The highly conserved LAMMER/CLK2 protein kinases prevent germ cell overproliferation in Drosophila

AbstractGerm cells undergo proper mitotic amplification before entering meiosis. The mitosis/meiosis switch drives the germ cells to leave the potential stem cell pool and become terminally differentiated. This important process is tightly controlled in the spermatogenesis of all animals. However, a unifying mechanism has yet to be unraveled. Drosophila spermatogenesis is an ideal system to dissect the regulatory program of the mitosis/meiosis switch. The timely accumulation of the pro-differentiation factor Bam has been shown to be central in this process. In a Drosophila genetic screen, we discovered that the mutations in Doa, a gene encoding a member of the highly conserved LAMMER/Cdc2-like kinase (CLK) family, cell-autonomously induced the germ cell overproliferation due to the failed transition from mitosis to meiosis. Additional Bam expression in Doa mutant germline promoted the differentiation from the mitotic to the meiotic state. Remarkably, the human or murine CLK2 could prevent the germline overproliferation and even restore the fertility of Doa mutant flies. Such rescuing activity of Doa or its human homolog requires a conserved residue in their predicted kinase catalytic domain. We propose that LAMMER/Cdc2-like kinase, represented by Doa and its mammalian homolog CLK2, is a critical and conserved component in the regulatory program of the mitosis-to-meiosis switch

Integrated estimation model of clean coal ash content for froth flotation based on model updating and multiple LS-SVMs

Clean coal ash content, a prominent product index describing coal froth flotation, is difficult to be measured online. This constraint leads to a lack of timely guidance during operation and impedes the optimal operation of the coal flotation process. To solve this problem, considering the fluctuation of working conditions, the heterogeneity of raw coal and the variation of feed coal classes, an integrated estimation model of clean coal ash content for coal flotation based on model updating and multiple least squares support vector machines (LS-SVMs) is proposed. First, a single estimation model for a single class of coal based on LS-SVM is built, and the internal parameters are optimized by gravitational search algorithm (GSA). Second, the model updating strategy is designed to solve the problem of the decline in single model accuracy. Furthermore, a multiple LS-SVMs model formed by several single models for different classes of coal is studied along with the model switching mechanism to address the problem of model mismatch. Finally, an industrial experiment and evaluation are conducted. The mean relative error between the estimated and actual values is 3.32%, and the correlation coefficient is 0.9331. The estimation accuracy and adaptability of the integrated model can meet the industrial requirements

Long-term variability in physiological measures in relation to mortality and epigenetic aging: prospective studies in the USA and China

Abstract Background Visit-to-visit body weight variability (BWV), pulse rate variability (PRV), and blood pressure variability (BPV) have been respectively linked to multiple health outcomes. The associations of the combination of long-term variability in physiological measures with mortality and epigenetic age acceleration (EAA) remain largely unknown. Methods We constructed a composite score of physiological variability (0-3) of large variability in BWV, PRV, and BPV (the top tertiles) in 2006/2008–2014/2016 in the Health and Retirement Study (HRS) and 2011–2015 in the China Health and Retirement Longitudinal Study (CHARLS). All-cause mortality was documented through 2018. EAA was calculated using thirteen DNA methylation-based epigenetic clocks among 1047 participants in a substudy of the HRS. We assessed the relation of the composite score to the risk of mortality among 6566 participants in the HRS and 6906 participants in the CHARLS by Cox proportional models and then investigated its association with EAA using linear regression models. Results A higher score of variability was associated with higher mortality risk in both cohorts (pooled hazard ratio [HR] per one-point increment, 1.27; 95% confidence interval [CI], 1.18, 1.39; P-heterogeneity = 0.344), after adjustment for multiple confounders and baseline physiological measures. Specifically, each SD increment in BWV, PRV, and BPV was related to 21% (95% CI: 15%, 28%), 6% (0%, 13%), and 12% (4%, 19%) higher hazard of mortality, respectively. The composite score was significantly related to EAA in second-generation clocks trained on health outcomes (e.g., standardized coefficient = 0.126 in the Levine clock, 95% CI: 0.055, 0.196) but not in most first-generation clocks trained on chronological age. Conclusions Larger variability in physiological measures was associated with a higher risk of mortality and faster EAA

Bearing Properties and Stability Analysis of the Slope Protection Framework Using Recycled Railway Sleepers

The slope protection framework developed using recycled railway sleepers offers a novel sustainable solution for slope protection. However, this has been inadequately reported, and its force and deformation, its protective effect, and the bonding characteristics between sleepers are still unclear. The slope protection framework project of a recycled railway sleeper embankment slope on the Beijing–Tongliao railway was numerically analyzed using three typical recycled railway sleeper slope protection structures. The bearing properties and the slope stability of rectangular, rhombic, and herringbone framework structures were determined. The results show that the stress state, stress level, and failure mode of the three types of slope protection structures are similar on average. The slope protection skeleton’s stress concentration position and failure area are all concentrated at the sleeper connection node at the slope base. The rectangular and rhombic framework structures have better stability than the herringbone framework. This study proposes applying a slope protection framework constructed entirely using recycled railway sleepers. Furthermore, it allows for proper disposal of recycled railway sleepers and a reduction in stone mining

Three RNA Binding Proteins Form a Complex to Promote Differentiation of Germline Stem Cell Lineage in <i>Drosophila</i>

<div><p>In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a <i>Drosophila</i> mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, we isolated a mutation in the gene <i>CG32364</i>, which encodes a putative RNA-binding protein (RBP) and is designated as <i>tumorous testis</i> (<i>tut</i>). In <i>tut</i> mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in <i>mei-P26</i> mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. We found that Tut binds preferentially a long isoform of <i>mei-P26</i> 3′UTR, and is essential for the translational repression of <i>mei-P26</i> reporter. Bam and Bgcn are both RBPs that have also been shown to repress <i>mei-P26</i> expression. Our genetic analyses indicate that <i>tut</i>, <i>bam</i>, or <i>bgcn</i> is required to repress <i>mei-P26</i> and to promote the differentiation of GSCs. Biochemically, we demonstrate that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Our <i>in vivo</i> and <i>in vitro</i> evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in <i>Drosophila</i> germline stem cell lineage.</p></div

Tut is required to repress <i>mei-P26</i> expression via <i>mei-P26</i> 3′UTR.

