured by existing IYCF monitoring systems.Financial support: P.B. was employed through an Australian Research Council Discovery Project (number 130101478)

Kinesin 5–independent poleward flux of kinetochore microtubules in PtK1 cells

Forces in the spindle that align and segregate chromosomes produce a steady poleward flux of kinetochore microtubules (MTs [kMTs]) in higher eukaryotes. In several nonmammalian systems, flux is driven by the tetrameric kinesin Eg5 (kinesin 5), which slides antiparallel MTs toward their minus ends. However, we find that the inhibition of kinesin 5 in mammalian cultured cells (PtK1) results in only minor reduction in the rate of kMT flux from ∼0.7 to ∼0.5 μm/min, the same rate measured in monopolar spindles that lack antiparallel MTs. These data reveal that the majority of poleward flux of kMTs in these cells is not driven by Eg5. Instead, we favor a polar “pulling-in” mechanism in which a depolymerase localized at kinetochore fiber minus ends makes a major contribution to poleward flux. One candidate, Kif2a (kinesin 13), was detected at minus ends of fluxing kinetochore fibers. Kif2a remains associated with the ends of K fibers upon disruption of the spindle by dynein/dynactin inhibition, and these K fibers flux

Merotelic kinetochore orientation occurs frequently during early mitosis in mammalian tissue cells and error correction is achieved by two different mechanisms

Merotelic kinetochore orientation is an error that occurs when a single kinetochore becomes attached to microtubules from two spindle poles rather than just to one pole. We obtained the first evidence that merotelic kinetochore orientation occurs very frequently during early mitosis in mammalian tissue cells and that two different correction mechanisms are critical for accurate chromosome segregation in cells possessing bipolar spindles and unperturbed chromosomes. Our data show that about 30% of prometaphase PtK1 cells possess one or more merotelically oriented kinetochores. This frequency is increased to over 90% in cells recovering from a nocodazole-induced mitotic block. A delay in establishing spindle bipolarity is responsible for the high frequency of merotelic orientations seen in cells recovering from nocodazole, but not in untreated cells. The frequency of anaphase cells with merotelically oriented lagging chromosomes is 1% in untreated cells and 18% in cells recovering from nocodazole. Prolonging metaphase by 2 hours reduced the frequency of anaphase cells with lagging chromosomes both for untreated and for nocodazole-treated cells. Surprisingly, anaphase lagging chromosomes represented a very small fraction of merotelic kinetochore orientations present in late metaphase. Our data indicate that two correction mechanisms operate to prevent chromosome missegregation due to merotelic kinetochore orientation. The first, a pre-anaphase correction mechanism increases the ratio of kinetochore microtubules attached to the correct versus incorrect pole and might eventually result in kinetochore reorientation before anaphase onset. The increase in microtubule ratio to opposite poles is the groundwork for a second mechanism, active in anaphase, that promotes the segregation of merotelically oriented chromosomes to the correct pole

Global trends and patterns of commercial milk-based formula sales:is an unprecedented infant and young child feeding transition underway?

OBJECTIVE: The marketing of infant/child milk-based formulas (MF) contributes to suboptimal breast-feeding and adversely affects child and maternal health outcomes globally. However, little is known about recent changes in MF markets. The present study describes contemporary trends and patterns of MF sales at the global, regional and country levels. DESIGN: Descriptive statistics of trends and patterns in MF sales volume per infant/child for the years 2008-2013 and projections to 2018, using industry-sourced data. SETTING: Eighty countries categorized by country income bracket, for developing countries by region, and in countries with the largest infant/child populations. SUBJECTS: MF categories included total (for ages 0-36 months), infant (0-6 months), follow-up (7-12 months), toddler (13-36 months) and special (0-6 months). RESULTS: In 2008-2013 world total MF sales grew by 40·8 % from 5·5 to 7·8 kg per infant/child/year, a figure predicted to increase to 10·8 kg by 2018. Growth was most rapid in East Asia particularly in China, Indonesia, Thailand and Vietnam and was led by the infant and follow-up formula categories. Sales volume per infant/child was positively associated with country income level although with wide variability between countries. CONCLUSIONS: A global infant and young child feeding (IYCF) transition towards diets higher in MF is underway and is expected to continue apace. The observed increase in MF sales raises serious concern for global child and maternal health, particularly in East Asia, and calls into question the efficacy of current regulatory regimes designed to protect and promote optimal IYCF. The observed changes have not been captured by existing IYCF monitoring systems