ABSTRACT
Viruses employ a series of diverse translational strategies to expand their coding capacity, which produces viral proteins with common domains and entangles virus-host interactions. P3N-PIPO, which is a transcriptional slippage product from the P3 cistron, is a potyviral protein dedicated to intercellular movement. Here, we show that P3N-PIPO from watermelon mosaic virus (WMV) triggers cell death when transiently expressed in Cucumis melo accession PI 414723 carrying the Wmr resistance gene. Surprisingly, expression of the P3N domain, shared by both P3N-PIPO and P3, can alone induce cell death, whereas expression of P3 fails to activate cell death in PI 414723. Confocal microscopy analysis revealed that P3N-PIPO targets plasmodesmata (PD) and P3N associates with PD, while P3 localizes in endoplasmic reticulum in melon cells. We also found that mutations in residues L35, L38, P41, and I43 of the P3N domain individually disrupt the cell death induced by P3N-PIPO, but do not affect the PD localization of P3N-PIPO. Furthermore, WMV mutants with L35A or I43A can systemically infect PI 414723 plants. These key residues guide us to discover some WMV isolates potentially breaking the Wmr resistance. Through searching the NCBI database, we discovered some WMV isolates with variations in these key sites, and one naturally occurring I43V variation enables WMV to systemically infect PI 414723 plants. Taken together, these results demonstrate that P3N-PIPO, but not P3, is the avirulence determinant recognized by Wmr, although the shared N terminal P3N domain can alone trigger cell death.IMPORTANCEThis work reveals a novel viral avirulence (Avr) gene recognized by a resistance (R) gene. This novel viral Avr gene is special because it is a transcriptional slippage product from another virus gene, which means that their encoding proteins share the common N-terminal domain but have distinct C-terminal domains. Amazingly, we found that it is the common N-terminal domain that determines the Avr-R recognition, but only one of the viral proteins can be recognized by the R protein to induce cell death. Next, we found that these two viral proteins target different subcellular compartments. In addition, we discovered some virus isolates with variations in the common N-terminal domain and one naturally occurring variation that enables the virus to overcome the resistance. These results show how viral proteins with common domains interact with a host resistance protein and provide new evidence for the arms race between plants and viruses.
Subject(s)
Plant Diseases , Potyvirus , Viral Proteins , Plant Diseases/virology , Potyvirus/genetics , Potyvirus/pathogenicity , Viral Proteins/genetics , Viral Proteins/metabolism , Cucumis melo/virology , Disease Resistance/genetics , Cell Death , Plasmodesmata/virology , Plasmodesmata/metabolism , Virulence , Cucurbitaceae/virology , Host-Pathogen Interactions , Endoplasmic Reticulum/virology , Endoplasmic Reticulum/metabolism , Mutation , Citrullus/virologyABSTRACT
14-3-3 proteins play vital roles in plant defense against various pathogen invasions. To date, how 14-3-3 affects virus infections in plants remains largely unclear. In this study, we found that Nicotiana benthamiana 14-3-3h interacts with TRANSLATIONALLY CONTROLLED TUMOR PROTEIN (TCTP), a susceptibility factor of potato virus Y (PVY). Silencing of Nb14-3-3h facilitates PVY accumulation, whereas overexpression of Nb14-3-3h inhibits PVY replication. The antiviral activities of 3 Nb14-3-3h dimerization defective mutants are significantly decreased, indicating that dimerization of Nb14-3-3h is indispensable for restricting PVY infection. Our results also showed that the mutant Nb14-3-3hE16A, which is capable of dimerizing but not interacting with NbTCTP, has reduced anti-PVY activity; the mutant NbTCTPI65A, which is unable to interact with Nb14-3-3h, facilitates PVY replication compared with the wild-type NbTCTP, indicating that dimeric Nb14-3-3h restricts PVY infection by interacting with NbTCTP and preventing its proviral function. As a counter-defense, PVY 6K1 interferes with the interaction between Nb14-3-3h and NbTCTP by competitively binding to Nb14-3-3h and rescues NbTCTP to promote PVY infection. Our results provide insights into the arms race between plants and potyviruses.
