The SR Splicing Factors: Providing Perspectives on Their Evolution,Expression, Alternative Splicing,and Function in Populus trichocarpa

Serine/arginine-rich (SR) proteins are important splicing factors in plant development and abiotic/hormone-related stresses. However, evidence that SR proteins contribute to the process in woody plants has been lacking. Using phylogenetics, gene synteny, transgenic experiments, and RNA-seq analysis, we identified 24 PtSR genes and explored their evolution, expression, and function in Popolus trichocarpa. The PtSR genes were divided into six subfamilies, generated by at least two events of genome triplication and duplication. Notably, they were constitutively expressed in roots, stems, and leaves, demonstrating their fundamental role in P. trichocarpa. Additionally, most PtSR genes (~83%) responded to at least one stress (cold, drought, salt, SA, MeJA, or ABA), and, especially, cold stress induced a dramatic perturbation in the expression and/or alternative splicing (AS) of 18 PtSR genes (~75%). Evidentially, the overexpression of PtSCL30 in Arabidopsis decreased freezing tolerance, which probably resulted from AS changes of the genes (e.g., ICE2 and COR15A) critical for cold tolerance. Moreover, the transgenic plants were salt-hypersensitive at the germination stage. These indicate that PtSCL30 may act as a negative regulator under cold and salt stress. Altogether, this study sheds light on the evolution, expression, and AS of PtSR genes, and the functional mechanisms of PtSCL30 in woody plants.     

Molecular Characterization,mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid,Toxoptera citricida (Kirkaldy)

Ryanodine receptors (RyRs) play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR), an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif) and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action.     

A Double-Edged Sword: The Two Faces of PARylation

Poly ADP-ribosylation (PARylation) is a post-translational modification process. Following the discovery of PARP-1, numerous studies have demonstrated the role of PARylation in the DNA damage and repair responses for cellular stress and DNA damage. Originally, studies on PARylation were confined to PARP-1 activation in the DNA repair pathway. However, the interplay between PARylation and DNA repair suggests that PARylation is important for the efficiency and accuracy of DNA repair. PARylation has contradicting roles; however, recent evidence implicates its importance in inflammation, metabolism, and cell death. These differences might be dependent on specific cellular conditions or experimental models used, and suggest that PARylation may play two opposing roles in cellular homeostasis. Understanding the role of PARylation in cellular function is not only important for identifying novel therapeutic approaches; it is also essential for gaining insight into the mechanisms of unexplored diseases. In this review, we discuss recent reports on the role of PARylation in mediating diverse cellular functions and homeostasis, such as DNA repair, inflammation, metabolism, and cell death.     

NAD(H) Regulates the Permeability Transition Pore in Mitochondria through an External Site

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD⁺ on: (1) the Ca²⁺-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca²⁺-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca²⁺-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca²⁺-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD⁺ increased the CRC of liver, heart, and brain mitochondria 1.5–2.5 times, insignificantly affecting the rate of Ca²⁺-uptake and the free Ca²⁺ concentration in the medium. NAD(H) suppressed the Ca²⁺-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca²⁺-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.     

A Regulatory Axis between Epithelial Splicing Regulatory Proteins and Estrogen Receptor α Modulates the Alternative Transcriptome of Luminal Breast Cancer

Epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) control the splicing pattern during epithelial to mesenchymal transition (EMT) in a physiological context and in cancer, including breast cancer (BC). Here, we report that ESRP1, but not ESRP2, is overexpressed in luminal BCs of patients with poor prognosis and correlates with estrogen receptor α (ERα) levels. Analysis of ERα genome-binding profiles in cell lines and primary breast tumors showed its binding in the proximity of ESRP1 and ESRP2 genes, whose expression is strongly decreased by ERα silencing in hormone-deprived conditions. The combined knock-down of ESRP1/2 in MCF-7 cells followed by RNA-Seq, revealed the dysregulation of 754 genes, with a widespread alteration of alternative splicing events (ASEs) of genes involved in cell signaling, metabolism, cell growth, and EMT. Functional network analysis of ASEs correlated with ESRP1/2 expression in ERα+ BCs showed RAC1 as the hub node in the protein–protein interactions altered by ESRP1/2 silencing. The comparison of ERα- and ESRP-modulated ASEs revealed 63 commonly regulated events, including 27 detected in primary BCs and endocrine-resistant cell lines. Our data support a functional implication of the ERα-ESRP1/2 axis in the onset and progression of BC by controlling the splicing patterns of related genes.     