<p>(A) Physical interaction of Tut protein and <i>mei-P26</i> 3′UTR detected by RNA immunoprecipitation (RIP). TAP-Tut or TAP-GFP and <i>mei-P26</i> 3′UTR were co-expressed in S2 cells. IgG beads were used to enrich TAP-Tut or TAP-GFP followed by TEV digestion to release Tut or GFP and bound RNA. RNA was extracted and reverse transcribed. The quantity of Actin5C mRNA, short (M3US) or long (M3UL) isoform of <i>mei-P26</i> 3′UTR was determined by real-time PCR. The Y axis represents the ratio of RIP/Input, which was normalized to 1 for TAP-GFP. Error bar indicates SD. ***, p<0.0001 in <i>t</i> test. n.s., not significant. (B–B″) Genotype: <i>bam-Gal4/Y; UAS-GFP-mei-P26 3′UTR (2k)/+</i>. A genomic region of 2 kb in length downstream of <i>mei-P26</i> stop codon was selected to cover both short and long isoforms (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004797#pgen.1004797.s002" target="_blank">Figure S2A</a>). Yellow dots outline Bam-expressing spermatogonia. GFP was repressed in most Bam-expressing spermatogonia. (C–C″) Genotype: <i>bam-Gal4/Y; UAS-GFP-mei-P26 3′UTR (2k)/+; bam^BG/bam^Δ86</i>. Orange dots outline GFP-positive spermatogonia. (D–D″) Genotype: <i>bam-Gal4/Y; UAS-GFP-mei-P26 3′UTR (2k)/+; tut¹/tut³</i>. Yellow dotted outline indicates Bam-expressing spermatogonia. GFP was de-repressed in <i>tut</i> mutant even in the presence of Bam. Scale bars: 25 µm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004797#pgen.1004797.s002" target="_blank">Figure S2</a>-<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004797#pgen.1004797.s003" target="_blank">S3</a>.</p

<i>tut</i> interacts with <i>bam</i> both genetically and physically.

<p>(A–B) Low magnification view of <i>tut³</i> (A) and <i>tutP-tut-GFP/+; tut³</i> (B) testes stained with DAPI. (C&C′) <i>tutP-tut-GFP/+; tut³</i> testis stained for GFP, germline marker Vasa, and hub cell marker FasIII. Yellow asterisk indicates hub and hereafter. Green arrowhead points to a GSC expressing weak Tut-GFP signal. (D–D″) Immunofluorescence images of <i>tutP-tut-GFP/+; tut³</i> testis. Tut-GFP and Bam were both expressed in spermatogonia. (E–I) <i>tut^4/+</i> (E), <i>bam^Δ86</i>^/+ (F), <i>tut⁴</i> (G), <i>tut⁴</i>, <i>bam^Δ86/+</i> (H), and <i>tut⁴</i>, <i>bam^BG/+</i> (I) testes stained with DAPI. Red arrowheads point to over-proliferational cysts. (J&J′) Immunofluorescence images of <i>tut⁴</i>, <i>bam^BG/+</i> testis. Note the branched fusome. (K–L) Bam and Tut coimmunoprecipitated from S2 cells expressing tagged proteins. Flag-Bam and Myc-Tut were over-expressed in S2 cells and the cell lysates were used for anti-Myc (K) or anti-Flag (L) immunoprecipitation. Western analysis with corresponding antibodies was performed to detect the presence of Flag-Bam and Myc-Tut. Asterisk indicates a nonspecific band. Scale bars: 25 µm (A, B, E–I); 200 µm (C,D,J). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004797#pgen.1004797.s004" target="_blank">Figure S4</a>.</p

Transcription factor ISL1 is essential for pacemaker development and function

The sinoatrial node (SAN) maintains a rhythmic heartbeat; therefore, a better understanding of factors that drive SAN development and function is crucial to generation of potential therapies, such as biological pacemakers, for sinus arrhythmias. Here, we determined that the LIM homeodomain transcription factor ISL1 plays a key role in survival, proliferation, and function of pacemaker cells throughout development. Analysis of several Isl1 mutant mouse lines, including animals harboring an SAN-specific Isl1 deletion, revealed that ISL1 within SAN is a requirement for early embryonic viability. RNA-sequencing (RNA-seq) analyses of FACS-purified cells from ISL1-deficient SANs revealed that a number of genes critical for SAN function, including those encoding transcription factors and ion channels, were downstream of ISL1. Chromatin immunoprecipitation assays performed with anti-ISL1 antibodies and chromatin extracts from FACS-purified SAN cells demonstrated that ISL1 directly binds genomic regions within several genes required for normal pacemaker function, including subunits of the L-type calcium channel, Ank2, and Tbx3. Other genes implicated in abnormal heart rhythm in humans were also direct ISL1 targets. Together, our results demonstrate that ISL1 regulates approximately one-third of SAN-specific genes, indicate that a combination of ISL1 and other SAN transcription factors could be utilized to generate pacemaker cells, and suggest ISL1 mutations may underlie sick sinus syndrome