Subject(s)
Potyvirus , Virus Diseases , Humans , 14-3-3 Proteins , Dimerization , Viral Proteins/geneticsABSTRACT
Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops in China. In October 2023, a new bacterial disease was discovered on tomato plants in a 0.3-acre farm's greenhouse (35.514806N, 118.996106E) in Longshan Town, Shandong Province, China. Over 50% of the tomato plants showed symptoms of stem rot, leaf wilt, or plant death. Three diseased tomato plants were collected for pathogen isolation and purification. Two leaf samples, each about 1 cm2, were cut from the junction area of healthy and diseased parts and disinfected with 75% alcohol for 60 s, followed by 0.1% HgCl2 for 90 s, and then washed three times with sterilized H2O. The samples were subsequently ground with 1.0 mL sterilized H2O. The ground samples were diluted to 10-4, 10-5, and 10-6 and then were plated on a potato dextrose agar (PDA) plate, respectively. White mucous bacterial colonies appeared at 28â for 24~48 h, no fungal colony was observed. Six bacterial colonies were randomly selected for gram staining and found to be gram-negative. To further determine their species classification, fragments of the 16SrDNA, hsp60, gyrB, and rpoB genes were separately amplified using previously reported PCR conditions and with primer pairs, including 27F/1492R (Wu et al., 2023), HSP60-F /HSP60-R (Gül et al., 2023), gyrB UP-1 / gyrB UP-2r (Yamamoto et al., 1995) and rpoB CM81-F / rpoB CM32b-R (Brady et al., 2008). Sequence analysis showed that the obtained sequences of the 16SrDNA, hsp60, gyrB, and rpoB genes among these six colonies were identical and 100%, 100%, 99.31%, and 99.36% similar to those of Enterobacter mori accessions OP601841 (with a coverage of 100%), MT199160 (83%), OP676246 (100%), and MN594495 (100%), at nucleotide level, respectively. Sequences of the above four genes of 23LSFQ were submitted to GenBank under the accession numbers PP461247, PP474090, PP136037, and PP136038, respectively. We selected one of these six colonies, 23LSFQ, for further analysis. The phylogenetic tree based on the concatenated sequences of the above four genes using the maximum likelihood method with MEGAX software showed that 23LSFQ is grouped with E. mori LMG25706 (NCBI: txid980518). To determine the pathogenicity of 23LSFQ , we sprayed 23LSFQ (OD600=0.8) onto five 30-day-old healthy plants of the tomato cultivars Alisa Craig, Jinpeng NO.1, and Chaobei, respectively. These seedlings were incubated in a chamber at 28°C with a 16 h light/ 8h dark photoperiod and 60% relative humidity. The leaves of the inoculated plants became curled and wilted at two days post inoculation (dpi) and appeared necrotic at 10 dpi. The symptoms were similar to those observed in field-infected tomato plants. No symptoms were observed on the plants inoculated with water. We further sequenced the re-isolated bacteria from the symptomatic inoculated seedlings. Results showed that they belong to E. mori. The experiment was repeated three times. E. mori has been found to cause diseases on peaches (Ahmad et al., 2021), watermelons (Wu et al., 2023), Canna indica, (Zhang et al., 2023), and strawberries (Ji et al., 2023). E. cloacae has been found to cause diseases on tomatoes in Heilongjiang province (Jin et al., 2023). This is the first report of E. mori causing leaf yellowing and wilting on tomatoes in China. These results are significant for the safe production and disease control of greenhouse tomatoes.
