Maternal mRNA deadenylation and allocation via Rbm14 condensates facilitate vertebrate blastula development.

Early embryonic development depends on proper utilization and clearance of maternal transcriptomes. How these processes are spatiotemporally regulated remains unclear. Here we show that nuclear RNA-binding protein Rbm14 and maternal mRNAs co-phase separate into cytoplasmic condensates to facilitate vertebrate blastula-to-gastrula development. In zebrafish, Rbm14 condensates were highly abundant in blastomeres and markedly reduced after prominent activation of zygotic transcription. They concentrated at spindle poles by associating with centrosomal γ-tubulin puncta and displayed mainly asymmetric divisions with a global symmetry across embryonic midline in 8- and 16-cell embryos. Their formation was dose-dependently stimulated by m6 A, but repressed by m5 C modification of the maternal mRNA. Furthermore, deadenylase Parn co-phase separated with these condensates, and this was required for deadenylation of the mRNAs in early blastomeres. Depletion of Rbm14 impaired embryonic cell differentiations and full activations of the zygotic genome in both zebrafish and mouse and resulted in developmental arrest at the blastula stage. Our results suggest that cytoplasmic Rbm14 condensate formation regulates early embryogenesis by facilitating deadenylation, protection, and mitotic allocation of m6 A-modified maternal mRNAs, and by releasing the poly(A)-less transcripts upon regulated disassembly to allow their re-polyadenylation and translation or clearance.

Human amnion mesenchymal stem cells promote endometrial repair via paracrine, preferentially than transdifferentiation.

BACKGROUND: Intrauterine adhesion (IUA) is one of the most severe causes of infertility in women of childbearing age with injured endometrium secondary to uterine performance. Stem cell therapy is effective in treating damaged endometrium. The current reports mainly focus on the therapeutic effects of stem cells through paracrine or transdifferentiation, respectively. This study investigates whether paracrine or transdifferentiation occurs preferentially in treating IUA. METHODS: Human amniotic mesenchymal stem cells (hAMSCs) and transformed human endometrial stromal cells (THESCs) induced by transforming growth factor beta (TGF-ß1) were co-cultured in vitro. The mRNA and protein expression levels of Fibronectin (FN), Collagen I, Cytokeratin19 (CK19), E-cadherin (E-cad) and Vimentin were detected by Quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and Immunohistochemical staining (IHC). The Sprague-Dawley (SD) rats were used to establish the IUA model. hAMSCs, hAMSCs-conditional medium (hAMSCs-CM), and GFP-labeled hAMSCs were injected into intrauterine, respectively. The fibrotic area of the endometrium was evaluated by Masson staining. The number of endometrium glands was detected by hematoxylin and eosin (H&E). GFP-labeled hAMSCs were traced by immunofluorescence (IF). hAMSCs, combined with PPCNg (hAMSCs/PPCNg), were injected into the vagina, which was compared with intrauterine injection. RESULTS: qPCR and WB revealed that FN and Collagen I levels in IUA-THESCs decreased significantly after co-culturing with hAMSCs. Moreover, CK19, E-cad, and Vimentin expressions in hAMSCs showed no significant difference after co-culture for 2 days. 6 days after co-culture, CK19, E-cad and Vimentin expressions in hAMSCs were significantly changed. Histological assays showed increased endometrial glands and a remarkable decrease in the fibrotic area in the hAMSCs and hAMSCs-CM groups. However, these changes were not statistically different between the two groups. In vivo, fluorescence imaging revealed that GFP-hAMSCs were localized in the endometrial stroma and gradually underwent apoptosis. The effect of hAMSCs by vaginal injection was comparable to that by intrauterine injection assessed by H&E staining, MASSON staining and IHC. CONCLUSIONS: Our data demonstrated that hAMSCs promoted endometrial repair via paracrine, preferentially than transdifferentiation.

