Definition of germ layer cell lineage alternative splicing programs reveals a critical role for Quaking in specifying cardiac cell fate

Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.     

Response of rats to the presence of stressed conspecifics as a function of time of day

The physiological response of nonrestrained rats to the presence of immobilized conspecifics during the beginning of the active period and the inactive period was studied. In immobilized animals concentrations of serum corticosterone (SCS), serum glucose, and liver glycogen, and the activity of liver tyrosine aminotransferase (TAT) during both the active and the inactive periods, were consistent with earlier studies. In nonrestrained rats the presence of immobilized conspecifics induced a significant increase in SCS during the active period, whereas it had no effect during the inactive period. The level of TAT was significantly elevated in the nonrestrained rats only during the inactive period and remained unchanged during the active period. The results demonstrate a physiological influence of stressed rats on unstressed conspecifics and provide evidence for regulation of TAT activity that is dependent on the situation and the time of day.     

Pre- and peri-ovulatory distribution of viable spermatozoa in the pig oviduct: a scanning electron microscope study

Using sexually mature animals, the distribution of spermatozoa has been examined at the utero-tubal junction and in the distal and proximal portions of the oviduct isthmus. Mating occurred during early oestrus and, with one exception, specimens were prepared shortly before or after ovulation. Distinct reservoirs of spermatozoa were identified in furrows between the terminal folds of the isthmus, and particularly within the troughs and transverse ridges of this region. The density of spermatozoa diminished steeply from the utero-tubal junction towards the isthmus, especially in the pre-ovulatory specimens. The membranes of most spermatozoa in the isthmus were intact up to the time of ovulation, suggesting that the acrosome reaction is a peri- or post-ovulatory event. Whilst the flagella of spermatozoa in the reservoirs were usually straight or only slightly curved, those on the surface of the epithelial folds were undulating (S-shaped). Specific microenvironments may therefore exist in the distal portion of the isthmus to regulate sperm motility; droplets of secretion were a notable feature in this region. In specimens prepared 24 hr after ovulation, spermatozoa were almost absent from the utero-tubal junction and isthmus. However, denuded eggs were observed in the proximal portion of the isthmus in this animal, and they had spermatozoa associated with the zona pellucida. Arguments are presented for a peri-ovulatory endocrine regulation of sperm redistribution and capacitation.     

Pathological and immunological evaluation of different regimens of praziquantel treatment in a mouse model of Schistosoma mansoni infection

BackgroundOne of the considerable challenges of schistosomiasis chemotherapy is the inefficacy of praziquantel (PZQ) at the initial phase of the infection. Immature schistosomes are not susceptible to PZQ at the curative dose. Here, we investigated the efficacy of different PZQ regimens administered during the initial stage of Schistosoma mansoni infection in mice.Methodology/Principal findingsTwo months-old mice were individually infected with 80 S. mansoni cercariae and divided into one infected-untreated control group (IC) and four PZQ-treated groups: PZQ at 100 mg/kg/day for five consecutive days (group PZQ1), PZQ at 100 mg/kg/day for 28 days (group PZQ2), PZQ at 18 mg/kg/day for 28 days (group PZQ3) and a single dose of PZQ at 500 mg/kg (group PZQ4). The treatment started on day one post-infection (p.i), and each group of mice was divided into two subgroups euthanized on day 36 or 56 p.i, respectively. We determined the mortality rate, the parasitological burden, the hepatic and intestinal granulomas, the serum levels of Th-1, Th-2, and Th-17 cytokines, and gene expression. The treatment led to a significant (p < 0.001) reduction of worm burden and egg counts in the intestine and liver in groups PZQ2 and PZQ3. On 56^th day p.i, there was a significant reduction (p < 0.001) of the number and volume of the hepatic granulomas in groups PZQ2 and PZQ3 compared to group PZQ1 or PZQ4. Moreover, in group PZQ3, the serum levels of IFN-γ, TNF-α, IL-13, and IL-17 and their liver mRNA expressions were significantly reduced while IL-10 and TGF-β gene expression significantly increased. The highest mortality rate (81.25%) was recorded in group PZQ2.Conclusion/SignificanceThis study revealed that the administration of PZQ at 18 mg/kg/day for 28 consecutive days was the optimal effective posology for treating S. mansoni infection at the initial stage in a murine model.     

Chlorophyll fluorescence images demonstrate variable pathways in the effects of plasma membrane excitation on electron flow in chloroplasts of <Emphasis Type=Italic>Chara</Emphasis> cells

Chlorophyll fluorescence Imaging and Microscopy PAM fluorometry were applied to study spatial dynamics of photosystem II quantum yield ( DF/F_m￠ ) left( {Delta F/F_m^prime } right) and non-photochemical quenching (NPQ) in resting and electrically stimulated Chara corallina cells in the absence and presence of the hydrophilic electron acceptor methyl viologen (MV) in the external medium. Electrical excitation of the plasma membrane temporarily enhanced the heterogeneity of photosynthetic patterns under physiological conditions (in the absence of MV), but irreversibly eliminated these patterns in the presence of MV. These findings suggest that the action potential (AP) of the excitable plant cell affects the spatial patterns of photosynthesis and chlorophyll fluorescence through different pathways operated in the absence and presence of MV. Based on the extent of NPQ as an indicator of MV-dependent electron flow, it is supposed that MV cannot permeate into the chloroplasts of photosynthetically active “acid cell regions” but gains an immediate access to the stroma of these chloroplasts after triggering of an AP. The AP-triggered MV-dependent non-photochemical quenching in the chloroplasts of acidic cell regions was routinely observed at 0.1 mM Ca²⁺ in the medium but not at elevated (2 mM) external Ca²⁺ concentration. The results are interpreted in terms of competition between two permeant divalent ion species, Ca²⁺ and MV²⁺, for their passage through the voltage-gated calcium channels of the plasma membrane. It is proposed that the herbicidal activity of MV in characean cells, here serving as model object, can be manipulated by triggering AP and varying Ca²⁺ concentration in the environmental medium.     

Borrelia burgdorferi binds fibronectin through a tandem beta-zipper, a common mechanism of fibronectin binding in staphylococci, streptococci, and spirochetes

BBK32 is a fibronectin-binding protein from the Lyme disease-causing spirochete Borrelia burgdorferi. In this study, we show that BBK32 shares sequence similarity with fibronectin module-binding motifs previously identified in proteins from Streptococcus pyogenes and Staphylococcus aureus. Nuclear magnetic resonance spectroscopy and isothermal titration calorimetry are used to confirm the binding sites of BBK32 peptides within the N-terminal domain of fibronectin and to measure the affinities of the interactions. Comparison of chemical shift perturbations in fibronectin F1 modules on binding of peptides from BBK32, FnBPA from S. aureus, and SfbI from S. pyogenes provides further evidence for a shared mechanism of binding. Despite the different locations of the bacterial attachment sites in BBK32 compared with SfbI from S. pyogenes and FnBPA from S. aureus, an antiparallel orientation is observed for binding of the N-terminal domain of fibronectin to each of the pathogens. Thus, these phylogenetically and morphologically distinct bacterial pathogens have similar mechanisms for binding to human fibronectin.