A replication-coupled repair based model of centromere inheritance.

<p>(A) A replication-coupled repair based model for propagation of CENP-A^CaCse4 chromatin at an early replicating centromere. During S phase, replication forks, originating from proximal conserved early origins, stall at the kinetochore. The stalling of replication forks at the centromere leads to accumulation of single stranded (ss) DNA. The homologous recombination proteins Rad51 and Rad52 are possibly recruited via ssDNA to the stalled replication forks at the centromere. A transient Rad51/Rad52-CENP-A^CaCse4 complex is stabilized by one or more cell-cycle regulated proteins (chaperone?) at the centromere, thereby regulating the replication coupled deposition of CENP-A at the centromeres. The CENP-A^CaCse4 bound kinetochore is indicated by red circles whereas replisome is depicted by large purple circle. Functional origin locations are shown by blue filled ovals. <i>CEN</i>- centromere, KT- kinetochore. (B) Schematic depicts the effect of CENP-A^CaCse4/Rad51/Rad52 depletion on replication fork passage through the centromere. Depletion of CENP-A^CaCse4 causes kinetochore disintegrity. As a result forks are no longer stalled at the <i>CEN</i>-kinetochore barrier and fork stalling is weakened. Depletion of Rad51/Rad52 also causes improper kinetochore assembly. As a result fork stalling is weakened. (C) Schematic depicts the effect of deletion of a proximal origin on replication fork passage through the centromere. On deletion of a proximal origin, fork stalling at the centromere is reduced and concomitantly CENP-A^CaCse4 binding is reduced, leading to a weaker centromere.</p

Replication forks stall/terminate randomly during centromere replication.

<p>Schematic of a ∼30 kb region of chromosome 7 centered on the centromere (<i>CEN7</i>) is shown. The hatched rectangles denote the positions of the nearest neocentromere (<i>nCEN7</i>) hotspots as described earlier <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Thakur1" target="_blank">[38]</a>. The filled grey circles indicate the positions of the chromosomal origins identified during 2D analysis (<i>ORI7-LI</i> and <i>ORI7-RI</i>). Replication intermediates from this 30 kb region in asynchronously grown <i>C. albicans</i> cells were analyzed by 2D gel electrophoresis assays using overlapping restriction fragments (1–8). Arrowheads and numbers indicate the positions and the identities of the ORFs. Open rectangles indicate the fragments used for the <i>ARS</i> function assay. Schematic of replication intermediates indicates simple ‘Y’ arcs (broken line), specific termination (Double-Ys), joint molecules (Xs) and random termination signals (triangular smear). The dark grey zone at the inflection point of the ‘Y’ arc indicates replication fork stalling. The presence of Xs and triangular smear in fragments 1, 2, 4, 5 and 7 indicates replication fork stalling/termination. Bubble arcs are observed in fragments 3 and 6 signaling chromosomal origins of replication (<i>ORI7-LI</i> and <i>ORI7-RI</i>). The plate pictures in the lower panel show the results of an <i>ARS</i> function assay using the fragments (open rectangles) located within <i>ORI7-LI</i> and <i>ORI7-RI</i> in the wild-type. The corresponding 2-D signals in high contrast are shown in the inset. Both fragments show <i>ARS</i> activity.</p

Centromeric fork stalling/termination is CENP-A mediated and involves Rad51 and Rad52.

<p>(A) A line diagram of a 6 kb region of chromosome 7 centered on <i>CEN7</i> is shown. <i>CEN7</i> (black rectangle) and flanking regions (grey rectangles) that include the 5 kb <i>Eco</i>RI fragment (fragment 4 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen-1004344-g001" target="_blank">Figure 1</a>) used for 2D gel analysis are shown. Schematics of replication intermediates as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen-1004344-g001" target="_blank">Figure 1</a> are also shown. Quantification of the termination signals was performed as following: Relative intensity of termination (RIT) = random termination signal/1n spot. (B) Replication intermediates from the core <i>CEN7</i> region were determined by 2-D gel analysis at wild-type and depleted levels of CENP-A^CaCse4. (C) The 1n spot (schematic) and the termination signals (triangular smear) were quantified by Image Gauge software (Fujifilm) and RIT values were calculated as described above for wild-type and CENP-A^CaCse4 depleted condition. The RIT values, plotted on a bar graph, indicate a gradual decrease in the termination signal in CENP-A^CaCse4 repressed conditions as compared to wild-type. The values represent the mean of three independent 2D experiments ± SD. (D) Replication intermediates from the core <i>CEN7</i> region (black rectangle) were determined by 2D gel analysis for the wild-type, <i>rad51</i>, and <i>rad52</i> mutants. (E) RIT values were calculated for wild-type and <i>rad51</i> and <i>rad52</i> mutants. The RIT values, plotted on a bar graph, indicate a decrease in the termination signal in <i>rad51</i> and <i>rad52</i> mutants as compared to wild-type. The values represent the mean of three independent 2D experiments ± SD.</p

Rad51 or Rad52 depletion affects kinetochore assembly.