IUA is the crucial cause of infertility in women of childbearing age, and no satisfactory treatment measures have been found in the clinic. hAMSCs can effectively treat intrauterine adhesions through paracrine and transdifferentiation mechanisms. This study confirmed in vitro and in vivo that amniotic mesenchymal stem cells preferentially inhibited endometrial fibrosis and promoted epithelial repair through paracrine, thus effectively treating intrauterine adhesions. The level of fibrosis marker proteins in IUA-THESCs decreased significantly after co-culturing with hAMSCs for 2 days in vitro. However, the level of epithelial marker proteins in hAMSCs increased significantly, requiring at least 6 days of co-culture. hAMSCs-CM had the same efficacy as hAMSCs in inhibiting fibrosis and promoting endometrial repair in IUA rats, supporting the idea that hAMSCs promoted endometrial remodeling through paracrine in vivo. In addition, GFP-labeled hAMSCs continuously colonized the endometrial stroma instead of the epithelium and gradually underwent apoptosis. These findings prove that hAMSCs ameliorate endometrial fibrosis of IUA via paracrine, preferentially than transdifferentiation, providing the latest insights into the precision treatment of IUA with hAMSCs and a theoretical basis for promoting the "cell-free therapy" of MSCs.

Analysis of Pickled Cucumber Products, Based on Microbial Diversity and Flavor Substance Detection.

Changes to the microbial community during pickled cucumber fermentation were studied using the 16S rDNA technique. The changes of volatile organic compounds (VOCs) during pickled cucumber fermentation were studied by gas chromatograph-ion mobility spectrometry. At the phylum level, Cyanophyta and Proteobacteria were the dominant flora in the natural fermentation group, and Firmicutes were the dominant flora in the added-bacteria fermentation group. At the generic level, the addition of Lactobacillus led to changes in the community of the bacteria in the added-bacterial fermentation group and decreased the species abundance of other bacteria. In total, 75 volatile organic compounds were identified from naturally fermented pickled cucumber, and 60 volatile organic compounds were identified from fermented pickled cucumber with bacterial addition. The main metabolites were esters, aldehydes, acids, alcohols, ketones, alkanes, nitriles, and alkenes. These metabolites will bring their unique aroma components to the pickled cucumber. Metabolomic analysis of the O2PLS model showed that Weissella and Lactobacillus were closely and positively correlated with nine alcohols, six esters, five aldehydes, four acids, three ketones, and one pyrazine. Pseudomonas and norank_f_Mitochondria show a close positive correlation with four kinds of alcohols, two kinds of esters, one kind of aldehyde, and one kind of nitrile.

Fertilization can accelerate the pace of soil microbial community response to rest-grazing duration in the three-river source region of China.

Overgrazing leads to grassland degradation and productivity decline. Rest-grazing during the regreen-up period can quickly restore grassland and fertilization is a common restoration strategy. However, the effects of rest-grazing time and fertilization on soil microorganisms are unclear in the alpine grasslands. Therefore, the experiment of rest-grazing time and fertilization was carried out to explore the response of soil microorganisms to rest-grazing time and fertilization measures. A field control experiment with rest-grazing time and fertilization as factors have been conducted from the time when grass returned to green till the livestock moved to the summer pasture in Dawu Town of Maqin County of China. The primary treatment we established was the five rest-grazing time, including rest-grazing time of 20 days, 30 days, 40 days, 50 days, and traditional grazing was used as a check group. At the same time, the secondary treatment was nitrogen addition of 300 kg·hm-2 in each primary treatment. The results showed that the total phospholipid fatty acid (total PLFA), actinomyces (Act), and arbuscular mycorrhizal fungi (AMF) showed an ever-increasing biomass with the increase of rest-grazing time and the highest was at 50 days of rest-grazing, and they were all significantly higher than CK. In addition, soil microbial biomass carbon-nitrogen ratio (MBC/MBN) had great influence on the change of microbial community. Applying nitrogen fertilizer can increase the maximum value of biomass of all PLFA groups and the biomass of all PLFA groups changed in an "inverted V" shape with the increase of rest-grazing time. Besides, instead of MBC/MBN, NO3 --N was positively correlated with the biomass of all PLFA groups, which actively regulated the trend of microbial functions. The longer rest-grazing time is more conducive to the biomass of all PLFA groups. However, applying nitrogen fertilizer could break this pattern, namely, the 30 days rest-grazing would be beneficial to the biomass of all PLFA groups. These findings provide key information that rest-grazing during the regreen-up period is benefiscial to the all PLFA groups and fertilization could change the response of microorganisms to rest-grazing, which provide reference measures for the restoration of degraded alpine meadows.

Cloud-Based English Multimedia for Universities Test Questions Modeling and Applications.