ABSTRACT
Triacylglycerol lipase (TGL) plays critical roles in providing energy for seed germination and plant development. However, the role of TGL in regulating plant virus infection is largely unknown. In this study, we adopted affinity purification coupled with mass spectrometry and identified that a maize (Zea mays) pathogenesis-related lipase protein Z. mays TGL (ZmTGL) interacted with helper component-proteinase (HC-Pro) of sugarcane mosaic virus (SCMV). Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation assays confirmed that ZmTGL directly interacted with SCMV HC-Pro in vitro and in vivo. The 101-460 residues of SCMV HC-Pro were important for its interaction with ZmTGL. ZmTGL and SCMV HC-Pro co-localized at the mitochondria. Silencing of ZmTGL facilitated SCMV infection, and over-expression of ZmTGL reduced the RNA silencing suppression activity, most likely through reducing HC-Pro accumulation. Our results provided evidence that the lipase hydrolase activity of ZmTGL was associated with reducing HC-Pro accumulation, activation of salicylic acid (SA)-mediated defense response, and inhibition of SCMV infection. We show that ZmTGL inhibits SCMV infection by reducing HC-Pro accumulation and activating the SA pathway.
Subject(s)
Potyvirus , Zea mays , Lipase/genetics , Lipase/metabolism , Plant Diseases , Potyvirus/physiology , Salicylic Acid/metabolism , Zea mays/geneticsABSTRACT
The Editors of Medical Science Monitor wish to inform you that the above manuscript has been retracted from publication due to concerns with the credibility and originality of the study, the manuscript content, and the Figure images. Reference: Ting Cao, Teng Ma, Yang Xu, Yanping Tian, Qiyan Cai, Baichuan Li, Hongli Li. Caffeine Treatment Promotes Differentiation and Maturation of Hypoxic Oligodendrocytes via Counterbalancing Adenosine 1 Adenosine Receptor-Induced Calcium Overload. Med Sci Monit, 2019; 25: 1729-1739. DOI: 10.12659/MSM.915147.
ABSTRACT
AIM: This study aims to identify and synthesize available research reporting parental mental health outcomes related to contact with a stillborn baby. BACKGROUND: Stillbirth is devastating events for parents. The effects of contact with the stillborn baby on parental mental health are uncertain. METHODS: This was a systematic review and meta-analysis carried out by searching six international electronic databases including PubMed, EMBASE, Cochrane, Web of Science, PsycINFO and CNKI databases from inception to 15 January 2023. Review Manager software was used for data analysis. RESULTS: Ten studies were included (n = 3974). Contact with a stillborn baby increased the risks of anxiety, depression and post-traumatic stress disorder in the short term and increased the risks of anxiety and post-traumatic stress disorder in the long term. Parents who had contact with a stillborn baby were more satisfied with their decision. Subgroup analysis showed that seeing a stillborn baby had no significant effect on anxiety or depression, but holding a stillborn baby increased the risks of anxiety. CONCLUSIONS: Caregivers should respect the parents' decision on whether to have contact with the stillborn baby and provide parents with continuous information, emotional and behavioural support after they have contact with stillborn babies.
Subject(s)
Mental Health , Stillbirth , Female , Pregnancy , Humans , Stillbirth/epidemiology , Stillbirth/psychology , Parents/psychology , Emotions , Anxiety/epidemiologyABSTRACT
Cucurbits are economically important crops worldwide. The genomic data of many cucurbits are now available. However, functional analyses of cucurbit genes and noncoding RNAs have been impeded because genetic transformation is difficult for many cucurbitaceous plants. Here, we developed a set of tobacco ringspot virus (TRSV)-based vectors for gene and microRNA (miRNA) function studies in cucurbits. A TRSV-based expression vector could simultaneously express GREEN FLUORESCENT PROTEIN (GFP) and heterologous viral suppressors of RNA silencing in TRSV-infected plants, while a TRSV-based gene silencing vector could knock down endogenous genes exemplified by PHYTOENE DESATURASE (PDS) in Cucumis melo, Citrullus lanatus, Cucumis sativus, and Nicotiana benthamiana plants. We also developed a TRSV-based miRNA silencing vector to dissect the functions of endogenous miRNAs. Four representative miRNAs, namely, miR159, miR166, miR172, and miR319, from different cucurbits were inserted into the TRSV vector using a short tandem target mimic strategy and induced characteristic phenotypes in TRSV-miRNA-infected plants. This TRSV-based vector system will facilitate functional genomic studies in cucurbits.
