Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane

Protein phosphorylation is a fundamental post-translational modification in all organisms. In photoautotrophic organisms, protein phosphorylation is essential for the fine-tuning of photosynthesis. The reversible phosphorylation of the photosystem II (PSII) core and the light-harvesting complex of PSII (LHCII) contribute to the regulation of photosynthetic activities. Besides the phosphorylation of these major proteins, recent phosphoproteomic analyses have revealed that several proteins are phosphorylated in the thylakoid membrane. In this study, we utilized the Phos-tag technology for a comprehensive assessment of protein phosphorylation in the thylakoid membrane of Arabidopsis. Phos-tag SDS-PAGE enables the mobility shift of phosphorylated proteins compared with their non-phosphorylated isoform, thus differentiating phosphorylated proteins from their non-phosphorylated isoforms. We extrapolated this technique to two-dimensional (2D) SDS-PAGE for detecting protein phosphorylation in the thylakoid membrane. Thylakoid proteins were separated in the first dimension by conventional SDS-PAGE and in the second dimension by Phos-tag SDS-PAGE. In addition to the isolation of major phosphorylated photosynthesis-related proteins, 2D Phos-tag SDS-PAGE enabled the detection of several minor phosphorylated proteins in the thylakoid membrane. The analysis of the thylakoid kinase mutants demonstrated that light-dependent protein phosphorylation was mainly restricted to the phosphorylation of the PSII core and LHCII proteins. Furthermore, we assessed the phosphorylation states of the structural domains of the thylakoid membrane, grana core, grana margin, and stroma lamella. Overall, these results demonstrated that Phos-tag SDS-PAGE is a useful biochemical tool for studying in vivo protein phosphorylation in the thylakoid membrane protein

Reconstructing Bohr's Reply to EPR in Algebraic Quantum Theory

Halvorson and Clifton have given a mathematical reconstruction of Bohr's reply to Einstein, Podolsky and Rosen (EPR), and argued that this reply is dictated by the two requirements of classicality and objectivity for the description of experimental data, by proving consistency between their objectivity requirement and a contextualized version of the EPR reality criterion which had been introduced by Howard in his earlier analysis of Bohr's reply. In the present paper, we generalize the above consistency theorem, with a rather elementary proof, to a general formulation of EPR states applicable to both non-relativistic quantum mechanics and algebraic quantum field theory; and we clarify the elements of reality in EPR states in terms of Bohr's requirements of classicality and objectivity, in a general formulation of algebraic quantum theory.Comment: 13 pages, Late

Identification and classification of genes regulated by phosphatidylinositol 3-kinase- and TRKB-mediated signalling pathways during neuronal differentiation in two subtypes of the human neuroblastoma cell line SH-SY5Y

<p>Abstract</p> <p>Background</p> <p>SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signalling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is unclear. To investigate this mechanism, we compared the gene expression profiles in SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which show a different phenotype during RA-mediated differentiation.</p> <p>Findings</p> <p>SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. After exposing cells to a PI3K inhibitor, LY294002, we identified 386 genes and categorised these genes into two clusters dependent on the PI3K signalling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster, including 158 neural genes, was greatly reduced in SK-N-SH cells and partially impaired in SH-SY5Y-E cells, which is consistent with a defect in the neuronal phenotype of these cells. Additional stimulation with BDNF induced a set of neural genes that were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in differentiated SH-SY5Y-A cells.</p> <p>Conclusion</p> <p>We identified gene clusters controlled by PI3K- and TRKB-mediated signalling pathways during the differentiation of two subtypes of SH-SY5Y cells. The TRKB-mediated bypass pathway compensates for impaired neural function generated by defects in several signalling pathways, including PI3K in SH-SY5Y-E cells. Our expression profiling data will be useful for further elucidation of the signal transduction-transcriptional network involving PI3K or TRKB.</p

X-rays from the Power Sources of the Cepheus A Star-Forming Region

We report an observation of X-ray emission from the exciting region of Cepheus A with the Chandra/ACIS instrument. What had been an unresolved X-ray source comprising the putative power sources is now resolved into at least 3 point-like sources, each with similar X-ray properties and differing radio and submillimeter properties. The sources are HW9, HW3c, and a new source that is undetected at other wavelengths "h10." They each have inferred X-ray luminosities >= 10^31 erg s^-1 with hard spectra, T >= 10^7 K, and high low-energy absorption equivalent to tens to as much as a hundred magnitudes of visual absorption. The star usually assumed to be the most massive and energetic, HW2, is not detected with an upper limit about 7 times lower than the detections. The X-rays may arise via thermal bremsstrahlung in diffuse emission regions associated with a gyrosynchrotron source for the radio emission, or they could arise from powerful stellar winds. We also analyzed the Spitzer/IRAC mid-IR observation from this star-formation region and present the X-ray results and mid-IR classifications of the nearby stars. HH 168 is not as underluminous in X-rays as previously reported.Comment: Accepted in the ApJ, 30 pages, 11 figures, in one .pdf fil