This study constructs a cloud computing-based college English multimedia test question modeling and application through an in-depth study of cloud computing and college English multimedia test questions. The emergence of cloud computing technology undoubtedly provides a new and ideal method to solve test data and paper management problems. This study analyzes the advantages of the Hadoop computing platform and MapReduce computing model and builds a distributed computing platform based on Hadoop using universities' existing hardware and software resources. The study analyzes the advantages of the Hadoop computing platform and the MapReduce computing model. The UML model of the system is given, the system is implemented, the system is tested functionally, and the results of the analysis are given. Multimedia is the critical link to realizing the optimization of English test questions. The proper use of multimedia test questions will undoubtedly become an inevitable trend in the development of English test questions in the future, which requires every worker on the education front to continuously analyze and study the problems arising from multimedia teaching, summarize the experience of multimedia teaching, and explore new methods of multimedia teaching, so that multimedia teaching can better promote the optimization of English test questions in colleges and universities and better serve the education teaching.

C:N:P stoichiometry in plant, soil and microbe in Sophora moorcroftiana shrubs across three sandy dune types in the middle reaches of the Yarlung Zangbo River.

The alpine sandy dune ecosystem is highly vulnerable to global climate change. Ecological stoichiometry in plants and soils plays a crucial role in biogeochemical cycles, energy flow and functioning in ecosystems. The alpine sandy dune ecosystem is highly vulnerable to global climate change. However, the stoichiometric changes and correlations of plants and soils among different types of sandy dunes have not been fully explored. Three sandy dune types (moving dune, MD; semifixed dune, SFD; and fixed dune, FD) of the Sophora moorcroftiana shrub in the middle reaches of the Yarlung Zangbo River were used as the subjects in the current study. Plant community characteristics, soil physicochemical properties, carbon (C), nitrogen (N), and phosphorus (P) contents of leaves, understorey herbs, litter, and soil microbes were evaluated to explore the C:N:P stoichiometry and its driving factors. Sandy dune type significant affected on the C:N:P stoichiometry in plants and soils. High soil N:P ratio was observed in FD and high plant C:P and N:P ratios in SFD and MD. The C:N ratio decreased with sand dune stabilization compared with other stoichiometric ratios of soil resources. Leaf C:P and N:P ratios in S. moorcroftiana were higher than those in the understorey herb biomass, because of the low P concentrations in leaves. C, N and P contents and stoichiometry of leaves, understorey herbs, litter and microbe were significantly correlated with the soil C, N and P contents and stoichiometry, with a higher correlation for soil N:P ratio. P was the mainly limiting factor for the growth of S. moorcroftiana population in the study area and its demand became increasingly critical with the increase in shrub age. The variation in the C:N:P stoichiometry in plants and soils was mainly modulated by the soil physicochemical properties, mainly for soil moisture, pH, available P and dissolved organic C. These findings provide key information on the nutrient stoichiometry patterns, element distribution and utilization strategies of C, N and P and as well as scrubland restoration and management in alpine valley sand ecosystems.

Fibrogranular materials function as organizers to ensure the fidelity of multiciliary assembly.

Multicilia are delicate motile machineries, and how they are accurately assembled is poorly understood. Here, we show that fibrogranular materials (FGMs), large arrays of electron-dense granules specific to multiciliated cells, are essential for their ultrastructural fidelity. Pcm1 forms the granular units that further network into widespread FGMs, which are abundant in spherical FGM cores. FGM cores selectively concentrate multiple important centriole-related proteins as clients, including Cep131 that specifically decorates a foot region of ciliary central pair (CP) microtubules. FGMs also tightly contact deuterosome-procentriole complexes. Disruption of FGMs in mouse cells undergoing multiciliogenesis by Pcm1 RNAi markedly deregulates centriolar targeting of FGM clients, elongates CP-foot, and alters deuterosome size, number, and distribution. Although the multicilia are produced in correct numbers, they display abnormal ultrastructure and motility. Our results suggest that FGMs organize deuterosomes and centriole-related proteins to facilitate the faithful assembly of basal bodies and multiciliary axonemes.

Wdr47, Camsaps, and Katanin cooperate to generate ciliary central microtubules.

The axonemal central pair (CP) are non-centrosomal microtubules critical for planar ciliary beat. How they form, however, is poorly understood. Here, we show that mammalian CP formation requires Wdr47, Camsaps, and microtubule-severing activity of Katanin. Katanin severs peripheral microtubules to produce central microtubule seeds in nascent cilia. Camsaps stabilize minus ends of the seeds to facilitate microtubule outgrowth, whereas Wdr47 concentrates Camsaps into the axonemal central lumen to properly position central microtubules. Wdr47 deficiency in mouse multicilia results in complete loss of CP, rotatory beat, and primary ciliary dyskinesia. Overexpression of Camsaps or their microtubule-binding regions induces central microtubules in Wdr47-/- ependymal cells but at the expense of low efficiency, abnormal numbers, and wrong location. Katanin levels and activity also impact the central microtubule number. We propose that Wdr47, Camsaps, and Katanin function together for the generation of non-centrosomal microtubule arrays in polarized subcellular compartments.