Subject(s)
Citrullus/genetics , Cucumis sativus/genetics , Genetic Vectors , MicroRNAs/genetics , Nepovirus/genetics , Nicotiana/genetics , Citrullus/virology , Cucumis sativus/virology , Gene Knockdown Techniques , Genetic Engineering , Green Fluorescent Proteins , Oxidoreductases/genetics , Plant Proteins/genetics , RNA Interference , RNA, Plant/genetics , Nicotiana/virologyABSTRACT
Chloroplasts play an indispensable role in the arms race between plant viruses and hosts. Chloroplast proteins are often recruited by plant viruses to support viral replication and movement. However, the mechanism by which chloroplast proteins regulate potyvirus infection remains largely unknown. In this study, we observed that Nicotiana benthamiana ribosomal protein large subunit 1 (NbRPL1), a chloroplast ribosomal protein, localized to the chloroplasts via its N-terminal 61 amino acids (transit peptide), and interacted with tobacco vein banding mosaic virus (TVBMV) nuclear inclusion protein b (NIb), an RNA-dependent RNA polymerase. Upon TVBMV infection, NbRPL1 was recruited into the 6K2-induced viral replication complexes in chloroplasts. Silencing of NbRPL1 expression reduced TVBMV replication. NbRPL1 competed with NbBeclin1 to bind NIb, and reduced the NbBeclin1-mediated degradation of NIb. Therefore, our results suggest that NbRPL1 interacts with NIb in the chloroplasts, reduces NbBeclin1-mediated NIb degradation, and enhances TVBMV infection.
Subject(s)
Chloroplast Proteins/genetics , Plant Diseases/genetics , Potyvirus/physiology , Viral Proteins/genetics , Chloroplast Proteins/metabolism , Plant Diseases/virology , Potyvirus/enzymology , Nicotiana , Viral Proteins/metabolismABSTRACT
Yap is the key effector of Hippo signaling; however, its role in embryonic stem cells (ESCs) remains controversial. Here, we identify two Yap splicing isoforms (Yap472 and Yap488), which show equal expression levels but heterogeneous distribution in ESCs. Knockout (KO) of both isoforms reduces ESC self-renewal, accelerates pluripotency exit, but arrests terminal differentiation, while overexpression of each isoform leads to the reverse phenotype. The effect of both Yap isoforms on self-renewal is Teads-dependent and mediated by c-Myc. Nonetheless, different isoforms are found to affect overlapping yet distinct genes, and confer different developmental potential to Yap-KO cells, with Yap472 exerting a more pronounced biological effect and being more essential for neuroectoderm differentiation. Constitutive activation of Yaps, particularly Yap472, dramatically upregulates p53 and Cdx2, inducing trophectoderm trans-differentiation even under self-renewal conditions. These findings reveal the combined roles of different Yap splicing isoforms and mechanisms in regulating self-renewal efficiency and differentiation potential of ESCs.
Subject(s)
Embryonic Development , Embryonic Stem Cells , Cell Differentiation/genetics , Embryonic Stem Cells/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
Potyviral coat protein (CP) is involved in the replication and movement of potyviruses. However, little information is available on the roles of CP-coding sequence in potyviral infection. Here, we introduced synonymous substitutions to the codon C574G575C576 coding conserved residue arginine at position 192 (R192) of tobacco vein banding mosaic virus (TVBMV) CP. Substitution of the codon C574G575C576 to A574G575A576 or A574G575G576, but not C574G575A576, C574G575T576, or C574G575G576, reduced the replication, cell-to-cell movement, and accumulation of TVBMV in Nicotiana benthamiana plants, suggesting that C574 was critical for replication of TVBMV. Nucleotides 531 to 576 of the TVBMV CP-coding sequence were predicted to form a stem-loop structure, in which four consecutive C-G base pairs (C576-G531, C532-G575, C574-G533, and C534-G573) were located at the stem. Synonymous substitutions of R178-codon C532G533C534 to A532G533A534 and A532G533G534, but not C532G533A534, C532G533T534, or C532G533G534, reduced the replication levels, cell-to-cell, and systemic movement of TVBMV, suggesting that C532 was critical for TVBMV replication. Synonymous substitutions disrupting base pairs C576-G531 and C534-G573 did not affect viral accumulation. After three serial-passage inoculations, the accumulation of spontaneous mutant viruses was restored, and codons A532G533A534, A532G533G534, A574G575A576, or A574G575G576 of mutants were each separately changed to C532G533A534, C532G533G534, C574G575A576, or C574G575G576. Synonymous mutation of R178 and R192 also reduced viral accumulation in N. tabacum plants. Therefore, we concluded that the two consecutive C532-G575 and C574-G533 base pairs played critical roles in TVBMV replication via maintaining the stability of the stem-loop structures formed by nucleotides 531 to 576 of the CP-coding sequence.
