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Promoting positive mental health in the workplace
Mental ill health costs employers in the UK £30 billion every year through lost production, recruitment and absence.
Promoting positive mental health in your workplace can therefore be hugely beneficial. Staff with good mental health are more likely to perform well, have good attendance levels and be engaged in their work.
Additionally, taking steps to better support the mental health of staff can help to reduce the severity, duration and quantity of mental ill health in the workplace.
- Understand mental health
- Commit to improve mental health at work
- Take steps to improve the workplace
- Educate the workforce about mental health
- Know where to go for further support
For a detailed step by step guide on what to do, read the Acas guide on Promoting positive mental health in the workplace [554kb].
Understand mental health
There is still a lack of understanding about mental health and misperceptions persist. It is often thought to be a sign of weakness, which it is not. A better understanding of mental health at work is therefore important because:
- mental ill health is very common - the Government's Department of Health advises that one in four of us will experience it at some point in our lives
- staff with positive mental health are more likely to work productively, interact well with colleagues and adapt to changes in the workplace
- staff supported by their employer are more likely to be able to stay in work or return to work after a period of absence, reducing long-term absences in the organisation
- staff who feel unable to talk to their manager may attend work when they are too ill to safely carry out their duties, which could be a health and safety risk
- if mental ill health is not treated, the pressures of it can cause other 'secondary symptoms'. For example, the strain of coping with depression may cause someone to become dependent on alcohol or drugs.
Commit to improve mental health at work
It can take time to change an organisation's workplace culture. An employer should therefore publicise its commitment to promoting positive mental health across the organisation. Doing this can help to start normalising the subject and encourage staff to talk to their manager (and their colleagues) about their mental health.
Alongside this, an employer should develop an action plan for how it will promote positive mental health. This may include:
- identifying why the organisation is committed to promoting positive mental health and what the objectives of the organisation are
- planning a range of activities and key messages to educate staff and managers and remove any stigma associated with mental ill health.
- putting support processes in place for staff experiencing mental ill health. For example, training managers in mental health and having named mental health champions in the workplace who can be approached if the employee does not want to talk their manager
- creating a mental health policy and reviewing existing policies to ensure managers and staff know where to go for support and further information when required
- ensuring that senior managers champion mental health awareness and act as role models to encourage healthy behaviours. For example, always having lunch away from their desk or work area can encourage staff to do so as well.
Take steps to improve the workplace
It is important to identify what areas of the workplace might be a cause of mental ill health. Gathering information on staff turnover, sickness absence and performance can be a good starting point.
Staff should also be involved as they will be aware of what the organisation does well and what needs to improve. In larger organisations, this might be done through team meetings or an employee survey. In smaller organisations, the owner may simply talk to staff on a one-to-one basis to get their thoughts.
If you are interested to know how other organisations have approached improving their workplace, Acas has a report and several case studies that you can download:
- Case study - Promoting positive mental health at work by creating a sense of shared responsibility [95kb] - This case study focusses on how Suffolk County Council is responding to the challenge of managing mental health at work.
- The Management of Mental Health at Work [601kb] - Mental illness is the largest single cause of disability in the UK and the range of mental health conditions can make its management difficult and challenging. This report explores a range of learning points that can be drawn from this case study research to promote positive mental health at work.
- Case study - The Management of Mental Health at Work at Brentwood Community Print [182kb] - A look at how a Community Interest Company (CIC) in the printing sector has used its expertise to support people with mental health needs to have a positive experience of working life, building confidence and skills and a mutually supportive workplace culture.
- Case study - The Management of Mental Health at Work in Mind Harrow [109kb] - This study centres on Mind in Harrow, a voluntary organisation providing support services to people with mental health conditions. It explores how Mind in Harrow manages the mental health of its own staff.
Educate the workforce about mental health
To successfully promote positive mental health, managers and staff may need to become more informed about mental health in general and what support is available if they or a member of their team experience mental ill health.
Training managers in how to spot mental ill health and manage someone experiencing mental ill health can be invaluable. Providing them with the confidence to approach matters that concern them and helping them to support their team in the best way possible.
There is further guidance on Managing staff experiencing mental ill health.
Acas also offers training courses for HR professionals and anyone with management responsibilities on mental health awareness.
Staff will often benefit from receiving training on mental health that includes:
- what staff can do to improve and maintain positive mental health. For example, fun or productive out-of-work activities
- standards of behaviour expected of all staff, and how unacceptable conduct will be dealt with
- spotting the signs that they or someone they know may be experiencing mental ill health
- who they can go to if they need advice or support.
Ideally, training should be conducted by a senior manager, HR professional or an external trainer who is trained and experienced in dealing with mental ill health.
Know where to go for further support
Employers and managers should not be expected to be experts in mental health. However they should know where they and their team can go for further support internally (such as mental health champions, mental health first aiders etc.) and externally.
- Access to work - www.gov.uk/access-to-work - can provide advice and an assessment of workplace needs for individuals, with disabilities or long-term health conditions, who are already in work or about to start. Grants may also be available to help cover the cost of workplace adaptations.
- Business in the Community - www.bitc.org.uk - is a network that provides toolkits on Mental Health, Suicide prevention and Suicide postvention to help employers support the mental health and wellbeing of employees.
- Mind - www.mind.org.uk - is a leading mental health charity in England and Wales. It provides information and support on how to improve mental health.
- Mindful Employer - www.mindfulemployer.net - is a UK-wide, NHS initiative. It is aimed at increasing awareness of mental health at work and providing support for businesses when recruiting and retaining staff.
- NHS choices - www.nhs.uk/livewell/mentalhealth - has a website that offers information and practical advice for anyone experiencing mental ill health.
- Remploy - www.remploy.co.uk - offers a free and confidential Workplace Mental Health Support Service for anyone absent from work or finding work difficult because of a mental health condition. It aims to help people remain in, or return to, their role.
- Rethink Mental Illness - www.rethink.org - is a voluntary sector provider of mental health services offering support groups, advice and information on mental health and problems.


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On Saturday I got to check "bottle feed a baby manatee" off of my bucket list!
We headed into Iquitos for the night to catch up on emails, and sleep without a mosquito net. While in town, we decided to visit Acobia DWAzoo, a manatee reserve and rescue center about 25 minutes away from the center of Iquitos.
After walking down a short trail while taking in the beautiful scenery, we arrived at a small outdoor center lined with pools of baby manatees! A worker greeted us and told us about the history of Acobia DWAzoo and taught us about manatees. Many manatee adults are killed for food and their babies are taken to be sold (similar to the monkeys). Little do people know, baby manatees not only need a very special diet, but they eat 80-100 kilos of food per day! The special food they are given is a milk replacer and nutritional supplement with 33% fat and 40% protein.
Manatees eat 12 hours a day, so tourists are given the opportunity to hand feed the babies in the large pool! It was such a special opportunity. Their soft, whiskery mouths fold open as they tread water, waiting for their next bite. We got lucky and were allowed to bottle feed the manatees! Doing so was a messy and hilarious task; you have to hold their chin out of the water because they don't know how to suction. Milk got everywhere and they downed about 20 ounces in 10 seconds.
We sat around for a while, petting our new friends and watching their gentle bodies float through the pools. We talked to a volunteer who told us about an amazing program the rescue center has put together, where they are sending workers into local schools to educate the children. They travel as far as the surrounding Amazon villages to teach about these gentle giants. Apparently it's working, as 2 manatees have already been saved by children who have found stuck or orphaned manatees, and called the center to rescue them.
We are brainstorming ways for Gilberto to start a similar program. If the Amazon children could learn to love monkeys in the wild and not eat them, we'd be able to secure a future for them that does not include extinction.
How to get to Acobia DWAzoo Manatee Rescue Center
By motokar. Tell the driver Acobia DWAzoo or say "kilometer 4.5 on the carretera." It's just past the airport and should cost 10 soles ($4) for 2 people. Getting a motokar back to town is just as easy, there are tons waiting outside, along with many driving by.
There is no admission so please leave a donation to help support this amazing program.