Proteomic Analysis of Roots Response to Potassium Deficiency and the Effect of TaHAK1-4A on K+ Uptake in Wheat

Potassium (K⁺) is essential for plant growth and stress responses. A deficiency in soil K⁺ contents can result in decreased wheat quality and productivity. Thus, clarifying the molecular mechanism underlying wheat responses to low-K⁺ (LK) stress is critical. In this study, a tandem mass tag (TMT)-based quantitative proteomic analysis was performed to investigate the differentially abundant proteins (DAPs) in roots of the LK-tolerant wheat cultivar “KN9204” at the seedling stage after exposure to LK stress. A total of 104 DAPs were identified in the LK-treated roots. The DAPs related to carbohydrate and energy metabolism, transport, stress responses and defense, and post-translational modifications under LK conditions were highlighted. We identified a high-affinity potassium transporter (TaHAK1-4A) that was significantly up-regulated after the LK treatment. Additionally, TaHAK1-4A was mainly expressed in roots, and the encoded protein was localized in the plasma membrane. The complementation assay in yeast suggested that TaHAK1-4A mediates K⁺ uptake under extreme LK conditions. The overexpression of TaHAK1-4A increased the fresh weight and root length of Arabidopsis under LK conditions and improved the growth of Arabidopsis athak5 mutant seedlings, which grow poorly under LK conditions. Moreover, silencing of TaHAK1-4A in wheat roots treated with LK stress decreased the root length, dry weight, K⁺ concentration, and K⁺ influx. Accordingly, TaHAK1-4A is important for the uptake of K⁺ by roots exposed to LK stress. Our results reveal the protein metabolic changes in wheat induced by LK stress. Furthermore, we identified a candidate gene potentially relevant for developing wheat lines with increased K⁺ use efficiency.     

Glucocorticoid Regulates the Synthesis of Porcine Muscle Protein through m6A Modified Amino Acid Transporter SLC7A7

The occurrence of stress is unavoidable in the process of livestock production, and prolonged stress will cause the decrease of livestock productivity. The stress response is mainly regulated by the hypothalamic-pituitary-adrenal axis (HPA axis), which produces a large amount of stress hormones, namely glucocorticoids (GCs), and generates a severe impact on the energy metabolism of the animal body. It is reported that m⁶A modification plays an important role in the regulation of stress response and also participates in the process of muscle growth and development. In this study, we explored the effect of GCs on the protein synthesis procession of porcine skeletal muscle cells (PSCs). We prove that dexamethasone affects the expression of SLC7A7, a main amino acid transporter for protein synthesis by affecting the level of m⁶A modification in PSCs. In addition, we find that SLC7A7 affects the level of PSC protein synthesis by regulating the conduction of the mTOR signaling pathway, which indicates that the reduction of SLC7A7 expression may alleviate the level of protein synthesis under stress conditions.     

Characterization in Effective Stimulation on the Magnitude,Gating, Frequency Dependence,and Hysteresis of INa Exerted by Picaridin (or Icaridin), a Known Insect Repellent

Picaridin (icaridin), a member of the piperidine chemical family, is a broad-spectrum arthropod repellent. Its actions have been largely thought to be due to its interaction with odorant receptor proteins. However, to our knowledge, to what extent the presence of picaridin can modify the magnitude, gating, and/or the strength of voltage-dependent hysteresis (Hys_(V)) of plasmalemmal ionic currents, such as, voltage-gated Na⁺ current [I_Na], has not been entirely explored. In GH₃ pituitary tumor cells, we demonstrated that with exposure to picaridin the transient (I_Na(T)) and late (I_Na(L)) components of voltage-gated Na⁺ current (I_Na) were differentially stimulated with effective EC₅₀’s of 32.7 and 2.8 μM, respectively. Upon cell exposure to it, the steady-state current versus voltage relationship I_Na(T) was shifted to more hyperpolarized potentials. Moreover, its presence caused a rightward shift in the midpoint for the steady-state inactivate curve of the current. The cumulative inhibition of I_Na(T) induced during repetitive stimuli became retarded during its exposure. The recovery time course from the I_Na block elicited, following the conditioning pulse stimulation, was satisfactorily fitted by two exponential processes. Moreover, the fast and slow time constants of recovery from the I_Na block by the same conditioning protocol were noticeably increased in the presence of picaridin. However, the fraction in fast or slow component of recovery time course was, respectively, increased or decreased with an increase in picaridin concentrations. The Hys_(V)’s strength of persistent I_Na (I_Na(P)), responding to triangular ramp voltage, was also enhanced during cell exposure to picaridin. The magnitude of resurgent I_Na (I_Na(R)) was raised in its presence. Picaritin-induced increases of I_Na(P) or I_Na(R) intrinsically in GH₃ cells could be attenuated by further addition of ranolazine. The predictions of molecular docking also disclosed that there are possible interactions of the picaridin molecule with the hNa_V1.7 channel. Taken literally, the stimulation of I_Na exerted by the exposure to picaridin is expected to exert impacts on the functional activities residing in electrically excitable cells.     