Quality of life of children with neurodevelopmental disorders and their parents during the COVID-19 pandemic : a 1-year follow-up study

This study aimed to reveal changes in the quality of life (QOL) of children with neurodevelopmental disorders and their parents, and the interaction between their QOL and parental mental state during the coronavirus 2019 (COVID-19) pandemic. Eighty-nine school-aged children and parents participated in surveys in May 2020 (T1) and May 2021 (T2). The parents completed questionnaires that assessed their QOL, depression, parenting stress, and living conditions. Children's temporary mood status was evaluated using the self-reported visual analog scale (VAS). Children's QOL and VAS at T2 were higher than their QOL at T1. Parents' QOL at T2 was lower than their QOL at T1. Severe parental depression at T1 had a synergistic effect on severe parenting stress and severe depressive state at T2. Additionally, children's high QOL at T1 had a synergistic effect on low parenting stress and children's high QOL at T2. Furthermore, children's low VAS scores and parents' low QOL at T2 were associated with deterioration of family economic status. Children and parents' QOL changed during the prolonged COVID-19 pandemic. Improvement in children's QOL was influenced by reduced maternal depressive symptoms. Public support for parental mental health is important to avoid decreasing QOL.Peer reviewe

Directional cell expansion requires NIMA-related kinase 6 (NEK6)-mediated cortical microtubule destabilization;

　Plant cortical microtubules align perpendicular to the growth axis to determine the direction of cell growth. However, it remains unclear how plant cells form well-organized cortical microtubule arrays in the absence of a centrosome. In this study, we investigated the functions of Arabidopsis NIMA-related kinase 6 (NEK6), which regulates microtubule organization during anisotropic cell expansion. Quantitative analysis of hypocotyl cell growth in the nek6-1 mutant demonstrated that NEK6 suppresses ectopic outgrowth and promotes cell elongation in different regions of the hypocotyl. Loss of NEK6 function led to excessive microtubule waving and distortion, implying that NEK6 suppresses the aberrant cortical microtubules. Live cell imaging showed that NEK6 localizes to the microtubule lattice and to the shrinking plus and minus ends of microtubules. In agreement with this observation, the induced overexpression of NEK6 reduced and disorganized cortical microtubules and suppressed cell elongation. Furthermore, we identified five phosphorylation sites in β-tubulin that serve as substrates for NEK6 in vitro. Alanine substitution of the phosphorylation site Thr166 promoted incorporation of mutant β-tubulin into microtubules. Taken together, these results suggest that NEK6 promotes directional cell growth through phosphorylation of β-tubulin and the resulting destabilization of cortical microtubules

Characterization of tryptophan oxidation affecting D1 degradation by FtsH in the photosystem II quality control of chloroplasts

Photosynthesis is one of the most important reactions for sustaining our environment. Photosystem II (PSII) is the initial site of photosynthetic electron transfer by water oxidation. Light in excess, however, causes the simultaneous production of reactive oxygen species (ROS), leading to photo-oxidative damage in PSII. To maintain photosynthetic activity, the PSII reaction center protein D1, which is the primary target of unavoidable photo-oxidative damage, is efficiently degraded by FtsH protease. In PSII subunits, photo-oxidative modifications of several amino acids such as Trp have been indeed documented, whereas the linkage between such modifications and D1 degradation remains elusive. Here, we show that an oxidative post-translational modification of Trp residue at the N-terminal tail of D1 is correlated with D1 degradation by FtsH during high-light stress. We revealed that Arabidopsis mutant lacking FtsH2 had increased levels of oxidative Trp residues in D1, among which an N-terminal Trp-14 was distinctively localized in the stromal side. Further characterization of Trp-14 using chloroplast transformation in Chlamydomonas indicated that substitution of D1 Trp-14 to Phe, mimicking Trp oxidation enhanced FtsH-mediated D1 degradation under high light, although the substitution did not affect protein stability and PSII activity. Molecular dynamics simulation of PSII implies that both Trp-14 oxidation and Phe substitution cause fluctuation of D1 N-terminal tail. Furthermore, Trp-14 to Phe modification appeared to have an additive effect in the interaction between FtsH and PSII core in vivo. Together, our results suggest that the Trp oxidation at its N-terminus of D1 may be one of the key oxidations in the PSII repair, leading to processive degradation by FtsH