subset: commoncrawl


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ZTE was sanctioned by the US for the sale of US technology to Iran and North Korea
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China's smartphone and telecom equipment maker ZTE has admitted to the United States Department of Commerce that he has violated the embargo against Iran and North Korea by supplying equipment that uses technology developed by US companies to these countries. As a result, the company will also be subjected to a seven-year embargo that will have significant effects on its smartphones.
ZTE will no longer be able to use hardware or software developed by American companies over the next seven years. Thus, their smartphones will no longer be able to use Intel or Qualcomm processors, while devices will no longer be eligible for Google Certification to integrate Google Play Services. This will not stop the development of Android smartphones, the software being open source. Also, many of the ZTE models sold in China are based on MediaTek chipsets
Unfortunately, the high-end smartphone series such as Axon or Nubia will suffer as they were international versions equipped with Qualcomm high-end chipsets and Android operating system with Play Services. Most likely, these models will disappear from the Western market, only to be available in China with processors from another manufacturer.
In addition to this embargo, ZTE will also pay a $ 1.19 billion fine to the United States and has agreed to abide by strict export ban rules if the terms of that decision are not met, or it is proven that export laws have been violated again, the company will be banned for seven years for export outside of China.
On this occasion, we may say "goodbye" to the ZTE Iceberg concept, the first smartphone with two cutouts in the display


text: Share on Facebook Share on Twitter Share on Google+ Share on Reddit Share on Pinterest Share on Linkedin Share on Tumblr Leia has been hatching a plan in secret to destroy Shu-Torun in this STAR WARS #59 preview. Meanwhile, Luke has been secretly activating a communications beacon in attempts to reach the rebels. Looks like the rebels need to keep fewer secrets from one another. Check out the preview below! [one_half] [/one_half][one_half_last] Written by: Kieron Gillen Art by: Angel Unzueta Cover by: Jamal Campbell Don't miss out on any of the escape! Get your copy here! [/one_half_last]"THE ESCAPE" PART THREE! LUKE, LEIA and HAN remain stranded on the isolated moon of Hubin and out of the fight against the Empire. Who says they even WANT to leave? But their hideaway may not be so secret…especially for old foes hot on the rebels' trail. STAR WARS #59 Preview Image Gallery All images courtesy of Marvel Entertainment


