Blockade of Pachytene piRNA Biogenesis Reveals a Novel Requirement for Maintaining Post-Meiotic Germline Genome Integrity

<div><p>Piwi-interacting RNAs are a diverse class of small non-coding RNAs implicated in the silencing of transposable elements and the safeguarding of genome integrity. In mammals, male germ cells express two genetically and developmentally distinct populations of piRNAs at the pre-pachytene and pachytene stages of meiosis, respectively. Pre-pachytene piRNAs are mostly derived from retrotransposons and required for their silencing. In contrast, pachytene piRNAs originate from ∼3,000 genomic clusters, and their biogenesis and function remain enigmatic. Here, we report that conditional inactivation of the putative RNA helicase MOV10L1 in mouse spermatocytes produces a specific loss of pachytene piRNAs, significant accumulation of pachytene piRNA precursor transcripts, and unusual polar conglomeration of Piwi proteins with mitochondria. Pachytene piRNA–deficient spermatocytes progress through meiosis without derepression of LINE1 retrotransposons, but become arrested at the post-meiotic round spermatid stage with massive DNA damage. Our results demonstrate that MOV10L1 acts upstream of Piwi proteins in the primary processing of pachytene piRNAs and suggest that, distinct from pre-pachytene piRNAs, pachytene piRNAs fulfill a unique function in maintaining post-meiotic genome integrity.</p> </div

Post-natal disruption of <i>Mov10l1</i> leads to post-meiotic spermiogenic arrest.

<p>(A) Timeline of mouse spermatogenesis, with blue lines and tan histograms representing developmental expression patterns of three mouse Piwi proteins (MIWI, MIWI2, MILI) and MOV10L1, and pre-pachytene and pachytene piRNA populations, respectively. Crosses on lines mark the time point of spermatogenic arrest in the respective mouse mutant (4 ubiquitous null mutants and 2 <i>Mov10l1</i> conditional mutants). Arrows on the timeline bar indicate the onset of Cre expression in the different <i>Mov10l1</i> mutants generated. Disruption of <i>Mov10l1</i> by Prm-Cre did not cause spermiogenic arrest (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003038#pgen.1003038.s008" target="_blank">Table S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003038#pgen.1003038.s003" target="_blank">Figure S3</a>). (B–D) Histology of testes from adult wild-type (B), <i>Mov10l1</i>^fl/- Neurog3-Cre (C), and <i>Mov10l1</i>^fl/- Hspa2-Cre (D) mice. H&E staining was performed on testis sections as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003038#s4" target="_blank">Materials and Methods</a>. Abbreviations: Pa, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids.</p

Northern blot analysis of individual pachytene piRNAs in conditional <i>Mov10l1</i> mutant testes.

<p>RNAs were prepared from adult testes and hybridized with radiolabeled probes specific for pachytene piR1, piR2, piR3, and a pre-pachytene piRNA. Hybridization with a let7g miRNA probe served as a loading control. The three individual pachytene piRNAs were absent in <i>Mov10l1</i>^fl/- Neurog3-Cre testes, sharply reduced in abundance in <i>Mov10l1</i>^fl/- Hspa2-Cre testes, and present in <i>Mov10l1</i>^fl/- Prm-Cre testes, while the level of the pre-pachytene piRNA was not affected in either of these mutant testes.</p

<i>Mov10l1</i> is required for biogenesis of pachytene piRNAs.

<p>(A) Depletion of pachytene piRNAs in mutant testes from <i>Mov10l1</i>^fl/- Neurog3-Cre adult mice. Total RNAs were ³²P-end-labelled and separated by denaturing polyacrylamide gel electrophoresis. 18S and 28S ribosomal RNAs served as a loading control. (B) MILI is devoid of pachytene piRNAs in <i>Mov10l1</i>^fl/- Neurog3-Cre testes. MILI was immunoprecipitated from testicular extracts. One-tenth of the immunoprecipitated material was used for detection of associated piRNAs, and the remainder was used for Western blotting (WB) analysis of MILI. (C) MIWI immunoprecipitation on <i>Mov10l1</i>^fl/- Neurog3-Cre and wild-type (+/+) testes, and serial dilutions (1∶2) of MIWI IP complexes. (D) Depletion of pachytene piRNAs in mutant testes from <i>Mov10l1</i>^fl/- Hspa2-Cre adult mice. (E) MILI is loaded with pre-pachytene piRNAs in postnatal day 10 <i>Mov10l1</i>^fl/- Neurog3-Cre testes.</p

Massive DNA damage in piRNA-deficient round spermatids.

