NPTX1 Regulates Neural Lineage Specification from Human Pluripotent Stem Cells

SummaryNeural induction is the first fundamental step in nervous system formation. During development, a tightly regulated niche modulates transient extracellular signals to influence neural lineage commitment. To date, however, the cascade of molecular events that sustain these signals in humans is not well understood. Here we show that NPTX1, a secreted protein, is rapidly upregulated during neural induction from human pluripotent stem cells (hPSCs). By manipulating its expression, we were able to reduce or initiate neural lineage commitment. A time-course transcriptome analysis and functional assays show that NPTX1 acts in part by binding the Nodal receptor cofactor TDGF1, reducing both Nodal and BMP signaling. Our findings identify one of the earliest genes expressed upon neural induction and provide insight into human neural lineage specification

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Nucleolar asymmetry and the importance of septin integrity upon cell cycle arrest

Cell cycle arrest can be imposed by inactivating the anaphase promoting complex (APC). In S. cerevisiae this arrest has been reported to stabilize a metaphase-like intermediate in which the nuclear envelope spans the bud neck, while chromatin repeatedly translocates between the mother and bud domains. The present investigation was undertaken to learn how other features of nuclear organization are affected upon depletion of the APC activator, Cdc20. We observe that the spindle pole bodies and the spindle repeatedly translocate across the narrow orifice at the level of the neck. Nevertheless, we find that the nucleolus (organized around rDNA repeats on the long right arm of chromosome XII) remains in the mother domain, marking the polarity of the nucleus. Accordingly, chromosome XII is polarized: TelXIIR remains in the mother domain and its centromere is predominantly located in the bud domain. In order to learn why the nucleolus remains in the mother domain, we studied the impact of inhibiting rRNA synthesis in arrested cells. We observed that this fragments the nucleolus and that these fragments entered the bud domain. Taken together with earlier observations, the restriction of the nucleolus to the mother domain therefore can be attributed to its massive structure. We also observed that inactivation of septins allowed arrested cells to complete the cell cycle, that the alternative APC activator, Cdh1, was required for completion of the cell cycle and that induction of Cdh1 itself caused arrested cells to progress to the end of the cell cycle

Dynamics of Chromatin and the Nucleolus in Arrested Cells.

<p>(A) Time-lapse images of chromatin and the nucleolus. A <i>MET3-CDC20</i> strain expressing the tagged histone, Htb2-mRFP, and the tagged nucleolar protein, Gar1-GFP, was arrested by addition of methionine for 4 hours before imaging. Note that the chromatin mass (red) shifted between the mother (M) and bud (B) domains repeatedly and changed shape in doing so. When most of the chromatin was in the bud domain, a narrow tail (T) extended across the mother domain. When the chromatin was retracting from the bud domain, the trailing edge often had a flattened surface (F) (rectangle). The nucleolus (Nu—green) always remained in the mother domain. In the inserts, the green signal is absent so that the red can be more uniformly detected. The blue images are bright-field. Strain: ATY3175. (B) As in Fig 2A except that the nucleolus and chromatin in the mother domain were further extended. In this situation, the tail in the mother domain defined a narrow ribbon that ends in a red patch (*) at its extremity. Strain: ATY3175. (C) Time-lapse images of chromatin, the spindle and the spindle pole body. A <i>MET3-CDC20</i> strain expressing Htb2-mRFP, Tub1-GFP and the SPB protein, Spc42-CFP, was arrested by addition of methionine for 4 hours and then imaged. The orientation of the spindle (Sp) shifted and the entire spindle frequently moved from one domain to the other along with chromatin. As shown in the lower images, the SPBs (arrow) remained associated with both extremities of the spindle. Strain: ATY7135. (D-E) Localization of centromeres (D) and kinetochores (E). The tagged variant histone, Cse4-GFP, and the tagged kinetochore protein, Ndc10-GFP, were localized in cycling cells (upper panels) and in a <i>MET3-CDC20</i> strain after arrest for 4 hours by addition of methionine (lower panels). Cycling cells: In most unbudded cells (Unb), both Cse4-GFP and Ndc10-GFP formed single clusters, while they had doubled in cells with small buds (Sm) or medium-sized buds (Md) and in cells in anaphase (Ana). Arrested cells: Note the frequent presence of additional fluorescent foci (fragments) for both Cse4-GFP and Ndc10-GFP. Both cells expressing Cse4-GFP with the tag at an internal position and cells with the tag at the C-terminus exhibited comparable fragmentation. Strains: ATY6196, ATY6882, ATY6833. (F) Quantitation of the number of foci detected in preparations equivalent to Fig 2D and 2E. Strains: ATY6196 and ATY6882.</p

Organization of the Nuclear Envelope in Arrested Cells.