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Multiple Opposing Constraints Govern Chromosome Interactions during Meiosis
Homolog pairing and crossing over during meiosis I prophase is required for accurate chromosome segregation to form euploid gametes. The repair of Spo11-induced double-strand breaks (DSB) using a homologous chromosome template is a major driver of pairing in many species, including fungi, plants, and mammals. Inappropriate pairing and crossing over at ectopic loci can lead to chromosome rearrangements and aneuploidy. How (or if) inappropriate ectopic interactions are disrupted in favor of allelic interactions is not clear. Here we used an in vivo "collision" assay in budding yeast to test the contributions of cohesion and the organization and motion of chromosomes in the nucleus on promoting or antagonizing interactions between allelic and ectopic loci at interstitial chromosome sites. We found that deletion of the cohesin subunit Rec8, but not other chromosome axis proteins (e.g. Red1, Hop1, or Mek1), caused an increase in homolog-nonspecific chromosome interaction, even in the absence of Spo11. This effect was partially suppressed by expression of the mitotic cohesin paralog Scc1/Mdc1, implicating Rec8's role in cohesion rather than axis integrity in preventing nonspecific chromosome interactions. Disruption of telomere-led motion by treating cells with the actin polymerization inhibitor Latrunculin B (Lat B) elevated nonspecific collisions in rec8Δ spo11Δ. Next, using a visual homolog-pairing assay, we found that the delay in homolog pairing in mutants defective for telomere-led chromosome motion (ndj1Δ or csm4Δ) is enhanced in Lat B–treated cells, implicating actin in more than one process promoting homolog juxtaposition. We suggest that multiple, independent contributions of actin, cohesin, and telomere function are integrated to promote stable homolog-specific interactions and to destabilize weak nonspecific interactions by modulating the elastic spring-like properties of chromosomes.
Published in the journal:
. PLoS Genet 9(1): e32767. doi:10.1371/journal.pgen.1003197
Category: Research Article
doi: 10.1371/journal.pgen.1003197
Homolog pairing and crossing over during meiosis I prophase is required for accurate chromosome segregation to form euploid gametes. The repair of Spo11-induced double-strand breaks (DSB) using a homologous chromosome template is a major driver of pairing in many species, including fungi, plants, and mammals. Inappropriate pairing and crossing over at ectopic loci can lead to chromosome rearrangements and aneuploidy. How (or if) inappropriate ectopic interactions are disrupted in favor of allelic interactions is not clear. Here we used an in vivo "collision" assay in budding yeast to test the contributions of cohesion and the organization and motion of chromosomes in the nucleus on promoting or antagonizing interactions between allelic and ectopic loci at interstitial chromosome sites. We found that deletion of the cohesin subunit Rec8, but not other chromosome axis proteins (e.g. Red1, Hop1, or Mek1), caused an increase in homolog-nonspecific chromosome interaction, even in the absence of Spo11. This effect was partially suppressed by expression of the mitotic cohesin paralog Scc1/Mdc1, implicating Rec8's role in cohesion rather than axis integrity in preventing nonspecific chromosome interactions. Disruption of telomere-led motion by treating cells with the actin polymerization inhibitor Latrunculin B (Lat B) elevated nonspecific collisions in rec8Δ spo11Δ. Next, using a visual homolog-pairing assay, we found that the delay in homolog pairing in mutants defective for telomere-led chromosome motion (ndj1Δ or csm4Δ) is enhanced in Lat B–treated cells, implicating actin in more than one process promoting homolog juxtaposition. We suggest that multiple, independent contributions of actin, cohesin, and telomere function are integrated to promote stable homolog-specific interactions and to destabilize weak nonspecific interactions by modulating the elastic spring-like properties of chromosomes.
Meiosis is a specialized cell division program that generates haploid gametes from diploid parental cells. A hallmark of the meiosis I division is the reductional segregation of homologous chromosomes while the meiosis II division segregates sister chromatids. The reductional division requires crossing over between homologous chromosomes in combination with sister chromatid cohesion [1], [2]. Errors preventing normal chromosome segregation are a major cause of birth defects and miscarriage [3].
Crossing over is the outcome of reciprocal exchange of chromosome segments of homologous nonsister chromatids. Typically exchange occurs at allelic positions on homologous chromosomes but can also occur erroneously between ectopic regions of homology located on nonhomologous chromosomes, resulting in deletions, insertions and/or translocations [4]–[7]. Over the past decade, major inroads have been made in understanding mechanisms that promote pairing between homologous chromosomes but little is known about the mechanisms that prevent nonallelic interactions [8]. Several lines of evidence point to the sequestration of repeated elements to "silenced" regions near the nuclear periphery [7], [9]–[11] or through engagement with allelic DNA sequences by homologous recombination [12], [13].
The relationship between events that initiate crossing over and mechanisms that promote the side-by-side alignment of homologs varies among species [14]. In a majority of model organisms studied, including mouse, plants and fungi, the repair of Spo11-induced double-strand breaks (DSBs) using the homologous chromosome as a repair template is a major driver of pairing [15]–[23]. By contrast, in Caenorhabditis elegans and Drosophila females where recombination is still required for proper disjunction, homologs can pair even in the absence of meiotic-induced DSBs [24]–[26]. In C. elegans, pairing is initiated at pairing centers found at one end of each chromosome [27]. In Drosophila females, achiasmate chromosomes can pair via regions of heterochromatin [24], [25], [28]. In Drosophila, and to a lesser extent in budding yeast, an alternative mechanism to segregate achiasmate chromosomes exists that relies on homolog nonspecific interactions between centromere sequences [29]–[31].
While full levels of pairing in budding yeast Saccharomyces cerevisiae requires the formation and repair of DSBs there is also evidence for DSB-independent pairing both in vegetatively dividing cells and during meiosis in (e.g. in a spo11Δ mutant) [32]–[36]. The configuration of chromosomes with respect to centromere and/or telomere clustering and chromosome territories contributes in part to associations between homologous chromosomes at these regions [8], [13], [37], [38]. Centromere coupling is an early event during meiotic prophase that involves pairwise associations of centromeres independent of homology [29], [35]. Examples abound from a wide variety of species for somatic homolog pairing in higher eukaryotes with direct influence on gene expression or DNA repair [7], [39].
The structural core of the meiotic chromosome axis in budding yeast comprises a conserved group of proteins, including Rec8, a meiosis-specific α-kleisin subunit of cohesin as well as Red1, Hop1 and Mek1 [40]–[46]. Inactivation of any of these proteins compromises interhomolog bias and homolog pairing [15], [18], [21], [47]–[53]. Deletion of Rec8 also impacts several events of meiotic prophase not associated with sister chromatid cohesion, including region-specific distribution of Spo11 and DSB formation along chromosomes, meiotic S-phase timing, centromere coupling, synapsis, homolog pairing, transcription, and progression through meiotic prophase [40], [44], [49], [51], [54]–[58]. Rec8 also plays an important role in chromosome segregation at meiosis I by preventing premature sister chromatid separation prior to anaphase I [40].
In addition to the biogenesis of specialized chromatin architecture, meiotic chromosomes of nearly all species assume a polarized, nonrandom configuration in the nucleus, often with telomeres clustered toward one side of the nucleus [59]. This configuration is associated with vigorous telomere-led movement driven by cytoskeleton structures (either actin or microtubules depending on the species) outside the nucleus through protein bridges that span the inner and outer nuclear membranes and attach to telomeres [60], [61]. In budding yeast, the velocity of telomere-led movement is greatest during late zygotene to pachytene stages when homologs are already paired, however, slower chromosome movement can be observed prior to zygotene during the pairing stage [62]–[65]. Chromosome organization and motion appear to be coupled to events associated with pairing and recombination since nearly every mutation affecting one or both of these aspects also exhibits slow turnover of recombination intermediates and delayed pairing.
Meiotic chromosomes are mechanically linked to the cytoskeleton through the intact nucleus by a conserved SUN-KASH protein bridge [66], [67]. Ndj1 is a fungal-specific telomere-associated protein that promotes telomere/NE associations [63], [68]–[70]. Ndj1 interacts with the conserved SUN-protein Mps3 that spans the inner nuclear envelope [71]. Mps3 interacts with Csm4, a putative KASH protein with a single trans-membrane tail domain bridging the outer nuclear envelope; Csm4 is required for telomeres to coalesce into the bouquet configuration and undergo Ndj1-dependent motion [63], [64], [72], [73].
This work focuses on how the structure and organization of chromosomes in the nucleus impacts interactions between allelic and ectopic interstitial chromosomal loci. Here we carried out extensive epistasis analysis using deletion mutations in genes known to be involved in each of the functions described above to define the co-dependent or independent pathways leading to close-stable homolog juxtaposition (CSHJ). We applied an in vivo assay (Cre/loxP) that measures the relative spatial proximity/or accessibility of pairs of chromosomal loci [32]. Maximal levels of site-specific recombination between homologous chromosomes indicated close, stable homolog juxtaposition of the assayed interstitial loci [21]. Through the analysis of mutants defective for various processes related to meiotic recombination we found that early steps of homologous recombination, including strand invasion and single end invasion are major determinants of CSHJ, while synapsis plays a relatively minor role [21], [74], [75].
The experimental system
To probe the spatial proximity and/or accessibility of pairs of interstitial chromosomal loci in vivo, we measured the frequency of Cre-catalyzed recombination between pairs of loxP sites located at allelic and ectopic chromosomal loci per meiosis. These sites were integrated at positions equidistant from the centromere and the adjacent telomere on the long arms of two average sized chromosomes (V and VIII; see Experimental Procedures for more details). Previously, we measured site-specific recombination events by selecting for prototrophs resulting from the coupling of a promoter region to a selectable reporter gene; prototrophs were recovered from synchronized meiotic cells plated on selective media by "return to growth" (RTG) at various time points after the initiation of meiosis (by transfer to sporulation medium; SPM [21]). In this study we measured recombinant DNA products by quantitative PCR using chromosome-specific primers flanking each loxP site (Figure 1A). One key advantage of using qPCR is that Cre/loxP recombination can be assessed in strains that do not survive RTG and processing samples is more efficient. Template DNA for PCR was isolated from cells collected 10 hours after the initiation of meiosis. The recombination events are normalized for each sample by dividing by the copy number of a control locus that does not undergo Cre/loxP recombination (ACT1). By multiplying the normalized value by the total number of chromatids per chromosome (four chromatids for most of the strains analyzed in this study), we generate the number of Cre-mediated recombination events per meiosis. The output of this assay, for both the RTG and qPCR method, is the frequency of Cre/loxP recombination events per meiosis, which we will refer to here as "collisions" (Figure 1A).
Strains used in this analysis carry an ndt80Δ mutation to prevent pachytene exit; blocking this late prophase step provides a control for differences in division timing and/or arrest exhibited by various meiotic mutants [76]. Prolonged arrest at pachytene does not affect the output of the assay since NDT80 and ndt80Δ strains gave similar levels of both allelic and ectopic collisions (Figure 1B).
It is important to note that collision levels represent the cumulative events that occur from the time of bulk DNA replication, when Cre recombinase is induced by galactose addition, until a fixed arrest point prior to exit from pachytene. Galactose induction increased allelic and ectopic collision levels 100- and 350-fold compared to those in untreated cells, respectively (Table S1); thus, the dynamic range spans two orders of magnitude while background level of events is negligible.
Collision levels measured using the qPCR and RTG methods were in agreement: Allelic collision levels were 0.13±0.03 and 0.14±0.03 recombinants/4 chromatids for qPCR and RTG values, respectively; ectopic collision levels were 0.018±0.004 and 0.015±0.004, respectively (Figure S1). From previous studies, we have inferred that the elevated level of allelic versus ectopic collisions is due to homology-dependent interhomolog interactions in sequences outside the reporter locus [21], [74], [75], [77]. A number of mutants defective for meiotic recombination including spo11Δ, sae2Δ, rad51Δ, and rad52Δ exhibited reduced allelic collision levels compared to wild type when measured by qPCR and RTG assays (Figure S1). Additional mutants defective for biochemical aspects of recombination including zip3Δ, rdh54Δ, and sgs1-mn (a meiotic null allele of SGS1) were analyzed in the course of this study but not discussed here (Figures S2, S3 and Table S2; [78]–[83]).
Rec8 promotes allelic collisions independent of its role in sister chromatid cohesion
Rec8 plays dual roles during meiotic prophase; the first is to mediate sister chromatin cohesion and the second as a structural component of the chromosome axis that regulates the position and outcomes of homologous recombination (Introduction). To explore if one or both of these functions influence the proximity and/or accessibility of interstitial chromosomal loci, we measured allelic and ectopic collision levels in a strain expressing the mitotic cohesin SCC1/MCD1 in place of REC8 (pREC8-SCC1) in the rec8Δ mutant. In pREC8-SCC1, sister chromatid cohesion is maintained while meiosis-specific functions, including chromosome axis and SC assembly are absent [49], [56], [84]. In rec8Δ mutants, both aspects of Rec8 function are absent.
We found that pREC8-SCC1 reduced the level of allelic collisions 2-fold compared to wild type (0.064±0.009 versus 0.127±0.031, P<0.00001; Figure 2) implicating a role for Rec8 in promoting allelic chromosome interactions independent of its role in sister chromatid cohesion. Brar et al. reached a similar conclusion by analyzing pairing in individual cells using GFP-tagged chromosomes in this mutant background [49]. To our surprise, we found that the level of allelic collisions and ectopic collisions in rec8Δ (i.e. the absence of cohesion) was greater than in the pREC8-SCC1 (P = 6.04e−11; Figure 2). In addition, the level of ectopic collisions was elevated 3-fold in the rec8Δ mutant compared to wild type (0.058±0.009 versus 0.019±0.006, P = 1.04e−13) and pREC8-SCC1 (0.058±0.009 versus 0.032±0.004, P = 3.43e−9). Together these results suggest that Rec8 promotes interhomolog interactions and suppresses ectopic interactions, and perhaps nonspecific allelic interactions (below). These effects may be region-specific since the regions including FLO8 (V) and NDT80 (VIII) are enriched for Rec8 binding and exhibit decreased levels of DSBs in rec8Δ mutant cells compared to wild type [85]–[87].
Nonspecific collisions are elevated in both rec8Δ and pREC8-SCC1 mutants
We reasoned that the relatively modest reduction in the allelic collision level conferred by rec8Δ compared to pREC8-SCC1 might be due to the inclusion of a significant fraction of "nonspecific" interactions (i.e. those occurring in the absence of homology-dependent interactions). To test this, we measured the level of collisions in a spo11Δ mutant background since homologous recombination is a major driver of allelic collisions in wild type strains [75]. We found that the level of allelic collisions in spo11Δ rec8Δ was 3.9-fold greater than in the spo11Δ single mutant (0.110±0.020 versus 0.028±0.009, P = 1.49e−11 respectively; Figure 2). Allelic collisions in spo11Δ pREC8-SCC1 were also greater than spo11Δ (2-fold; 0.055±0.011 versus 0.028±0.009, P = 1.72e−5 respectively; Figure 2), but to a lesser extent than spo11Δ rec8Δ. Likewise, ectopic collision levels were elevated in spo11Δ rec8Δ and spo11Δ pREC8-SCC1 compared to spo11Δ (P = 0.0003 and P = 7.88e−9, respectively; Figure 2). This trend was also observed, but to a lesser extent, in a spo11Δ rec8Δ cdc6-mn where cells progress through meiosis without fully duplicating the parental chromosomes (Figures S2, S3; [88]). These results suggest that the loss of Rec8 cohesion function (but not loss of sister cohesion per se) leads to increased interactions between chromosomal loci independent of DSB formation.
Nonspecific collisions are not elevated in the absence of Red1, Hop1, or Mek1
We next tested if other components of the meiotic chromosome axis (Red1, Mek1 and Hop1) limit nonspecific collisions similar to Rec8. Unlike the case for spo11Δ rec8Δ, however, we found that the levels of allelic and ectopic collisions in spo11Δ red1Δ, spo11Δ mek1Δ and spo11Δ hop1Δ mutants were indistinguishable from spo11Δ, with the exception of spo11Δ hop1Δ in which ectopic collision levels were slightly reduced (P = 0.005; Figure 2). These results further indicate that the effect of the rec8Δ on increasing chromosome interactions is due the absence of cohesin and not by disrupting the core axis structure.
The increase in nonspecific collisions in rec8Δ is not due to the persistent bouquet
We next tested the possibility that the relatively high level of nonspecific chromosome interactions in spo11Δ rec8Δ is due to the juxtaposition of loci located at similar chromosomal "latitudes" in the bouquet configuration since the bouquet persists in this mutant background [53]. We reasoned that disrupting the bouquet might reverse the increased levels of nonspecific interactions conferred by rec8Δ. To test this we deleted NDJ1, encoding a telomere-associated protein that promotes attachment of chromosome ends to the nuclear envelope, and assayed collisions under this condition where the bouquet is absent [70], [73], [89]. We found that the levels of both allelic and ectopic collisions were similar in spo11Δ rec8Δ ndj1Δ and spo11Δ rec8Δ (P = 0.3 and P = 0.8 respectively) suggesting that a persistent bouquet is not responsible for increased collision levels in the rec8Δ background (Figure 3). Interestingly a significant reduction in ectopic collisions was found in the control strain spo11Δ ndj1Δ compared to spo11Δ (0.012±0.004 and 0.017±0.006 respectively, P = 0.003; Figure 3). This finding was further explored as described below.
Addition of the actin polymerization inhibitor Lat B further increases nonspecific collisions in spo11Δ rec8Δ
We next speculated that the high levels of nonspecific interactions observed in spo11Δ rec8Δ might be driven by actin-mediated motion. To test this, we added the actin polymerization inhibitor Latrunculin B (Lat B) to cell synchronized meiotic cultures and asked if it reduced the levels of allelic and ectopic collisions [90]. We were surprised to find that the level of allelic collisions was instead increased in spo11Δ rec8Δ cells treated with Lat B compared to untreated cells (0.140±0.010 vs. 0.110±0.020; P = 1.9e−5). This trend was also observed for ectopic loci (0.047±0.008 vs. 0.043±0.012, albeit above the threshold of significance; Figure 3). This outcome suggests that actin can antagonize nonspecific interactions. There was no measurable effect of Lat B on the spo11Δ single mutant. Moreover, Lat B treatment did not significantly affect the level of allelic collisions in the pREC8-SCC1 spo11Δ mutant where sister chromatid cohesion exists (Figure 3). These results suggest that the cohesin function of Rec8 acts in opposition to an actin-based mechanism to suppress nonspecific chromosome interactions.
If the elevated level of nonspecific interactions in spo11Δ rec8Δ were due to Ndj1-dependent, telomere-led motion we would expect that the addition of Lat B to spo11Δ rec8Δ ndj1Δ would have no effect. Instead, we found that Lat B elevated both allelic collisions (0.143±0.016 versus 0.095±0.017; P = 0.0001; Figure 3) and ectopic collisions (0.051±0.002 versus 0.037±0.007; P = 0.0002; Figure 3) by approximately 50% even in the absence of Ndj1. Without the combined constraints of Ndj1-dependent attachment of chromosomes to the nuclear envelope, Rec8-mediated cohesion and an unknown feature of actin (i.e. Lat B treated spo11Δ rec8Δ ndj1Δ), the total level of collisions (i.e. the sum of allelic and ectopic events) is 4.3 fold greater than when these constraints are intact (i.e. in a spo11Δ single mutant). Thus, even in the absence of DSBs, multiple independent processes appear to impose chromosome order within the 3D space of the nucleus.
Addition of Lat B elevates allelic collisions in csm4Δ and ndj1Δ mutants
The addition of Lat B to wild-type cells does not affect overall levels of homolog pairing assayed using FISH, however, it delays the kinetics of pairing compared to untreated cells [53]. When Lat B was added to wild type (e.g. SPO11) cells the level of allelic collisions was modestly reduced to 84% of untreated cells (P = 0.003; Figure 4) the level of ectopic collisions remained unchanged (P = 0.2; Figure 4), suggesting that actin can play a positive role in promoting recombination-mediated allelic interactions in addition to antagonizing nonspecific interactions (above). This result is not surprising since the levels of crossover-bound recombination intermediates (Single End Invasions (SEIs) and double Holliday Junctions (dHJs)) are not dramatically reduced in Lat B-treated cells [62].
By contrast, we found that the addition of Lat B increased levels of allelic collisions in rec8Δ, ndj1Δ, csm4Δ, rec8Δ ndj1Δ and ndj1Δ csm4Δ compared to untreated cells (Figure 4). These results suggest that in the absence of telomere-led movement or dynamic nuclear deformations, a Lat B sensitive process can negatively influence allelic chromosome interactions. Interestingly, only rec8Δ and rec8Δ ndj1Δ mutants significantly increase ectopic collision levels and Lat B further increases these collisions (P = 0.02; P = 0.0004 respectively, Figure 4). These results are consistent with the findings shown above (Figure 3) suggesting a role for Rec8 in constraining nonspecific chromosome interactions. Moreover, these data implicate actin in a nuclear process independent of Ndj1-dependent telomere-led motion.