<p>(A) Schematic representation of the expression of several proteins involved in chromatin remodeling during mouse spermiogenesis. (B) Phosphorylation of histone H2AX during normal spermatogenesis. Note the presence of γH2AX in three distinct types of germ cells: leptotene/zygotene spermatocytes (due to meiotic recombination), pachytene spermatocytes (XY body only, due to sex chromosome silencing), and elongating spermatids (due to chromatin remodeling). (C) Presence of γH2AX in round spermatids from <i>Mov10l1^fl</i>^/- Neurog3-Cre testes. (D, E) Double staining of γH2AX and TOP2B in seminiferous tubules from wild-type and <i>Mov10l1^fl</i>^/- Neurog3-Cre testes. (F, G) Double staining of γH2AX and PRM2 in seminiferous tubules from wild-type and <i>Mov10l1^fl</i>^/- Neurog3-Cre testes. Red channels and DAPI staining are also shown in separate panels (D–G). Pa, pachytene spermatocytes; RS, round spermatids; ES, elongating spermatids. Scale bar, 25 µm.</p

Synthesis of Sulfur-Containing Oxindoles by Photoinduced Alkene Difunctionalization via Sulfur 1,2-Relocation

Organosulfur compounds are prevalent in various natural products, which have been widely applied in agrochemicals and pharmaceuticals. Herein, a new approach for the efficient construction of sulfur-containing oxindoles by photoinduced alkene difunctionalization via sulfur 1,2-relocation is developed. The method exhibited a high functional group tolerance and broad substrate compatibility. A library of sulfur-containing oxindole derivatives were synthesized under mild conditions (metal-, photocatalyst-, and additive-free). Mechanistic investigations revealed this photochemical process was triggered by the formation of an EDA complex of oxindole enolates with a redox-active ester, and the in situ generation of alkenes from the C–S bond cleavage of β-sulfanyl radicals was a key step in this transformation

Changes in serum sodium concentration during hemodialysis is a predictor of mortality and cardio-cerebrovascular event

Previous study consistently showed that lower serum sodium (SNa) was associated with a greater risk of mortality in hemodialysis (HD) patients. However, few studies have focused on the change in SNa (ΔSNa = post-HD SNa – pre-HD SNa) during an HD session. In a retrospective cohort of maintenance HD adults, all-cause mortality and cardio-cerebrovascular event (CCVE) were followed up for a medium of 82 months. Baseline pre-HD SNa and ΔSNa were collected; time-averaged pre-HD SNa and ΔSNa were computed as the mean values within 1-year, 2-year and 3-year intervals after enrollment. Cox proportional hazards models were used to evaluate the relationships of pre-HD and ΔSNa with outcomes. Time-averaged pre-HD SNa were associated with all-cause mortality (2-year pre-HD SNa: HR [95% CI] 0.86 [0.74–0.99], p = 0.042) and CCVE (3-year pre-HD SNa: HR [95% CI] 0.83 [0.72–0.96], p = 0.012) with full adjustment. Time-averaged ΔSNa also demonstrated an association with all-cause mortality (3-year ΔSNa: HR [95% CI] 1.26 [1.03–1.55], p = 0.026) as well as with CCVE (3-year ΔSNa: HR [95% CI] 1.51 [1.21–1.88], p = Lower time-averaged pre-HD SNa and higher time-averaged ΔSNa were associated with a greater risk of all-cause mortality and CCVE in HD patients.</p

AD-MSCs can promote vessel formation <i>in vivo</i>.