Characterization of the far-red light absorbing light-harvesting chlorophyll a/b binding complex, a derivative of the distinctive Lhca gene family in green algae

Prasiola crispa, an aerial green alga, exhibits remarkable adaptability to the extreme conditions of Antarctica by forming layered colonies capable of utilizing far-red light for photosynthesis. Despite a recent report on the structure of P. crispa’s unique light-harvesting chlorophyll (Chl)-binding protein complex (Pc-frLHC), which facilitates far-red light absorption and uphill excitation energy transfer to photosystem II, the specific genes encoding the subunits of Pc-frLHC have not yet been identified. Here, we report a draft genome sequence of P. crispa strain 4113, originally isolated from soil samples on Ongul Island, Antarctica. We obtained a 92 Mbp sequence distributed in 1,045 scaffolds comprising 10,244 genes, reflecting 87.1% of the core eukaryotic gene set. Notably, 26 genes associated with the light-harvesting Chl a/b binding complex (LHC) were identified, including four Pc-frLHC genes, with similarity to a noncanonical Lhca gene with four transmembrane helices, such as Ot_Lhca6 in Ostreococcus tauri and Cr_LHCA2 in Chlamydomonas reinhardtii. A comparative analysis revealed that Pc-frLHC shares homology with certain Lhca genes found in Coccomyxa and Trebouxia species. This similarity indicates that Pc-frLHC has evolved from an ancestral Lhca gene with four transmembrane helices and branched out within the Trebouxiaceae family. Furthermore, RNA-seq analysis conducted during the initiation of Pc-frLHC gene induction under red light illumination indicated that Pc-frLHC genes were induced independently from other genes associated with photosystems or LHCs. Instead, the genes of transcription factors, helicases, chaperones, heat shock proteins, and components of blue light receptors were identified to coexpress with Pc-frLHC. Those kinds of information could provide insights into the expression mechanisms of Pc-frLHC and its evolutional development

Endobronchial dental prosthesis retrieval by a snare technique using a flexible bronchoscope and fluoroscopy: Two case reports and technical tips

We describe two cases in each of which a dental prosthesis, presenting as an endobronchial foreign body (FB), was successfully retrieved using a snare technique employing a flexible bronchoscope and fluoroscopy that enabled us to avoid the need for rigid bronchoscope and thoracotomy. In one case, the FB was peripherally lodged and bronchoscopically invisible. In the other case, the FB was observed in the right intermediate bronchus, but the grasping basket and forceps were unable to retrieve it. The combination of a flexible bronchoscope and fluoroscopy extended the application of the snare technique to bronchoscopically invisible FBs and facilitated placement of an encircling loop around the FB. Since dental prostheses are rigid and irregular in shape, the snare loop technique can be used. For patients in a stable condition with a dental prosthesis FB, using the snare technique with a flexible bronchoscope and fluoroscopy is a good option. We provide technical tips based on our experiences

Expression of Conjoined Genes: Another Mechanism for Gene Regulation in Eukaryotes

From the ENCODE project, it is realized that almost every base of the entire human genome is transcribed. One class of transcripts resulting from this arises from the conjoined gene, which is formed by combining the exons of two or more distinct (parent) genes lying on the same strand of a chromosome. Only a very limited number of such genes are known, and the definition and terminologies used for them are highly variable in the public databases. In this work, we have computationally identified and manually curated 751 conjoined genes (CGs) in the human genome that are supported by at least one mRNA or EST sequence available in the NCBI database. 353 representative CGs, of which 291 (82%) could be confirmed, were subjected to experimental validation using RT-PCR and sequencing methods. We speculate that these genes are arising out of novel functional requirements and are not merely artifacts of transcription, since more than 70% of them are conserved in other vertebrate genomes. The unique splicing patterns exhibited by CGs reveal their possible roles in protein evolution or gene regulation. Novel CGs, for which no transcript is available, could be identified in 80% of randomly selected potential CG forming regions, indicating that their formation is a routine process. Formation of CGs is not only limited to human, as we have also identified 270 CGs in mouse and 227 in drosophila using our approach. Additionally, we propose a novel mechanism for the formation of CGs. Finally, we developed a database, ConjoinG, which contains detailed information about all the CGs (800 in total) identified in the human genome. In summary, our findings reveal new insights about the functionality of CGs in terms of another possible mechanism for gene regulation and genomic evolution and the mechanism leading to their formation