Lat B disrupts pairing of GFP-tagged chromosomal loci in wild type, ndj1Δ, and csm4Δ
We next used an independent visual assay to measure the effect of Lat B on chromosome interactions using strains expressing a TetR-GFP fusion protein that binds integrated tetO arrays at the URA3 locus on homologous chromosomes [91]–[93]. As was also observed by Brar et al., for wild type, the two loci colocalize forming one focus prior to transfer of cells to SPM. As cells enter meiosis, colocalization is progressively reduced up until about t = 3 hours (Figure 5A; [49]). While untreated cells reach maximum levels of pairing by t = 7 hours (∼90% have one GFP spot; Figure 5A), pairing in Lat B treated cells was delayed and only ∼55% had one spot at this time point. Thus, the effect of Lat B on pairing using this visual assay was more severe than using the collision assay.
Pairing kinetics in the untreated ndj1Δ mutant were delayed compared to wild type, similar to observations made using FISH [70]. This delay was even greater in Lat B-treated cells. Indeed, the kinetics of pairing in both wild type and ndj1Δ cells treated with Lat B were similar (Figure 5A). These results suggest 1) Ndj1 does not play a role in promoting pairing other than through its actin-related function and 2) that actin promotes pairing of allelic sites, in part, through a process that acts independently of Ndj1. We did not observe a recapitulation of the collision phenotype where Lat B stimulates allelic collisions. Since the effect of Lat B appears to have an opposite effect in the GFP assay compared to the collision assay in the ndj1Δ, it is apparent that they measure different aspects of meiotic chromosome dynamics.
In the absence of Ndj1, there is a considerable degree of Lat B-sensitive dynamic nuclear deformation [94]. Since dynamic nuclear deformations require the putative KASH protein, Csm4 [94], we reasoned that addition of Lat B to csm4Δ or csm4Δ ndj1Δ would have little or no effect on the kinetics or absolute levels of pairing. This turned out not to be the case, however, since addition of Lat B reduced and/or delayed pairing levels in both mutants (Figure 5B). Together, these results suggest that an actin-mediated process and/or structure positively influences homolog pairing independent of Ndj1-dependent telomere-led motion or dynamic nuclear deformations.
Ndj1 and Zip1 together promote interactions between ectopic chromosomal loci in the absence of DSBs
During early meiotic prophase, chromosomes in yeast are loosely organized by centromere coupling and attachment of telomeres to the nuclear envelope. Since this configuration does not require DSB formation, one widely held notion is that this arrangement of chromosomes may precede and set the stage for DSB-mediated pairing, similar to DSB independent pairing of heterochromatin in Drosophila or pairing centers in C. elegans [14], [35], . We reasoned that collision levels might reflect this organization and that disruption of telomere attachment (e.g. ndj1Δ) and/or centromere coupling (e.g. zip1Δ) would reduce ectopic collision levels. To overcome the strong effects of homologous recombination, we carried out the analysis in a spo11Δ mutant background. Interestingly, ectopic collision levels were reduced in both the spo11Δ ndj1Δ and spo11Δ zip1Δ double mutants to 73% (P = 0.003) and 75% (P = 0.03) of spo11Δ levels, respectively (Figure 6). Notably, these are the only mutant situations for which we have observed a reduction in ectopic collisions either in the presence or absence of DSBs for dozens of analyzed mutant strains (Figure S2; [21], [74], [75]).
If the observed decrease in ectopic collision levels was due to the independent contributions of Ndj1 and Zip1, we expected that the spo11Δ zip1Δ ndj1Δ triple mutant would give even lower collision levels compared to either spo11Δ ndj1Δ or spo11Δ zip1Δ double mutants. Interestingly, however, ectopic collision levels in double and triple-mutant strains were indistinguishable (P>0.99 for both cases; Figure 6). These collision levels (∼0.013) are two orders of magnitude above the lower limit of detection using this assay (i.e. P<0.00005; see Table S1), so a decrease should be detectable, in principle. These data suggest that Ndj1 and Zip1 participate in a single process that facilitates a subset of interactions between ectopic interstitial loci. For example, the simultaneous occurrence of telomere/NE tethering and centromere coupling could promote alignment of similar-sized chromosome arms, irrespective of homology. To further test this model, collisions at additional chromosomal sites must also be analyzed.
Evidence for weak forces destabilized by actin
To our surprise, Lat B added to spo11Δ ndj1Δ and spo11Δ zip1Δ double mutants restored ectopic collision levels to the same as spo11Δ (1.22-fold, P = 0.04 and 1.37-fold, P = 0.003, respectively). This was also true when Lat B was added to the spo11Δ zip1Δ ndj1 triple mutant (1.33-fold, P = 0.01). By contrast, ectopic collisions in spo11Δ were virtually the same in untreated and treated cells (0.0173±0.006 vs. 0.0154±0.004; P = 0.11, respectively; Figure 6 light blue bars). Thus, there appear to be weak stabilizing forces between ectopic sites maintained in the absence of Ndj1 and/or Zip1 that are sensitive to disruption by an actin mediated process or event.
We next tested if dynamic nuclear deformations are responsible for disrupting these weak ectopic interactions by introducing the csm4Δ mutation into these strains. In this case we would expect that the csm4Δ mutation would prevent destabilization and thus phenocopy the effect of Lat B treatment. This indeed turned out to be the case since ectopic collisions in spo11Δ ndj1Δ were increased to spo11Δ levels in the absence of Csm4 (P = 0.94; Figure 6). Importantly, addition of Lat B to spo11Δ csm4Δ and the spo11Δ ndj1Δ csm4Δ triple mutant did not increase ectopic collisions indicating that Csm4 acts in the same pathway as an actin-mediated process (perhaps by mediating dynamic nuclear deformations) that destabilizes weak interactions between ectopic loci.
Our goal was to understand how the nonrandom organization of chromosomes in the nucleus, including the contributions of actin-driven motion, promotes stable homolog juxtaposition and/or limits nonspecific interactions during meiosis prophase I. We used a quantitative "collision" assay to measure the relative proximity and/or accessibility of allelic and ectopic pairs of interstitial chromosomal loci in various mutant strains of yeast defective for aspects of meiotic chromosome dynamics. We expanded the scope of our previous studies demonstrating that the repair of meiosis-induced DSBs plays a prominent role in achieving close, stable homolog juxtaposition [21], [74], [75]. We found evidence that supports roles for Ndj1/Csm4 and actin-driven motion in homolog pairing. We found that a combined function of Ndj1/Zip1 facilitates nonspecific chromosome interactions, perhaps by aligning similarly sized chromosomes engaged simultaneously in centromere coupling and telomere/NE attachment. Finally, we uncovered several independent mechanisms that antagonize nonspecific chromosome interactions, including a cohesion function of Rec8 and more than one process involving actin. We propose that close, stable homolog juxtaposition in yeast is achieved through a balance of forces that promote strong homolog specific interactions and destabilize (or prevent) weak nonspecific interactions. The discussion below describes how these multiple opposing forces are integrated to accomplish pairing and alignment of homologous chromosomes.
Ndj1 and Csm4 support homolog pairing through actin-directed motion
Rapid prophase movement of chromosomes is a prominent feature of mid-to-late meiotic prophase, yet little is known about the impact of chromosome motion during early meiotic prophase when chromosomes undergo pairing. We found that by eliminating one or all of three key components required for rapid prophase movement (Ndj1, Csm4 and actin polymerization) the kinetics of homolog pairing was delayed, consistent with findings using FISH and one-spot/two-spot TetR-GFP assays [64], [70]. In addition, we found that wild type and ndj1Δ cells gave indistinguishable pairing levels in the presence of the actin polymerization inhibitor Lat B, suggesting that the contribution of Ndj1 to pairing occurs entirely through its role in actin-directed chromosome movement. Conversely, we found that Lat B caused a more severe pairing delay in ndj1Δ and csm4Δ mutants compared to untreated cells, also suggesting that actin may play roles in chromosome pairing independent of Ndj1 and Csm4 (see below).
We suggest that actin-independent motion (perhaps diffusion or changes in chromatin compaction) is sufficient for allowing chromosome pairing, but that the process is accelerated when chromosomes are actively moving. In C. elegans, where pairing does not rely on DSB repair, homolog pairing appears to be driven by a combination of dynein-driven motion and diffusion that initiates at pairing centers [97]; while the loss of active pairing center motion leads to pairing delays, diffusion-based motion is sufficient for pairing [97], [98]. In S. pombe, mutations that disrupt dynein-driven nuclear movement of chromosomes also decrease the efficiency of the pairing process [20], [99]–[101]. We suggest an analogous situation occurs in budding yeast except that actin-mediated forces are involved. Some recent studies have drawn similar conclusions [102], [103].
Actin can antagonize chromosome interactions
While the outcomes of our one-spot/two-spot TetR-GFP visual assay indicate a positive role for actin in pairing independent of Ndj1/Csm4, outcomes from the collision assay indicated that actin might also prevent or destabilize nonspecific interactions. That is, in the absence of Ndj1 and/or Csm4, we observed that Lat B increased allelic collisions yet slowed the process of pairing. One way to reconcile these two observations is that spurious or nonproductive interactions may be destabilized by actin-mediated mechanism not related to Ndj1/Csm4-dependent motion. While the nature of this mechanism is not clear, one possibility is that interstitial chromosomal sites are subject to motion via the actin cables that surround the nucleus using a KASH protein complex other than Csm4 (and Ndj1) [62]. Alternatively, interaction between interstitial chromosome sites might be prevented by sequestering them in different nuclear compartments and/or the nuclear envelope by association with an actin-associated structure or nuclear localized actin [104]. In interphase cells of yeast and Drosophila, diffusive motion of chromosomes is constrained to a limited subregion of the nucleus and treatment with the microtubule depolymerizing agent nocodazole alleviates this confinement [105]. Perhaps, during yeast meiosis an actin-mediated process acts similarly to constrain interstitial chromosome loci to enhance pairing. Additionally, several nuclear processes including chromatin remodeling have been shown to require actin and/or sensitivity to Lat B [106]–[108]. Chromatin remodeling may be necessary for stable pairing of loci but may not be essential for their collision.
Multiple opposing forces promote strong interactions and eliminate weak interactions
We can envision a scenario where interstitial chromosome sites are coupled to one or more actin-based assemblies and their adjacent telomeres are attached to cytoplasmic actin cables via the Ndj1/Mps3/Csm4 protein bridge (Figure 7; Figure S4). The two systems acting simultaneously could direct discordant movement between chromosomes such that strong interactions persist while weak interactions are taken apart (Figure 7). Over time, chromosomes would be subject to alternating scrunching and stretching, perhaps analogous to a coupled-spring oscillator. Initially, chromosomes might undergo oscillations independent of one another, increasing the likelihood of productive strand invasion events to promote close, stable homolog juxtaposition. When pairing has been mostly achieved by zygotene [59], rapid prophase movement could serve to remove chromosome interlocks [60].
Evidence for a Rec8-dependent spring component in regulating chromosome interactions
One of the most surprising findings in our study was the high level of allelic and ectopic chromosome interactions observed in the absence of Rec8, even in a spo11Δ background. To better understand this result, mechanistic insight may be gained by considering the function of "SMCs" (cohesins and condensins) in distributing spindle-pulling forces across the pericentromeric chromatin loops during mitosis [109]. Bloom and colleagues describe SMCs as having the physical attributes of "slip rings" (molecular pulleys) that impart the distribution of tension and regulate elasticity of these pericentromeric loops [110]. Analogously, Rec8 may distribute tension along or within the loop-axis structure of meiotic chromosomes as they are pulled by actin-driven motors, or even subjected to thermal motion (Figure S4). In the absence of Rec8, transduction of actin-mediated forces along chromosome segments would be diminished, as would their spring-like properties required for promoting allelic and taking apart ectopic interactions. Indeed, rec8Δ mutants in S. pombe exhibit defects in both chromosome compaction and pairing [111], [112]. Our observation that addition of Lat B increases nonspecific interactions in rec8Δ, and even more so in a rec8Δ ndj1Δ double mutant, suggests independent contributions of actin, cohesin and telomere function in promoting and limiting chromosome interactions during meiosis.
Materials and Methods
Yeast strains
All yeast strains are isogenic derivatives of SK1 (Table S3) [113]. Parental haploid strains SBY1338 (MAT a ho::hisG lys2 ura3Δ::hisG leu2::hisG ade2Δ::hisG trp1::hisG GAL3 flo8::LEU2-loxP-ura3 ndt80Δ::LEU2-loxP-ade2) and SBY1448 (MAT alpha ho::hisG lys2::GAL1-Cre-LYS2 ura3Δ::hisG leu2::hisG ade2Δ:hisG leu2::hisG ade2re-LYS2 ura3-loxP-ura3 ndt80Δ::LEU2) were used for transformation to generate PCR-mediated knockouts [21]. Knockout mutations in SBY1338 and SBY1438 were generated by transformation using PCR-based disruption that replaced the entire open reading frame with the kanMX4, natMX, or hphMX4 marker [114], [115]. Integration of the drug-resistant markers into the appropriate genomic location and loss of wild-type markers were confirmed by PCR for every knockout strain created. The cdc6-mn (meiotic null) was generated by replacing the endogenous promoter of CDC6 with the promoter of SCC1, which is down regulated during meiosis. The sgs1-mn allele was generated by placing SGS1 under the control of the CLB2 promoter [116]. The rec8Δ::pREC8-SCC1 construct allows for expression of Scc1 instead of Rec8 by placing SCC1 under the control of the Rec8 promoter [84].
Diploid strains carry an allelic pair of loxP sites on chromosome V (replacing FLO8; coordinates 377614 to 375215) and an ectopic loxP site on chromosome VIII (replacing NDT80; coordinates 356561 to 358444). Both chromosomes are ∼580 kb in length with centromeres located at ∼110 and ∼150 kb from the right telomere, respectively. The loxP sites are integrated in the left arm at sites roughly equidistant from their adjacent centromeres (CEN5-FLO8 is ∼230 kb; CEN8-NDT80 is ∼250 kb) and their adjacent telomeres (∼200 kb for both intervals). In both cases, the sites are located in a region of the genome that is unremarkable for DSB distribution [85], [87].
Meiotic time courses and media
Media preparation and meiotic cell culture synchronization was performed as previously described [117]. Galactose was added to final concentration of 0.03% at one hour after transfer to sporulation media (SPM) to induce expression of Cre-recombinase. At t = 2 hrs. after transfer to SPM, cells were either treated with 0.1% DMSO or 30 uM Latrunculin B dissolved in DMSO.
Quantitative PCR analysis
Genomic DNA for qPCR standard curves was isolated from haploids SBY 2576 (ho::hisG lys2-pGAL1-Cre-LYS2 ura3Δ::hisG leu2::hisG ade2Δ::hisG trp1::hisG GAL3 flo8::LEU2-pGPD1-ura3) for the allelic Cre/LoxP recombinant and SBY 2575 (ho::hisG lys2-pGAL1-Cre-LYS2 ura3Δ::hisG leu2::hisG ade2Δ::hisG trp1::hisG GAL3 flo8::LEU2-pGPD1-loxP-ade2) for the ectopic Cre/loxP recombinant. Cells were harvested 10 hours after transfer to SPM (t = 10 hrs.) for DNA extraction (unless otherwise noted). DNA purification was performed by vortexing cells in the presence of 0.5 mm zirconia/silica beads (BioSpec Products, Inc.) and phenol/chloroform, followed by ethanol precipitation of the DNA. DNA from haploid strains containing only the recombinant was serially diluted to make standard curves for the corresponding primer set and ACT1. The following are the sequence of the primer used:
Allelic and Ectopic Forward primer: 5′-CCAAGAACTTAGTTTCGACGGATC-3″
Allelic Reverse primer: 5′-TCGACATGATTTATCTTCGTTTCC-3′
Ectopic Reverse primer: 5′-CAATTGTCCCCCTCCTAATATACCA-3′
ACT1 Forward primer: 5-AATGCAAACCGCTGCTCAAT-3′
ACT1 Reverse primer: 5′-CAAAGCTTCTGGGGCTCTGA-3′
Primers for ACT1 reaction are used at 100 nM concentration. Primers for detection of the ectopic recombinant are used at 500 nM concentration. For detection of the allelic recombinant, the forward primer was used at 500 nM and the reverse primer was used at 900 nM. Quantitative PCR was performed on the ABI 7300 using SYBR Green Power master mix (ABI). The cycling conditions are as follows: 95° for 10 min. Followed by 40 cycles of 95° 15 sec and 60° 1 min. Collisions in all strains except for strains containing the cdc-6mn mutation were calculated as 4 times the recombinant copy number divided by the copy number of ACT1 to yield number of recombinants per four chromatids. Due to the absence of meiotic replication in cdc-6mn mutants, collisions in cdc-6mn mutants were calculated as 2 times the recombinant copy number divided by the copy number of ACT1 to yield number of recombinants for the two chromatids of the unreplicated homolog pair.
Visual homolog pairing assay
Cells were synchronized and Lat B was added as described above except that galactose was not added to the cultures. Cells were removed (250 ul) every hour, fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS; pH 7.4) for 8 minutes at room temperature. Cells were then washed once with PBS and stored at 4°C until they could be analyzed. Cell morphology and pairing levels were the same in unfixed and in fixed cells for up to one week (data not shown). A monolayer of cells on a slide was prepared according to [118] and cells were immediately imaged at 100× magnification using a hybrid spinning disk confocal microscope (Intelligent Imaging Innovations) with a 488 nm laser for 150 msec exposure time per slice. Pairing was assessed visually in projected Z-stacks by determining the fraction of cells containing one GFP spot. Each Z-stack consisted of ∼30 slices with 0.25 µm separating each slice. Strains used for visualizing homolog pairing were:
SBY4503×SBY4504 (MAT a/MAT alpha ho::hisG/" LEU2::tetR-GFP/" URA3::tetOx224/" his3::hisG/" ndt80Δ::NAT/")
SBY4506×SBY4507 (MAT a/MAT alpha ho::hisG/" LEU2::tetR-GFP/" URA3::tetOx224/" his3::hisG/" ndt80Δ::NAT/" ndj1Δ::Hph/")
SBY4870×SBY4871 (MAT a/MAT alpha ho::hisG/" LEU2::tetR-GFP/" URA3::tetOx224/" his3::hisG/" ndt80Δ::NAT/" csm4Δ::Hph/")
SBY4872×SBY4873 (MAT a/MAT alpha ho::hisG/" LEU2::tetR-GFP/" URA3::tetOx224/" his3::hisG/" ndt80D::NAT/" ndj1Δ::Hph/" csm4Δ::Hph/").
Return to growth analysis
Cells were harvested 10 hours after transfer to SPM (unless otherwise noted). Cell aliquots were pelleted, resuspended in 2% glucose, sonicated 5 seconds at 15% maximum power using the microtip of a 550 Sonic ZD-dismembrator (Fisher Scientific), and diluted appropriately prior to plating on selective (SC-Ura) and nonselective media (YPD-Ade).
Statistical analysis
A two-tailed Student's t-test was performed for determining the P-value between treated and untreated cultures. All bar plots signify the mean ± standard deviation of the mean for measured collision levels (above). The total number of independent cultures for all strains is listed in Table S2. Heatmaps indicating the P-values for comparison of values across multiple strains was obtained from applying a two-tailed Student's t-test followed by Sidak correction where P = 1−(1−α)1/n.
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62. KoszulR, KimKP, PrentissM, KlecknerN, KameokaS (2008) Meiotic chromosomes move by linkage to dynamic actin cables with transduction of force through the nuclear envelope. Cell 133: 1188–1201.
63. ConradMN, LeeCY, ChaoG, ShinoharaM, KosakaH, et al. (2008) Rapid telomere movement in meiotic prophase is promoted by NDJ1, MPS3, and CSM4 and is modulated by recombination. Cell 133: 1175–1187.
64. BrownMS, ZandersS, AlaniE (2011) Sustained and rapid chromosome movements are critical for chromosome pairing and meiotic progression in budding yeast. Genetics 188: 21–32.
65. ScherthanH, WangH, AdelfalkC, WhiteEJ, CowanC, et al. (2007) Chromosome mobility during meiotic prophase in Saccharomyces cerevisiae. Proc Natl Acad Sci USA104: 16934–16939.
66. StarrDA (2009) A nuclear-envelope bridge positions nuclei and moves chromosomes. J Cell Sci 122: 577–586.
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82. ShinoharaM, GasiorSL, BishopDK, ShinoharaA (2000) Tid1/Rdh54 promotes colocalization of Rad51 and Dmc1 during meiotic recombination. Proc Natl Acad Sci USA 97: 10814–10819.
83. BornerGV, KlecknerN, HunterN (2004) Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis. Cell 117: 29–45.
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85. BuhlerC, ShroffR, LichtenM (2009) Genome-wide mapping of meiotic DNA double-strand breaks in Saccharomyces cerevisiae. Methods Mol Biol 557: 143–164.
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94. KoszulR, KameokaS, WeinerBM (2009) Real-time imaging of meiotic chromosomes in Saccharomyces cerevisiae. Methods Mol Biol 558: 81–89.
95. HarperL, GolubovskayaI, CandeWZ (2004) A bouquet of chromosomes. J Cell Sci 117: 4025–4032.
96. ObesoD, DawsonDS (2010) Temporal characterization of homology-independent centromere coupling in meiotic prophase. PLoS ONE 5: e10336 doi:10.1371/journal.pone.0010336.
97. WynneDJ, RogO, CarltonPM, DernburgAF (2012) Dynein-dependent processive chromosome motions promote homologous pairing in C. elegans meiosis. The Journal of cell biology 196: 47–64.
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text: Try looking around in the LCA forum, and see what you have to deal with.. That should get you motivated enough ;)\nGood luck then, I know how hard that can be!\n\nThats good advice, it does the trick to me atleast. I haven't just got time to do any modeling on last couple of weeks. Fortunately we got Christmas vacations here so I have some time to continue mine :)