<p>After EPCs were implanted with/without UC-MSCs or AD-MSCs into SCID mice for 2 weeks, formed vessels in matrigel were subjected to immune-rejection by allo-PBMCs. The results were demonstrated by gross observation of the matrigel grafts (A) or by pathological section observation after H&E staining (B) or immunofluorescence staining (green: CD31; red: SMAα; blue: DAPI. 400×)(C). Implanted cells in matrigel formed vessels that inosculated with host vasculature and were perfused with mouse blood. EPCs implanted with AD-MSCs formed more vessels than EPCs implanted alone or EPCs implanted with UC-MSCs (D). The vessels in EPC+AD-MSC grafts also had significantly larger lumen than other groups (D). After allo-PBMC injection, red blood cell leakage was very obvious in EPC grafts or EPC+UC-MSC grafts (indicated by red arrows). In EPC+AD-MSC grafts, the vessels remained stable, and the red blood cell infiltration could hardly be observed.</p

A Comparative Study of the Perturbed-Chain Statistical Associating Fluid Theory Equation of State and Activity Coefficient Models in Phase Equilibria Calculations for Mixtures Containing Associating and Polar Components

Vapor–liquid equilibria (VLE), liquid–liquid equilibria (LLE), and vapor–liquid–liquid equilibria (VLLE) for systems involving highly nonideal components, namely, water, alcohols, alkanes, ketones, aldehydes, esters, and ethers, were investigated to evaluate the perturbed-chain statistical associating fluid theory equation of state (PC-SAFT EOS) and two widely used activity coefficient models, that is, the universal quasichemical (UNIQUAC) and UNIQUAC functional-group activity coefficients (UNIFAC). Parameters used for the PC-SAFT EOS were taken from literature or estimated in this work, while those for UNIQUAC and UNIFAC were from commercial process simulator Aspen plus 8.4. It was found that all the three models yield reliable correlations/predictions for VLE calculations. However, UNIQUAC and UNIFAC were observed to be unreliable for LLE and VLLE calculations despite successful reproductions of experimental data in some cases. The calculated results deviate significantly from experimental data in many cases. Particularly, both models predict artificial liquid–liquid phase splitting for a number of miscible mixtures. Nonetheless, PC-SAFT EOS with the use of a single set of parameters reproduces experimental data quantitatively in most cases and provides reasonably accurate results in all other cases. This remarkable performance of PC-SAFT EOS potentially eliminates the need for various thermodynamic models and consequently the need for selecting a thermodynamic model when performing phase equilibria calculations using commercial software. This is important for practitioners, since (1) it remains unclear to select an appropriate model from the available models of a process simulator or thermodynamic package for a given phase equilibria calculation despite the presence of some type of rule of thumb and (2) it is also likely that none of the existing models is sufficiently accurate. In addition, it was shown that both pure-component parameters and binary interaction parameters for the PC-SAFT EOS are well-behaved for a homologous series, which allows for parametrization for weakly characterized components by interpolation or extrapolation, and consequently, facilitates the development of a practical tool for phase equilibria calculations

Characterization of EPCs, AD-MSCs and UC-MSCs.

<p>A, Expression patterns of endothelial makers on EPCs were analyzed by FACS. EPCs highly expressed CD31, vWF, CD144 and CD105, partly positive expressed VEGFR-2 and CD34, negative for CD90, CD45, CD14 and CD19. B, Biological function of EPCs was identified. a, Representative phase contrast images of cobblestone-like EPCs. b, EPCs bound with UEA-1 (red). c, EPCs incorporated DiI-Ac-LDL (green). d, EPCs formed vascular-like tubes on matrigel. C, Phenotype analysis of AD-MSCs and UC-MSCs by FACS. Both AD-MSCs and UC-MSCs were positive for CD29, CD90, CD73 and CD105, negative for VEGFR-2, CD14, CD31, CD34 and CD45. D, After 14 days of induction, AD-MSCs and UC-MSCs were differentiated into adipocytes and osteocytes. a, Adipogenic induction of AD-MSCs. b, Adipogenic induction of UC-MSCs. c, Osteogenic induction of AD-MSCs. d, Osteogenic induction of UC-MSCs. Adipogenesis was detected by the formation of neutral lipid vacuoles stainable with oil red O (red-orange). Osteogenesis was demonstrated by detection of alkaline phosphatase activity (brown).</p