<p>(A) Percentage of cells at each cell cycle stage was determined for wild-type, <i>rad51</i> and <i>rad52</i> mutants. At least 100 cells were counted at each stage. lb, large bud; elb, extended large bud. The extended large bud (elb) is an aberrant G2/M phenotype observed in <i>rad51</i> and <i>rad52</i> mutants only. (B) Using confocal microscopy, GFP-CENP-A^CaCse4 foci were scored in large budded cells of wild-type, <i>rad51</i> or <i>rad52</i> mutant strains. They were classified into three categories as shown in figure. n≥100. The percentage of large budded cells under each category was calculated for wild-type and mutant strains, and plotted. An increase in percentage of large budded cells with declustered GFP-CENP-A^CaCse4signals is observed in <i>rad51</i> or <i>rad52</i> mutant, which is an indicator of improper kinetochore assembly <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Thakur2" target="_blank">[44]</a>. Bar (white line), 5 µm. (C) Intensity of the GFP-CENP-A^CaCse4 spots was measured by the Image J software for wild-type, <i>rad51</i> or <i>rad52</i> mutant cells for the G2/M stage, n = 10 in each case. The normalized mean GFP intensity (with respect to background) was calculated for each cell and plotted. Legend shows the different categories of strains and stages and the average GFP-CENP-A^CaCse4 intensity ± S.E.M. Associated DIC images show the measurement technique for calculating GFP intensity. Bar (white line), 1 µm.</p

Replication-segregation interaction is evolutionarily conserved in unicellular organisms.

<p>(A) The phylogenetic tree reflects the evolutionary relationships of the corresponding taxa. The tree is drawn to scale with branch lengths in the units of the number of base substitutions per site in the 23S or 25S rRNA nucleotide sequences of the four species. (B) <i>CEN</i>-like loci or <i>CEN</i>s (green boxes) in prokaryotes and unicellular eukaryotes respectively are flanked by early replication origins (pink circles). The blue circles indicate the centromere factors influencing origin activity. The yellow circles indicate the origin/replication associated factors influencing <i>CEN</i> function. In the genome of the bacteria <i>B. subtilis</i>, the single replication origin is flanked by <i>CEN</i>-like <i>parS</i> sequence. The Spo0J (ParB) protein, binding to <i>parS</i>, organizes <i>ori</i> activity as well as recruits Smc proteins for proper segregation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Gruber1" target="_blank">[17]</a>. In <i>S. cerevisiae</i>, which has short ‘point’ centromeres, the Ctf19 complex directly recruits initiation factors for early firing of proximal origins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Natsume1" target="_blank">[20]</a>. Although early firing has been suggested for playing a role in <i>CEN</i> function, no <i>cis</i> factors has been identified. In <i>C. albicans</i>, which has ‘short regional’ <i>CEN</i>s, <i>CEN</i>s have been shown to govern early replication of proximal origins, although no <i>cis</i> factors were identified <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Koren1" target="_blank">[5]</a>. In this study we show that fork stalling at <i>CENs</i> from proximal origins recruit Rad51/Rad52 that, in turn, regulates CENP-A deposition. Finally in the ‘large regional’ centromeres of <i>S. pombe</i>, the centromeric heterochromatic protein Swi6 activates pericentric replication origins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Hayashi1" target="_blank">[16]</a>. The fork protection complex (FPC) that travels with the replisome negatively regulates Ams2 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Takayama2" target="_blank">[73]</a> that, in turn, regulates CENP-A deposition <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Takayama1" target="_blank">[10]</a>.</p

Rad51 and Rad52 aid in CENP-A^CaCse4 recruitment.