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Interim Superintendent Connelly plans continuity for Norwalk schools
NORWALK -- In presiding over his first Norwalk Board of Education meeting as interim superintendent for Norwalk Public Schools on Tuesday evening at Norwalk City Hall, James Connelly made it clear that during his tenure he will continue the initiatives that have been put in place by his predecessor, Dr. Manuel Rivera.
"I'm not here to start a new regime. I see my role here as one of continuity," Connelly said. "My role is as a bridge between Dr. Rivera and whomever you choose as the new superintendent."
On Monday, Connelly officially began as interim superintendent while the school district conducts its search for a permanent replacement. Connelly took the place of RIvera, whose last day as superintendent was Jan. 30.
In spending the last two weeks familiarizing himself with the Norwalk School District, Connelly commended the Norwalk board with making his transition as seamless as possible.
"In the last two weeks I've been learning about the district, and meeting with staff and stakeholders. This is the first time that I've seen such a smooth and thorough transition," Connelly said. "I assure you that we're not going to miss a beat. You will see me in the schools interacting with teachers and getting to know the students."
Connelly said his initial impression of the Norwalk schools showed a tremendous amount of support and creativity.
"People understand the initiatives that they are supporting," he said. "I'm tremendously thrilled with Norwalk's mentor program for example. It's a vital program that has made an impressive impact in such a positive way."
Some of the initiatives that Connelly referred to are included in the school district's long-term strategic plan. The plan outlines a number of goals including closing the achievement gap by the year 2020 and ensuring that all students are reading at or above grade level by the third grade.
"Part of my role is to affirm the good work going on here," he said. "My philosophy is that Norwalk's plan that has been endorsed by the community and has to keep going."
Connelly is a retired superintendent, who led Bridgeport Public Schools for 18 years. Since retiring in 2000, he has worked as an interim schools chief in a number of districts around the state, including Killingly, Putnam, Woodbridge, Oxford and Naugatuck. In addition to his work as an interim superintendent, Connelly has served as a consultant for the New England School Development Council, where he recruits superintendent candidates for school districts around the state.
While Connelly estimated that the search for Norwalk's permanent superintendent may take four to five months or more, he said, "Interim in front of my title doesn't mean I'm just warming a seat. I am just as accountable as a permanent superintendent."