<p>(A) Dynamics of the nuclear envelope: Time-lapse images of a <i>MET3-CDC20</i> strain expressing Htb2-mRFP and Nup49-GFP, after addition of methionine for 4 hours to deplete Cdc20. Note that the NE extends approximately equally into the mother and bud at all time points. The star (*) indicates a region of the nucleoplasm that is occupied by the nucleolus, judging from examination of other arrested strains. M and B designate the mother and bud domains respectively. In these and all other images, the scale bar is 5 microns. Unless indicated to the contrary, all images are projections of 5–10 Z-sections. In this figure and all others, the bud neck is designated as BN. Strain: ATY4435. (B) The bud neck: Distribution of Nup49-GFP. A <i>MET3-CDC20</i> strain expressing Htb2-mRFP and Nup49-GFP was arrested for 4 hr. The arrows indicate the frequent absence of Nup49-GFP from the bud neck. Strain: ATY4435. Exclusion of DiOC6 from the bud neck. A petite <i>MET3-CDC20</i> strain expressing Htb2-mRFP was arrested and stained with DiOC6 to detect lipids of the ER (and possibly other membranes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174306#pone.0174306.ref017" target="_blank">17</a>]). Note the near-absence of signal at the bud neck. This image is from a single 0.5 micron Z-section. Strain: ATY4435.</p

Polarity of Chromosome XII and the Nucleolus.

<p>(A) Distribution and coherence of the nucleolus. The top panels illustrate the relative behavior of a nucleolar protein (Gar1-GFP) and Htb2-mRFP during entry into anaphase of a normal cell cycle. Note that a limited amount of chromatin emerged into the bud domain at an early time-point (*) after being constricted at the level of the bud neck. The nucleolar signal then follows slowly, remaining in contact with the stretched chromatin mass. It subsequently is resolved into two equal parts that associate with the chromatin masses. Strain: ATY2416. The lower panels illustrate an intermediate time point when two nucleolar markers can be seen to traverse the neck during the normal cell cycle. These are the condensin, (Brn1-GFP) (that coats compacted rDNA and defines a narrow thread), and Sik1-mRFP, that occupies a broader territory. Both markers are compressed as the nucleolus traverses the bud neck region. Strain: ATY7473. (B) Schematic representation of nuclear domains and chromosome XII in arrested cells. The diagram illustrates the anticipated position of chromosome XII when the nucleolus (green) abuts on the bud neck. The position of the septin ring is also indicated, suggesting that this ring—in conjunction with the nucleolus—could control passage across the neck. The lacO or tetO loci that we have followed are from the following strains: locus A (ATY7633), locus B (ATY7634), locus C (ATY7733), locus D (ATY7241), locus E (ATY7637) and locus F (ATY7102). (C) Distribution and organization of telomere XII after arrest. Images of a <i>MET3-CDC20</i> strain that expresses Htb2-mRFP, Net1-CFP and a tetO tag at locus F (TelR). In the arrested cells, the tail portion of the chromatin and Net1-CFP (blue) were in the mother domain, whereas the mass of chromatin was in the bud domain. Note that when the tail was fully extended, the red extremity and the tetO locus were distal to Net1-CFP. The portion of the Htb2-mRFP signal corresponding to the nucleolus was relatively faint, perhaps because it is actively transcribed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174306#pone.0174306.ref036" target="_blank">36</a>]. Strain: ATY7102. (D) Distribution of rDNA: Images of a <i>MET3-CDC20</i> strain that expresses Htb2-mRFP, Net1-CFP and a tetO tag at locus D. Cells were arrested by addition of methionine for 4 hours and then imaged. Note that, by contrast to cells with a tagged telomere, the tagged locus abutted on the Net1-CFP signal and that—when the tail was maximally extended—this locus was not within the red telomeric region. Strain: ATY7241. (E) Distribution and organization of the telomeres of chromosome V: A <i>MET3-CDC20</i> strain expressing Htb2-mRFP, TelVL-tetO and TelVR-lacO along with YFP-tetR and CFP-lacI was arrested by addition of methionine for 4 hours. In some cells, both TelVL-YFP and TelVR-CFP were located in a single domain (left), while in other cells, the signals were each in a different domain (right). Strain: ATY7272. (F) Concanavalin staining of cells: Cells were stained with rhodamine-conjugated ConA for 15 minutes on ice. The cells were then washed and reincubated. The diagram indicates the staining pattern that was observed before and after reincubation to allow bud growth. (G) Localization of Gar1-GFP in <i>rpa190-3</i> ts mutant cells: <u>Images on the left</u>: A <i>MET3-CDC20 rpa190-3</i> strain expressing Gar1-GFP was labeled with rho-Con A and arrested by addition of methionine at 23°C. The cells were then shifted to 36°C (or not) and studied after 0–4 hours. The two upper images show that, before reincubation, Gar1-GFP remained in the mother domain (encircled by red) and reached as far as the bud neck. The three lower images are examples in which Gar1-GFP was also detected in the bud domain after a 4 hour reincubation. The bar graphs on the right quantitate the distribution of Gar1-GFP in the same ts cells and also in isogenic Ts+ cells. Note the stability of arrest in the Ts+ cells at both temperatures and the progressive loss of arrest in the ts cells after incubation at 36°C. The distribution of Gar1-GFP is symbolized as follows: M: only in the mother domain, N: contacts the neck or spans the neck, B: only in the bud, M > B: in both domains with more signal in the mother domain, M ~ B: approximately equal in both domains. In this figure and all others that quantitate distribution of markers, the data are given ± one standard deviation. Statistical significance was evaluated by Student’s <i>t</i>-test. In the lower right panel, pairs of bars (T0 hr, T4 hr) bearing the same number of stars have been compared to each other. *P < 0.005, **P < 0.05, and ***P < 0.05. Strains: ATY7760 and ATY3175. Parallel studies of <i>rpa190-3</i> cells that express a tagged histone (Htb2-GFP) did not show any indication of progression to or beyond anaphase.</p