<p>(A) Standard ChIP assays followed by quantitative real time PCR (qPCR) were performed in wild-type, <i>rad51</i> or <i>rad52</i> for CENP-A^CaCse4-Prot A for <i>CEN5</i> and <i>CEN7</i>. qPCR amplification from a non-centromeric (<i>non-CEN</i>) control was also performed to detect the background DNA elution in the ChIP assays. Enrichment of CENP-A^CaCse4 at the centromeres was calculated as a percentage of the total chromatin input and values were plotted as mean of three independent ChIP experiments ± SD. (B) Western blot analysis performed with the whole cell lysates from wild-type and homologous recombination (HR) and non-homologous end joining (NHEJ) mutants using anti-CENP-A^CaCse4 antibodies. PSTAIRE was used as a loading control. The relative levels of CENP-A^CaCse4 (CENP-A^CaCse4/PSTAIRE) was computed for each mutant and plotted in a bar graph. (C) Co-immunoprecipitation assays for the two sets of strains carrying Rad51-V5 and Rad52-V5 were performed using anti-V5 antibodies. Precipitates were analyzed by western blotting with anti- CENP-A^CaCse4 antibodies. In each case, untagged strains and no (-) antibody fractions were used as controls. Blue asterisk indicates a non-specific band.</p

Centromere proximal origins maintain centromere functioning.

<p>(A) Schematic showing the strategy of deletion of <i>ORI7-RI</i> (shown as red circles) and the chromosome loss assay. Since the strain RM100AH <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Sanyal1" target="_blank">[33]</a> is heterozygous for both <i>HIS1</i> and <i>ARG4</i> that mark the two chromosome 7 homologs, replacement of <i>ORI7-RI</i> by <i>URA3</i> will create a strain that can be used to assay the loss of the altered chromosome (by scoring simultaneous loss of Ura and Arg or Ura and His markers). (B) Dilutions of <i>ORI7-RI</i> deleted transformants (<i>ORI7-RI/</i>Δ<i>ORI7-RI</i>) were spotted on CM+5′-FOA plates to estimate the chromosome loss frequency. USN148 (Δ<i>ura3::imm434/</i>Δ<i>ura3::imm434</i>/CIp10) strain was used as the wild-type control to estimate the spontaneous loss rate of a chromosome. Subsequently, the 5′-FOA positive colonies were patched on YPDU. From YPDU these colonies were re-patched onto CM- Ura, CM-His and CM-Arg plates to assay for the loss of the altered chromosome 7 homolog. (C) Standard ChIP assays followed by quantitative real time PCR (qPCR) were performed in wild-type and CAKS105 (Δ<i>ORI7-RI/</i>Δ<i>ORI7-RI</i>) strain for enrichment of CENP-A^CaCse4-Prot A at the core <i>CEN7</i>. Enrichment of CENP-A^CaCse4 at the centromere was calculated as a percentage of the total chromatin input and values were plotted as mean of two independent experiments (three technical replicates for each experiment) ± SD. (D) Line diagrams depicting ∼15 kb region surrounding <i>CEN7</i> are shown (symbols as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen-1004344-g001" target="_blank">Figure 1</a>). Schematics depict the replication intermediates as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen-1004344-g001" target="_blank">Figure 1</a>. Bar (black line), 1 kb. The upper panel shows the 2-D image from the core <i>CEN7</i> region (fragment 4 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen-1004344-g001" target="_blank">Figure 1</a>) in the wild-type. The lower panel shows the 2-D image from the same fragment when <i>ORI7-RI</i> is deleted in CAKS105 (Δ<i>ORI7-RI/</i>Δ<i>ORI7-RI</i>).</p

Supplementary information files for Effects of COVID-19 home confinement on eating behaviour and physical activity: results of the ECLB-COVID19 international online survey

Supplementary files for article Effects of COVID-19 home confinement on eating behaviour and physical activity: results of the ECLB-COVID19 international online survey. Background: Public health recommendations and governmental measures during the COVID-19 pandemic have resulted in numerous restrictions on daily living including social distancing, isolation and home confinement. While these measures are imperative to abate the spreading of COVID-19, the impact of these restrictions on health behaviours and lifestyles at home is undefined. Therefore, an international online survey was launched in April 2020, in seven languages, to elucidate the behavioural and lifestyle consequences of COVID-19 restrictions. This report presents the results from the first thousand responders on physical activity (PA) and nutrition behaviours. Methods: Following a structured review of the literature, the “Effects of home Confinement on multiple Lifestyle Behaviours during the COVID-19 outbreak (ECLB-COVID19)” Electronic survey was designed by a steering group of multidisciplinary scientists and academics. The survey was uploaded and shared on the Google online survey platform. Thirty-five research organisations from Europe, North-Africa, Western Asia and the Americas promoted the survey in English, German, French, Arabic, Spanish, Portuguese and Slovenian languages. Questions were presented in a differential format, with questions related to responses “before” and “during” confinement conditions. Results: 1047 replies (54% women) from Asia (36%), Africa (40%), Europe (21%) and other (3%) were included in the analysis. The COVID-19 home confinement had a negative effect on all PA intensity levels (vigorous, moderate, walking and overall). Additionally, daily sitting time increased from 5 to 8 h per day. Food consumption and meal patterns (the type of food, eating out of control, snacks between meals, number of main meals) were more unhealthy during confinement, with only alcohol binge drinking decreasing significantly. Conclusion: While isolation is a necessary measure to protect public health, results indicate that it alters physical activity and eating behaviours in a health compromising direction. A more detailed analysis of survey data will allow for a segregation of these responses in different age groups, countries and other subgroups, which will help develop interventions to mitigate the negative lifestyle behaviours that have manifested during the COVID-19 confinement.</div