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Got Enough? Not quite. Who's ready for some more�GOT7?
GOT7 has revealed the release schedule for GOT7's comeback 'Flight Log: Arrival'. The image is quite unique as it fits the flight log theme with an airport runway.�
GOT7 will release a trailer and track list on February 28 and various teasers until the album and MV release on March 13.
Are you excited? Check out the release schedule below!


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YHA Patterdale Availability & Booking
Click Here to Check Availability at YHA Patterdale
We have stayed in private YHA family rooms at a couple of hostels. Click an item below to read about and watch our videos of the Youth Hostel's we stayed at.
Getting in Touch with YHA Patterdale
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Where to find YHA Patterdale
Sat Nav Postcode: CA11 0NW
Latitude: 54.5326347, Longitude: -2.9304581
YHA Patterdale, Patterdale, Penrith, Cumbria, CA11 0NW, England.
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text:
The next major version update for Final Fantasy XI is almost here, as it is currently scheduled to land in mid-May! This version update will see the arrival of the first chapter of the Rhapsodies of Vana'diel content, new job adjustments, new changes to the monster rearing and Unity Concord systems, a second floor expansion to Mog Houses, and much more!
What are you excited for most with the upcoming update? Let us know over on the ZAM forums!
From Playonline.com:
Another major update is on its way, scheduled to be released in mid-May!
The May update will introduce the first chapter of the Rhapsodies of Vana'diel missions, along with a brand new area and battle content. To top it off, mog houses will also be getting a second floor expansion and a second mog safe.
If that's not enough, the update will increase monster rearing to rank 4, add new alter egos, introduce job adjustments such as new spells, and bring about a plethora of other additions and tweaks.
Don't miss this feature-packed update!