Subject(s)
Plant Diseases , Potyvirus , Open Reading Frames , Potyvirus/genetics , RNA, Viral/genetics , Nicotiana , Virus ReplicationABSTRACT
Mixed virus infection has increasingly become a problem in the production of Solanaceae crops in recent years; therefore, a fast and accurate detection method is needed. In this study, a novel triplex immunostrip assay was developed for the simultaneous detection of tobacco mosaic virus (TMV), tobacco vein banding mosaic virus (TVBMV), and potato virus Y (PVY). The limits of detection of this novel immunostrip reached 200 ppb (ng/ml), 1 ppm (µg/ml), and 2 ppm for TMV, PVY, and TVBMV particles, respectively. Importantly, no cross-reactivity was observed among TMV, TVBMV, and PVY or to a nontarget virus. When the assay was applied to suspected virus-infected tobacco, tomato, and potato samples collected from fields in Southwest China, samples of single or mixed virus infection were successfully identified. In conclusion, the triplex immunostrip assay provides a fast and easy to use on-site detection method for field epidemiological studies of TMV, TVBMV, and PVY, and for managing diseases that are caused by them.
Subject(s)
Potyvirus , Tobacco Mosaic Virus , Plant Diseases , NicotianaABSTRACT
BACKGROUND: Due to insufficient basic medical knowledge and inappropriate learning strategies, students of 8-year medical programme encountered many obstacles in the initial stage of basic medicine learning. This study was to determine whether a prerequisite course can improve basic medicine learning performance and adjust learning strategies to adapt to basic medicine learning. METHODS: A prerequisite course of histology was constructed by a two-round modified Delphi study. Seventy-four students of 8-year medical programme were subjected to two groups: the prerequisite course group (PC group) and non-prerequisite course group (NPC group). The PC group take part in the prerequisite course by student-centred blended learning approach but NPC group not. The PC and NPC group underwent requisite histology teaching activities after prerequisite course. Examination of the prerequisite course and requisite histology course were carried out. Effect of the prerequisite course was evaluated by an empirical method using a questionnaire-based approach. RESULTS: The results of examinations showed students' scores of the PC group were significantly higher than those of students of NPC group in both prerequisite course and requisite histology examinations (P < 0.05). The results of questionnaires showed that students were satisfied with the prerequisite course, which was beneficial for uptake in medical knowledge, cultivation of clinical thinking and scientific research ability and adaptation in learning strategies (P < 0.01). Furthermore, our prerequisite course is conducive to subsequent courses learning, especially for pathology (P < 0.01). CONCLUSION: Our prerequisite course could effectively supplement knowledge of basic medicine, improve clinical thinking and scientific research ability and adapt their learning strategies. These findings suggest that the prerequisite course is useful and should be introduced in medical curriculum reform at the early stages of basic medical training.