Effects of home confinement on mental health and lifestyle behaviours during the COVID-19 outbreak: insights from the ECLB-COVID19 multicenter study

Although recognised as effective measures to curb the spread of the COVID-19 outbreak, social distancing and self-isolation have been suggested to generate a burden throughout the population. To provide scientific data to help identify risk factors for the psychosocial strain during the COVID-19 outbreak, an international cross-disciplinary online survey was circulated in April 2020. This report outlines the mental, emotional and behavioural consequences of COVID-19 home confinement. The ECLB-COVID19 electronic survey was designed by a steering group of multidisciplinary scientists, following a structured review of the literature. The survey was uploaded and shared on the Google online survey platform and was promoted by thirty-five research organizations from Europe, North Africa, Western Asia and the Americas. Questions were presented in a differential format with questions related to responses “before” and “during” the confinement period. 1047 replies (54% women) from Western Asia (36%), North Africa (40%), Europe (21%) and other continents (3%) were analysed. The COVID-19 home confinement evoked a negative effect on mental wellbeing and emotional status (P < 0.001; 0.43 ≤ d ≤ 0.65) with a greater proportion of individuals experiencing psychosocial and emotional disorders (+10% to +16.5%). These psychosocial tolls were associated with unhealthy lifestyle behaviours with a greater proportion of individuals experiencing (i) physical (+15.2%) and social (+71.2%) inactivity, (ii) poor sleep quality (+12.8%), (iii) unhealthy diet behaviours (+10%), and (iv) unemployment (6%). Conversely, participants demonstrated a greater use (+15%) of technology during the confinement period. These findings elucidate the risk of psychosocial strain during the COVID-19 home confinement period and provide a clear remit for the urgent implementation of technology-based intervention to foster an Active and Healthy Confinement Lifestyle AHCL)

COVID-19 home confinement negatively impacts social participation and life satisfaction: a worldwide multicenter study

Public health recommendations and governmental measures during the new coronavirus disease (COVID-19) pandemic have enforced numerous restrictions on daily living including social distancing, isolation, and home confinement. While these measures are imperative to mitigate spreading of COVID-19, the impact of these restrictions on psychosocial health is undefined. Therefore, an international online survey was launched in April 2020 to elucidate the behavioral and lifestyle consequences of COVID-19 restrictions. This report presents the preliminary results from more than one thousand responders on social participation and life satisfaction. Methods: Thirty-five research organizations from Europe, North-Africa, Western Asia, and the Americas promoted the survey through their networks to the general society, in 7 languages (English, German, French, Arabic, Spanish, Portuguese, and Slovenian). Questions were presented in a differential format with questions related to responses “before” and “during” confinement conditions. Results: 1047 participations (54% women) from Asia (36%), Africa (40%), Europe (21%), and others (3%) were included in the analysis. Findings revealed psychosocial strain during the enforced COVID-19 home confinement. Large decreases (p < 0.001) in the amount of social activity through family (−58%), friends/neighbors (−44.9%), or entertainment (−46.7%) were triggered by the enforced confinement. These negative effects on social participation were also associated with lower life satisfaction (−30.5%) during the confinement period. Conversely, the social contact score through digital technologies significantly increased (p < 0.001) during the confinement period with more individuals (+24.8%) being socially connected through digital technology. Conclusion: These preliminary findings elucidate the risk of psychosocial strain during the early COVID-19 home confinement period in 2020. Therefore, in order to mitigate the negative psychosocial effects of home confinement, implementation of national strategies focused on promoting social inclusion through a technology-based solution is strongly suggested