Jasplakinolide, an actin stabilizing agent, alters anaphase chromosome movements in crane-fly spermatocytes.

We added jasplakinolide to anaphase crane-fly spermatocytes and determined its effects on chromosome movement. Previous work showed that the actin depolymerizing agents cytochalasin D or latrunculin B blocked or slowed chromosome movements. We studied the effects of jasplakinolide, a compound that stabilizes actin filaments. Jasplakinolide had the same effect on movements of each half- bivalent in a separating pair of half-bivalents, but different half-bivalent pairs in the same cell often responded differently, even when the concentrations of jasplakinolide varied by a factor of two. Jasplakinolide had no effect on about 20% of the pairs, but otherwise caused movements to slow, or to stop, or, rarely, to accelerate. When cells were kept in jasplakinolide, stopped pairs eventually resumed movement; slowed pairs did not change their speeds. Confocal microscopy indicated that neither the distributions of spindle actin filaments nor the distributions of spindle microtubules were altered by the jasplakinolide. It is possible that jasplakinolide binds to spindle actin and blocks critical binding sites, but we suggest that jasplakinolide affects anaphase chromosome movement by preventing actin-filament depolymerization that is necessary for anaphase to proceed. Overall, our data indicate that actin is involved in one of the redundant mechanisms cells use to move chromosomes.

Microtubule-bundling protein Spef1 enables mammalian ciliary central apparatus formation.

Cilia are cellular protrusions containing nine microtubule (MT) doublets and function to propel cell movement or extracellular liquid flow through beating or sense environmental stimuli through signal transductions. Cilia require the central pair (CP) apparatus, consisting of two CP MTs covered with projections of CP proteins, for planar strokes. How the CP MTs of such '9 + 2' cilia are constructed, however, remains unknown. Here we identify Spef1, an evolutionarily conserved microtubule-bundling protein, as a core CP MT regulator in mammalian cilia. Spef1 was selectively expressed in mammalian cells with 9 + 2 cilia and specifically localized along the CP. Its depletion in multiciliated mouse ependymal cells by RNAi completely abolished the CP MTs and markedly attenuated ciliary localizations of CP proteins such as Hydin and Spag6, resulting in rotational beat of the ependymal cilia. Spef1, which binds to MTs through its N-terminal calponin-homologous domain, formed homodimers through its C-terminal coiled coil region to bundle and stabilize MTs. Disruption of either the MT-binding or the dimerization activity abolished the ability of exogenous Spef1 to restore the structure and functions of the CP apparatus. We propose that Spef1 bundles and stabilizes central MTs to enable the assembly and functions of the CP apparatus.

Nanog suppresses cell migration by downregulating thymosin ß4 and Rnd3.

Nanog, Sox2, and Oct4 are key transcription factors critical for the pluripotency and self-renewal of embryonic stem (ES) cells. Their downregulations lead to differentiation, accompanied with changes in cell motility. Whether these factors impact cell motility directly, however, is not clear. Here we addressed this question by initially assessing their effect in non-stem cells. We found that the ectopic expression of Nanog, Sox2, or Oct4 markedly inhibited ECV304 cell migration. Detailed examinations revealed that Nanog induced disorganizations of the actin cytoskeleton and peripheral localizations of focal adhesions. These effects required its DNA-binding domain and are thus transcription dependent. Furthermore, thymosin ß4 and Rnd3 were identified as its downstream targets. Their depletions in ECV304 cells by RNAi phenocopied the ectopic expression of Nanog in both cell motility and actin organization, whereas their ectopic expressions rescued the migration defect of Nanog overexpression. Both proteins were upregulated during mouse ES cell differentiation. Their levels in the pluripotent mouse P19 cells also increased upon Nanog ablation, coincident with an increase in cell motility. Moreover, persistent expression of Nanog in zebrafish embryos suppressed gastrulation and cell migration. These results indeed suggest a dual role of certain transcription factors in the orchestration of differentiation and motility.