Plasma Membrane H+-ATPase SmPHA4 Negatively Regulates the Biosynthesis of Tanshinones in Salvia miltiorrhiza

Salvia miltiorrhiza Bunge has been widely used in the treatment of cardiovascular and cerebrovascular diseases, due to the pharmacological action of its active components such as the tanshinones. Plasma membrane (PM) H⁺-ATPase plays key roles in numerous physiological processes in plants. However, little is known about the PM H⁺-ATPase gene family in S. miltiorrhiza (Sm). Here, nine PM H⁺-ATPase isoforms were identified and named SmPHA1–SmPHA9. Phylogenetic tree analysis showed that the genetic distance of SmPHAs was relatively far in the S. miltiorrhiza PM H⁺-ATPase family. Moreover, the transmembrane structures were rich in SmPHA protein. In addition, SmPHA4 was found to be highly expressed in roots and flowers. HPLC revealed that accumulation of dihydrotanshinone (DT), cryptotanshinone (CT), and tanshinone I (TI) was significantly reduced in the SmPHA4-OE lines but was increased in the SmPHA4-RNAi lines, ranging from 2.54 to 3.52, 3.77 to 6.33, and 0.35 to 0.74 mg/g, respectively, suggesting that SmPHA4 is a candidate regulator of tanshinone metabolites. Moreover, qRT-PCR confirmed that the expression of tanshinone biosynthetic-related key enzymes was also upregulated in the SmPHA4-RNAi lines. In summary, this study highlighted PM H⁺-ATPase function and provided new insights into regulatory candidate genes for modulating secondary metabolism biosynthesis in S. miltiorrhiza.     

Analysis of 235U Enrichment by Chemical Exchange in U(IV)‐U(VI) System on Anionite

Abstract

A theoretical study about ²³⁵U enrichment by the chemical exchange method in U(IV)‐U(VI) system on anion‐exchange resins is presented. The ²³⁵U isotope concentration profiles along the band were numerically calculated using an accurate mathematical model and simulations were carried out for the situation of product and waste withdrawal and feed supply. By means of numerical simulation, an estimation of the migration time, necessary for a desired enrichment degree, was obtained. The required migration distance, the production of uranium 3 at.% ²³⁵U per year and the plant configuration are calculated for different operating conditions. An analysis of the process scale for various experimental conditions is also presented.     

Blocking NHE Channels Reduces the Ability of In Vitro Capacitated Mammalian Sperm to Respond to Progesterone Stimulus

Few data exist about the presence and physiological role of Na+/H+ exchangers (NHEs) in the plasma membrane of mammalian sperm. In addition, the involvement of these channels in the ability of sperm to undergo capacitation and acrosomal reaction has not been investigated in any mammalian species. In the present study, we addressed whether these channels are implicated in these two sperm events using the pig as a model. We also confirmed the presence of NHE1 channels in the plasma membrane of ejaculated sperm by immunofluorescence and immunoblotting. The function of NHE channels during in vitro capacitation was analyzed by incubating sperm samples in capacitating medium for 300 min in the absence or presence of a specific blocker (DMA; 5-(N,N-dimethyl)-amiloride) at different concentrations (1, 5, and 10 µM); acrosome exocytosis was triggered by adding progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium and reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were evaluated after 0, 60, 120, 180, 240, 250, 270, and 300 min of incubation. NHE1 localized in the connecting and terminal pieces of the flagellum and in the equatorial region of the sperm head and was found to have a molecular weight of 75 kDa. During the first 240 min of incubation, i.e., before the addition of progesterone, blocked and control samples did not differ significantly in any of the parameters analyzed. However, from 250 min of incubation, samples treated with DMA showed significant alterations in total motility and the amplitude of lateral head displacement (ALH), acrosomal integrity, membrane lipid disorder, and MMP. In conclusion, while NHE channels are not involved in the sperm ability to undergo capacitation, they could be essential for triggering acrosome exocytosis and hypermotility after progesterone stimulus.     

Interleukin-21 in Viral Infections

Interleukin (IL)-21 is a cytokine that affects the differentiation and function of lymphoid and myeloid cells and regulates both innate and adaptive immune responses. In addition to regulating the immune response to tumor and viral infections, IL-21 also has a profound effect on the development of autoimmune and inflammatory diseases. IL-21 is produced mainly from CD4⁺ T cells—in particular, follicular helper T (Tfh) cells—which have a great influence on the regulation of antibody production. It is also an important cytokine for the activation of CD8⁺ T cells, and its role in recovering the function of CD8⁺ T cells exhausted by chronic microbial infections and cancer has been clarified. Thus, IL-21 plays an extremely important role in viral infections, especially chronic viral infections. In this review, I will introduce the findings to date on how IL-21 is involved in some typical viral infections and the potential of treating viral diseases with IL-21.