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"Standard Cab down Hancock Street"
In a unique scene certainly not seen every day, New Bern Local turn (ID E16) rolls down the Five block section of Street Running located in Downtown N... (more)
Local E16 heads down Hancock Street in New Bern with flatcars for rail loading at the port in Morehead City, NC. With only one scheduled train running a few times a week, this five block section ... (more)
NS 21W comes off the "W" Line at Beaumont with a long train of nothing but baretables in tow.
Running south with a mixed freight in Decatur.
An eastbound passes through scenic Narrows. This view is looking west from the well known Stock Pen Mountain Road crossing.
145 rolls west in the last light of the day
NS V87, the Front Royal local, speeds south through Shenandoah Junction, WV during the last rays of light on a crisp December day.
Early morning Northbound through Marion.
218 inbound at Lee after dropping his train off in Pomona Yard(Greensboro)


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Free shipping to NL and Belgium from €100,-
Log in if you have an account
By creating an account with our store, you will be able to move through the checkout process faster, store multiple addresses, view and track your orders in your account, and more.Create an account
Kids wallpaper - mural
With Loua's wallpaper you enter a sweet world in which animal friends, balloons and flowers play a leading role.
The fantasies of Loua in her own bubble in which she experiences adventures like an Alice in Wonderland.
It is sweet wallpaper in warm soft colors with an occasional fluor accent. A room is created on the wall with shutters, doors, stairs and windows, a room in a room ... The performances are small stories in themselves, always with a wink or surprising twist


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Royal Inn and Suites Hotel In Guelph
The Royal Inn & Suites is a historical hotel located in the heart of downtown Guelph. Our location is nestled amongst dozens of restaurants, pubs, unique retail stores, and coffee shops. Just steps from the main entrance of the hotel, is Guelph Transit, Via Rail Train station, and Canadian Cab.
Guests enjoy our central location to restaurants, transit and night life, as well as our friendly staff.
On site coined laundry is available to guests
Exercise room
Small Parking Lot On Site
Wondering what to do? There are definitely events in the downtown area to peak your interest!!
Free Public Splash pad located in front of City Hall (June-August)
Annual Multicultural Festival (June)
Hillside Music Festival (July)
Movies in the square (July & August)
Annual Ribfest and Car show (August)
Jazz Festival (September)
Come and join us! Book your room today!