Concrete Containing Waste Glass as an Environmentally Friendly Aggregate: A Review on Fresh and Mechanical Characteristics

The safe disposal of an enormous amount of waste glass (WG) in several countries has become a severe environmental issue. In contrast, concrete production consumes a large amount of natural resources and contributes to environmental greenhouse gas emissions. It is widely known that many kinds of waste may be utilized rather than raw materials in the field of construction materials. However, for the wide use of waste in building construction, it is necessary to ensure that the characteristics of the resulting building materials are appropriate. Recycled glass waste is one of the most attractive waste materials that can be used to create sustainable concrete compounds. Therefore, researchers focus on the production of concrete and cement mortar by utilizing waste glass as an aggregate or as a pozzolanic material. In this article, the literature discussing the use of recycled glass waste in concrete as a partial or complete replacement for aggregates has been reviewed by focusing on the effect of recycled glass waste on the fresh and mechanical properties of concrete

Fly Ash-Based Geopolymer Composites: A Review of the Compressive Strength and Microstructure Analysis

Geopolymer (GP) concrete is a novel construction material that can be used in place of traditional Portland cement (PC) concrete to reduce greenhouse gas emissions and effectively manage industrial waste. Fly ash (FA) has long been utilized as a key constituent in GPs, and GP technology provides an environmentally benign alternative to FA utilization. As a result, a thorough examination of GP concrete manufactured using FA as a precursor (FA-GP concrete) and employed as a replacement for conventional concrete has become crucial. According to the findings of current investigations, FA-GP concrete has equal or superior mechanical and physical characteristics compared to PC concrete. This article reviews the clean production, mix design, compressive strength (CS), and microstructure (Ms) analyses of the FA-GP concrete to collect and publish the most recent information and data on FA-GP concrete. In addition, this paper shall attempt to develop a comprehensive database based on the previous research study that expounds on the impact of substantial aspects such as physio-chemical characteristics of precursors, mixes, curing, additives, and chemical activation on the CS of FA-GP concrete. The purpose of this work is to give viewers a greater knowledge of the consequences and uses of using FA as a precursor to making effective GP concrete