Subject(s)
Learning , Students , China , Humans , Surveys and QuestionnairesABSTRACT
Potyviruses move to neighboring cells in the form of virus particles or a coat protein (CP)-containing ribonucleoprotein complex. However, the precise roles of RNA-binding residues in potyviral CP in viral cell-to-cell movement remain to be elucidated. In this study, we predicted the three-dimensional model of tobacco vein banding mosaic virus (TVBMV)-encoded CP and found nine residues presumably located in the CP RNA-binding pocket. Substitutions of the two basic residues at positions 192 and 225 (R192 and K225) with either alanine, cysteine, or glutamic acid abolished TVBMV cell-to-cell and systemic movement in Nicotiana benthamiana plants. These substitutions also reduced the replication of the mutant viruses. Results from the electrophoretic mobility shift assay showed that the RNA-binding activity of mutant CPs derived from R192 or K225 substitutions was significantly lower than that of wild-type CP. Analysis of purified virus particles showed that mutant viruses with R192 or K225 substitutions formed RNA-free virus-like particles. Mutations of R192 and K225 did not change the CP plasmodesmata localization. The wild-type TVBMV CP could rescue the deficient cell-to-cell movement of mutant viruses. Moreover, deletion of any of the other seven residues also abolished TVBMV cell-to-cell movement and reduced the CP RNA-binding activity. The corresponding nine residues in watermelon mosaic virus CP were also found to play essential roles in virus cell-to-cell movement. In conclusion, residues R192 and K225 in the CP RNA-binding pocket are critical for viral RNA binding and affect both virus replication and cell-to-cell movement.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Capsid Proteins , Nicotiana , Capsid Proteins/genetics , Potyvirus , RNA, Viral/genetics , Nicotiana/genetics , Virus ReplicationABSTRACT
Hippo signaling pathway is involved in many biological processes including the fate decision of embryonic stem cells (ESCs). Yes-associated protein (Yap) function as a key effector of Hippo pathway, but its role in ESCs is still controversial. So far, only two isoforms of Yap have been identified and they have both overlapping and distinct functions. Here, we identify six novel isoforms of mouse Yap, bringing the total number of isoforms to eight. According to the differences in the first exon, they are divided into two subtypes (a and b). Isoform-a and isoform-b exhibit different subcellular localizations. Moreover, isoform-a can fully reverse the impaired self-renewal phenotype induced by Yap knockout (KO). Upon overexpression, isoform-a moderately promotes mESCs self-renewal and markedly delays differentiation. On the contrary, no significant pro-self-renewal phenotype is observed when isoform-b overexpressed in wildtype (WT) mESCs or re-expressed in Yap KO cell lines. These finding not only help to clarify the role of Yap in mESCs, but also lay the foundation for advancing functional researches of Yap in other processes.
Subject(s)
Biological Phenomena , Mouse Embryonic Stem Cells , YAP-Signaling Proteins/genetics , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Embryonic Stem Cells/metabolism , Mice , Mouse Embryonic Stem Cells/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
Embryonic stem cells (ESCs) provide an ideal model for investigating developmental processes and are great sources for developing regenerative medicine. Harnessing apoptosis facilitates accurate recapitulation of signalling events during embryogenesis and allows efficient expansion of the ESCs during differentiation. Bcl2, a key regulator of intrinsic anti-apoptotic pathway, encodes two splicing isoforms. However, the identification and functional comparison of Bcl2 splicing isoforms in mouse ESCs (mESCs) remains to be elucidated. Here, we provide the evidence that both Bcl2 splicing variants are expressed in mESCs. Despite the structural difference, they have similar subcellular localisation. Both Bcl2α and Bcl2ß enhance differentiation efficiency of the ESCs and effectively improve the survival and growth of ESCs under serum-free conditions. However, the functional effect of Bcl2α was more potent than that of Bcl2ß. Moreover, only Bcl2α could maintain the long-term expansion and pluripotency of ESCs cultured in serum-free medium. Taken together, our results demonstrate previously unknown functional differences in Bcl2 alternative splicing isoforms in ESCs, and lay the foundation for future efforts to engineer ESCs for regenerative medicine.