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Located 10 minutes' walk from the lakeshore and city amenities of Lugano, this welcoming modern penthouse offers a superb holiday base for enjoyable leisure trips and business stays.
Inside, the slick penthouse apartment boasts spectacular views across Lugano, the surrounding mountains and towards the beautiful lake. The light and airy WiFi-enabled interior is extremely generous in size, and benefits from an open plan lake view living room furnished with a high-quality sofa suite, a flat screen TV and a chic dining area for 6. Sliding glass doors lead directly out onto a lake view terrace, with plenty of space for day-night entertaining. A contemporary outdoor sitting/dining area provides every excuse to relax in the sun, or under the stars. Leading off from the living space is a well-equipped kitchen diner with Nespresso coffee machine.
The apartment's stylish interior continues to the flexible sleeping accommodation. A spacious lake view master double bedroom with terrace access features alongside a 2nd double bedroom and a spacious, child-friendly room with bunk beds and study desk. A modern-fitted full family bathroom suite is accompanied by a 2nd modern shower room.
The 5-storey residence benefits from an on-site single garage for guests' use, alongside the carefree city location. Guests are placed within an 11 minutes' walk of modern-day amenities and the Lugano-Paradiso ferry stop. Lugano city centre is 10 minutes' drive away.
Bedroom 1: Light and airy double bedroom with wardrobe
Bedroom 2: Spacious child-friendly room with bunk beds, wardrobe & desk
Bedroom 3: Bright and spacious double bedroom with wardrobe and access to the lake view terrace
Bathroom 1: A modern fitted suite with bath tub, shower tap, wash basin, WC & bidet
Bathroom 2: A contemporary-fitted shower room with wash basin & WC
• Lake view living • Flat screen TV • Book-games library • Nespresso coffee machine • Access to the apartment is via 30 external steps and then a lift • Private roof terrace • Alfresco sitting/dining area
The spacious apartment residence is located within Lugano, along 'Via Cassarinetta'. Guests are within an 11 minutes' walk away from a host of local eateries and restaurants, a selection of supermarkets, Sixt car hire, Florida park and the Lugano-Paradiso ferry stop. Central Lugano can be reached by car in just 10 minutes.
Lugano city is a truly year-round destination (with its business links, Tennis Club, modern family Lido, designer shops, restaurants, casinos, buzzing nightlife and parks). Business travellers and groups can enjoy dipping in and out of all the city action. Lugano Golf club is just 14 minutes' drive away.
For those looking to sightsee some more, enchanting Lake Como is 39 minutes' drive away.
The holiday home is also situated within easy reach of Lugano Airport (9 minutes' drive away).
Please respect the rest times. Any noise from 10pm to 8am and from noon to 1pm, Sunday and public holidays is strictly prohibited.
- Lake View
- Smoking Not Permitted
- Car Not Necessary
- Pets Not Permitted
- Apartment
- Books/Games Library
- Child Friendly
- Coffee Machine
- Cooker Hob
- Dishwasher
- Electric Kettle
- Free Wifi
- Freezer
- Fridge
- Full Kitchen
- Hairdryer
- High Definition TV
- In Town
- Iron/Ironing Board
- Lift/Elevator
- Lounge/Diner
- Microwave
- Nespresso Machine
- Outside Dining Area
- Outside Table & Chairs
- Oven
- Private Parking Space
- Quiet
- Satellite TV
- Suitable for Elderly
- Terrace
- Toaster
- Very Quiet Location
- Very Sunny Position
- Waste Disposal
Activities & Attractions
Seasonal Weekly Tariffs
|Min stay
|Start date
|End date
|4 nights
|19 Oct 2020
|29 Oct 2020
|4 nights
|30 Oct 2020
|31 Oct 2020
|4 nights
|01 Nov 2020
|04 Nov 2020
|4 nights
|05 Nov 2020
|09 Nov 2020
|4 nights
|10 Nov 2020
|19 Dec 2020
|4 nights
|20 Dec 2020
|31 May 2021
|4 nights
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|31 Aug 2021
|4 nights
|01 Sep 2021
|31 Oct 2021
|4 nights
|01 Nov 2021
|19 Dec 2021
|4 nights
|20 Dec 2021
|07 Jan 2022
|Additional Service Charge: CHF215 (7 days & less) & CHF255 (7 days plus)


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Traditionalist Chesnutt shows his stuff after stellar performance by Clark
Houston Livestock Show and Rodeo, the Astrodome, Houston, March 1, 1997
By Brian Wahlert
HOUSTON - Country music's stylistic leaning and popularity tend to go in cycles.
Country becomes really popular, and then record labels and radio stations try to further increase that popularity by catering to the new, broader audience, which, presumably, wants a less traditional, more pop-rock bent.
Inevitably, what is called country gets so far away from what drew the broader audience in the first place that its popularity declines and much of the audience leaves for another format.
Then the country industry responds by going back to its roots. Eventually, people who are disenchanted with the commercialism and lack of real emotion in other musical formats latch on to country, and the cycle starts all over again.
Right now we're in the decline stage. Country fans seem to be tiring of the crassly commercial music and glitzy stage shows of many of country's top stars, and record labels are beginning to respond by cutting many artists from their rosters and signing distinctly traditional bands like BR5-49.
When the smoke clears, only a few of today's stars will remain, but the smart money says that the singers who performed at the Rodeo tonight will be two of the few.
Take Terri Clark, for example. Some might look at her cowboy hat and dismiss her as a traditionalist wannabe, but her music demonstrates that she's nothing but straight-ahead country.
On hard-core country ballads like "If I Were You" and "Just the Same" Clark's voice gets a chance to stretch out and shine. Her powerful, deep voice and great vocal control and these songs' beautiful fiddle-and-steel arrangements made them highlights of her show.
She's got the energetic, uptempo songs that get the crowd excited, too. Hits like "When Boy Meets Girl" and "Poor, Poor Pitiful Me" feature a big, electrified sound, but Clark's voice keeps them country.
Then there was "Twang Thang," a cheesy piece of country schlock with lyrics like, "It's gotta have a twang thang / It's gotta bend a heart strang." It's not worth Clark's talent, but her earnest delivery kept the song from being a total washout.
In addition to her ability as a singer and great traditional style, Clark also demonstrated an innocent, almost bewildered demeanor that endeared her to the audience.
"This is the biggest audience I've ever played for - definitely! I'm just a little bit nervous. I'm not afraid to tell you," she said with a slight quaver in her voice.
Then later, "I cannot believe I'm singing in front of this many people. I wish my mom was here to see this!"
The end result of her great performance and genuine personality was perhaps the best response to an opening act of this year's Rodeo. The crowd gave her a raucous standing ovation after her show, and she loved every second of it, waving and posing as she was driven around the Astrodome floor for her victory lap.
Few artists could follow that performance, especially in Houston, where the crowd knows real country music.
Mark Chesnutt is one of those few.
From the beginning of his career, Chesnutt has never been afraid to record traditional country music - in fact, it's what he does best. One of his earliest hits, "Too Cold at Home," featuring a gorgeous steel guitar, is still one of his greatest songs. A new ballad called "Let It Rain" looks poised to follow in that song's footsteps by becoming another huge hit.
Perhaps his best song, however, is his version of a Hank Williams Jr. record called "I'll Think of Something." Its heartbreaking lyrics of a man who would do anything to get over his lost love and Chesnutt's emotion-packed delivery made it one of the highlights of tonight's show.
Another great feature of Chesnutt's music is that he can go straight from a serious song of country heartbreak to loopy songs about a redneck who destroys a jukebox in a bar for playing a sad song that made him cry ("Bubba Shot the Jukebox") and a blue-collar worker who parties all weekend and dreads the beginning of the week ("It Sure Is Monday"). Chesnutt's sense of humor is a great added dimension to his music that too few artists have.
Chesnutt has also worked a Cajun flavor into some of his recent music, and this new part of his style suits him well. His most recent hit, "It's a Little Too Late," featured Cajun fiddle and accordion solos, and "Gonna Get a Life" was another big Cajun hit.
For several years, Chesnutt has been on the brink of superstardom, but he's never quite crossed that line. He headlined his own tour a couple of years back with generally miserable results - thousands of empty seats at most performances.
Even though he can't fill concert arenas on his own or sell at the multiplatinum level of far less talented but more commercial country stars like John Michael Montgomery and Tim McGraw, he still refuses to compromise his artistic integrity. And in Texas, at least, fans, like the 49,049 who came to see him at the Rodeo tonight, appreciate that.
©Country Standard Time • Jeffrey B. Remz, editor & publisher • firstname.lastname@example.com
About • Copyright • Newsletter • Our sister publication Standard Time


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Pressure Reducing Valve IR-720
The Model 720 Pressure Reducing Valve is a hydraulically operated, diaphragm actuated control valve that reduces higher upstream pressure to lower constant downstream pressure regardless of fluctuating demand or varying upstream pressure.
- Line pressure driven – Independent operation
- In-line serviceable – Easy maintenance
- Double chamber design
- Moderated valve reaction
- Protected diaphragm
- Flexible design – Easy addition of features
- Variety of accessories – Perfect mission matching
- "Y" or angle, wide body – Minimized pressure loss
- Semi-straight flow – Non-turbulent flow
- Stainless Steel raised seat – Cavitation damage resistant
- Obstacle free, full bore – Uncompromising reliability
- V-Port Throttling Plug – Low flow stability
Typical Applications
Pressure Reducing System for Municipal Networks.
Network design requires establishing various pressure zones due to topography, distances, demands, energy costs, reservoir availability, etc.


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NAME] - Working upwards!
The Counter stool PURE WORK GREY is a professional work chair, ideal for reception desks, trade fair stand or any office workstation with flexible working heights. The abrasion-resistant and padded upholstery of the seat, as well as the breathable mesh on the backrest ensure pleasant comfort even during long sitting periods of or on hot days. In addition, the integrated lumbar support can be adjusted individually in height. The wide, sturdy plastic chair base equipped with floor gliders ensures perfect stability. The foot ring can easily be height adjusted.
- The integrated lordose support is height-adjustable in an action radius of 10 cm and can be optimally adjusted within 7 steps
- Seat in abrasion-resistant fabric, backrest in breathable mesh
- Comfortable foot ring made of polished steel, height adjustable by simply turning
- The practical armrests, the frame of the backrest and also the sturdy chair base are in elegant grey
- Floor glider for a firm and soil-friendly stand
This item is supplied unassembled.
For assembly by the customer in just a few simple steps.
Of course, for gas springs, chair castors, footrests, etc., no construction is required.


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