Subject(s)
Mouse Embryonic Stem Cells/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Alternative Splicing , Animals , Cell Differentiation , Cell Line , Mice , Mouse Embryonic Stem Cells/cytology , Protein Isoforms/analysis , Protein Isoforms/genetics , Proto-Oncogene Proteins c-bcl-2/analysisABSTRACT
Embryonic stem (ES) cells are unique in their ability to self-renew indefinitely while maintaining pluripotency. Krüppel-like factor (Klf) 4 is an important member of the Klf family that is known to play a key role in pluripotency and somatic cell reprogramming. However, the identification and functional comparison of Klf4 splicing isoforms in mouse ESCs (mESCs) remains to be elucidated. Here, we identified three novel alternative splicing variants of Klf4 in mESCs-mKlf4-108, mKlf4-375 and mKlf4-1482-that are distinct from the previously known mKlf4-1449. mKlf4-1449 and mKlf4-1482 may stimulate the growth of ESCs, while mKlf4-108 can only promote the growth of ESCs in LIFlow/serum conditions. In addition, both mKlf4-1449 and mKlf4-1482 can inhibit the differentiation of mESCs. However, the ability of mKlf4-1482 to promote self-renewal and inhibit differentiation is not as strong as that of mKlf4-1449. In contrast, both mKlf4-108 and mKlf4-375 may have the ability to induce endodermal differentiation. Taken together, we have identified for the first time the existence of alternative splicing variants of mKlf4 and have revealed their different roles, which provide new insights into the contribution of Klf4 to the self-renewal and pluripotency of mouse ESCs.
Subject(s)
Alternative Splicing , Cell Self Renewal/genetics , Cell Self Renewal/physiology , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/physiology , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/physiology , Animals , Base Sequence , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Line , Codon, Nonsense , Kruppel-Like Factor 4 , Mice , Models, Biological , Poly A/genetics , Protein Isoforms/genetics , Protein Isoforms/physiologyABSTRACT
BACKGROUND We aimed to explore the involvement of adenosine 1 adenosine receptor (A1AR) in hypoxia-induced poor differentiation of oligodendrocytes (OLs), and the underlying mechanism of caffeine treatment in hypoxic injuries. MATERIAL AND METHODS Real-time polymerase chain reaction (RT-PCR) was used to assess the alterations of AR expression in cultured hypoxic OLs with or without caffeine treatment. Then, intracellular alterations of Ca²âº concentrations ([Ca²âº) were detected by confocal Fluo-3 imaging. The subsequent changes of myelin related protein expression were determined by western blot and immunofluorescence. RESULTS Three hours after hypoxia, significantly upregulated expression of A1AR was observed, accompanied with significantly decreased expression of oligodendrocyte transcription factor (Olig2). In addition, either hypoxia stimulation or 100 µM adenosine induced apparent elevation of resting [Ca²âº] in cultured OLs. However, pretreatment with DPCPX (A1AR selective antagonist) or caffeine abolished the [Ca²âº] increase, and the subsequent adenosine of high dose induced Ca²âº activity in developing OLs. Furthermore, caffeine or DPCPX improved the expression MBP and CNPase proteins after hypoxia stimulation, which resulted in the morphological maturation of OLs. CONCLUSIONS Caffeine treatment exerted protective effects on neonatal hypoxia injuries. It prevented Ca²âº overload injury, kept Ca²âº homeostasis in hypoxic developing OLs, and facilitated optimal expression of myelin related proteins by inhibiting A1AR in vitro. This study also provided experimental evidence for clinical application of caffeine in early treatment of neonatal hypoxia, and highlighted the potential significance of A1AR in anti-hypoxic drug discovery.
Subject(s)
Caffeine/pharmacology , Oligodendroglia/drug effects , Receptor, Adenosine A1/drug effects , Adenosine/metabolism , Animals , Caffeine/metabolism , Calcium/metabolism , Cell Differentiation/drug effects , Cells, Cultured , China , Homeostasis/drug effects , Hypoxia/drug therapy , Hypoxia/physiopathology , Myelin Sheath/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Calcium-Sensing/metabolism , Receptors, Purinergic P1/drug effects , Receptors, Purinergic P1/metabolismABSTRACT
AIMS AND OBJECTIVES: To determine midwives' preference for "Hands-on" and "Hands-off/poised" methods and to explore the impact factors. BACKGROUND: With the increasing rate of obstetric anal sphincter injuries, great controversy surrounds the "Hands-on" or "Hands-off" method during childbirth to decrease obstetric anal sphincter injuries incidence. Without regular records and related research, determining the use of this technique in China is difficult. DESIGN: This study used a quantitative study design, following the EQUATOR guidelines (STROBE). METHODS: A nationwide cross-sectional online survey was conducted using respondent-driven sampling across 31 provinces in China from 1st October 2017-31st December 2017. A total of 6,425 midwives were involved. Descriptive analyses, chi-square test and binary logistic regression were undertaken. RESULTS: A total of 5,225 questionnaires were returned; 55.8% of the participants preferred the "Hands-off/poised" method. The impact factors included hospital categories, total work experience in a birth unit in years, theoretical education and skill training. In the study, for situations with increased OASI risk, 100% of the midwives in the "Hands-off" group expressed willingness to change to the "Hands-on" method whether there was concern about impending obstetric anal sphincter injuries. CONCLUSION: The "Hands-off/poised" method is heavily practised by Chinese midwives. However, majority of the midwives adopt "Hands-on" method in the face of high risk for obstetric anal sphincter injuries. Further studies are needed to determine the association between obstetric anal sphincter injuries rate and perineal management for low-risk birth. RELEVANCE TO CLINICAL PRACTICE: The result indirectly illustrates the application of "Hands-off/poised" technique in China and provides evidence for the international midwifery organisation to understand the status of Chinese midwifery to some extent. It also provides the latest data for further study of these two methods and the study of the relationship between hands-off/poised method and obstetric anal sphincter injuries.
Subject(s)
Anal Canal/injuries , Delivery, Obstetric/adverse effects , Lacerations/prevention & control , Midwifery/methods , Adult , China , Cross-Sectional Studies , Delivery, Obstetric/methods , Female , Humans , Middle Aged , Perineum/injuries , Pregnancy , Surveys and QuestionnairesSubject(s)
Antibodies, Monoclonal , Potyvirus , Epitope Mapping , Epitopes , Antibodies, Neutralizing , Antibodies, ViralABSTRACT
The heightened vulnerability of premyelinating oligodendrocytes (PreOLs) in response to hypoxiaâ»ischemia may contribute to perinatal white matter injury and subsequent neurobehavioral dysfunction. Intracellular Ca2+ overload is considered a crucial mechanism predisposing PreOLs to ischemic injury. We previously reported that catalpol, an iridoid glycoside extracted from Rehmannia root, inhibits intracellular Ca2+ overload of PreOLs in an in vitro ischemia model. However, the exact underlying mechanisms remain elusive. In the present study, we aimed to investigate the protective effects of catalpol on PreOLs and to explore the underlying mechanisms involved in the modulation of intracellular Ca2+ homeostasis. Postnatal day 2 (P2) Sprague-Dawley (SD) rats subjected to bilateral common carotid artery ligation followed by exposure to 8% oxygen for 10 min were used as a rat model of neonatal hypoxiaâ»ischemia. We found that catalpol significantly improved behavioral functions and prevented PreOL loss and myelination deficit after hypoxiaâ»ischemia. Our in vitro studies also confirmed the direct effects of catalpol on oxygen-glucose deprivation (OGD)-induced cell death and arrested maturation of PreOLs. Moreover, we demonstrated that catalpol significantly inhibited intracellular Ca2+ overload and promoted the expression of Naâº/Ca2+ exchanger 3 (NCX3). Finally, we found that catalpol significantly reduced mitochondrial damage and subsequent extracellular signal-regulated kinase 1/2 (ERK1/2) and poly-ADP-ribose polymerase-1 (PARP-1) activation. Treatment with NCX3-preferring inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943) significantly reversed the protective effects of catalpol on PreOLs under OGD. Overall, our data suggest that catalpol protects PreOLs from ischemic injury through regulation of intercellular Ca2+ homeostasis via upregulation of NCX3 activity.