PLOS ONE: [sortOrder=DATE_NEWEST_FIRST, sort=Date, newest first, filterJournals=PLoSONE, q=subject:"Precipitation techniques"]PLOShttps://journals.plos.org/plosone/webmaster@plos.orgaccelerating the publication of peer-reviewed sciencehttps://journals.plos.org/plosone/search/feed/atom?sortOrder=DATE_NEWEST_FIRST&unformattedQuery=subject:%22Precipitation+techniques%22&sort=Date,+newest+first&filterJournals=PLoSONEAll PLOS articles are Open Access.https://journals.plos.org/plosone/resource/img/favicon.icohttps://journals.plos.org/plosone/resource/img/favicon.ico2024-03-28T17:13:39ZSubstrate preference of protein kinase B isoforms can vary depending on the cell lineMiguel S. PalmaSamantha R. PerezAida HusainDeepali Bhandari10.1371/journal.pone.02983222024-03-19T14:00:00Z2024-03-19T14:00:00Z<p>by Miguel S. Palma, Samantha R. Perez, Aida Husain, Deepali Bhandari</p>
Many proteins in higher eukaryotes, especially those with crucial functions, have multiple isoforms with redundant roles providing protection against potential functional deficiencies in one isoform. However, these isoforms can also have some unique roles. Protein kinase B, also known as Akt, is one such protein that has three isoforms encoded on different genes. Due to high sequence similarity and the general lack of specific reagents, most studies on Akt generalize their findings and do not distinguish between the isoforms. Using an established chemical genetic strategy and a set of known Akt substrates, this work explores substrate specificity of Akt isoforms under steady state conditions in two commonly used cell lines. This strategy can be applied to study any Akt isoform-specific substrates of interest in any cell line of choice as long as the cell line can be transfected.Antibody desolvation with sodium chloride and acetonitrile generates bioactive protein nanoparticlesLevi Collin NelemansVinicio Alejandro MeloMatej BuzgoEdwin BremerAiva Simaite10.1371/journal.pone.03004162024-03-14T14:00:00Z2024-03-14T14:00:00Z<p>by Levi Collin Nelemans, Vinicio Alejandro Melo, Matej Buzgo, Edwin Bremer, Aiva Simaite</p>
About 30% of the FDA approved drugs in 2021 were protein-based therapeutics. However, therapeutic proteins can be unstable and rapidly eliminated from the blood, compared to conventional drugs. Furthermore, on-target but off-tumor protein binding can lead to off-tumor toxicity, lowering the maximum tolerated dose. Thus, for effective treatment therapeutic proteins often require continuous or frequent administration. To improve protein stability, delivery and release, proteins can be encapsulated inside drug delivery systems. These drug delivery systems protect the protein from degradation during (targeted) transport, prevent premature release and allow for long-term, sustained release. However, thus far achieving high protein loading in drug delivery systems remains challenging. Here, the use of protein desolvation with acetonitrile as an intermediate step to concentrate monoclonal antibodies for use in drug delivery systems is reported. Specifically, trastuzumab, daratumumab and atezolizumab were desolvated with high yield (∼90%) into protein nanoparticles below 100 nm with a low polydispersity index (<0.2). Their size could be controlled by the addition of low concentrations of sodium chloride between 0.5 and 2 mM. Protein particles could be redissolved in aqueous solutions and redissolved antibodies retained their binding activity as evaluated in cell binding assays and exemplified for trastuzumab in an ELISA.Social vulnerability assessment under different extreme precipitation scenarios: A case study in Henan Province, ChinaXiaobing ZhouYongling ZhangWei WangXin LiHuanhuan YangYiting Sun10.1371/journal.pone.02999562024-03-08T14:00:00Z2024-03-08T14:00:00Z<p>by Xiaobing Zhou, Yongling Zhang, Wei Wang, Xin Li, Huanhuan Yang, Yiting Sun</p>
Extreme precipitation usually cause grievous losses&casualties, which varies greatly under different scenarios. This paper took Henan province as an example, it innovatively constructed three different extreme precipitation scenarios and built indicators system of social vulnerability from exposure, sensitivity and resilience based on MOVE framework. Social Vulnerability Indexs(SoVI) were then calculated by mathematical models under three different reoccurrence intervals. The results show that SoVI was low in the west and high in the north. High SoVI areas expanded to the middle and south as recurrence intervals increased. SoVI in each area of Henan province increased along with the recurrence intervals at different growth rates. The larger the recurrence interval was, the faster the SoVI increased. The results indicate SoVI is greatly affected by disaster levels, which need to be incorporated into social vulnerability. This study provides not only a new thought for social vulnerability assessment, but also a reference for the policymakers to formulate related risk management policies.The dispensability of 14-3-3 proteins for the regulation of human cardiac sodium channel Na<sub>v</sub>1.5Oksana IamshanovaAnne-Flore HämmerliElise RamayeArbresh SeljmaniDaniela Ross-KaschitzaNoëlia SchärzMaria EssersSabrina GuichardJean-Sébastien RougierHugues Abriel10.1371/journal.pone.02988202024-03-07T14:00:00Z2024-03-07T14:00:00Z<p>by Oksana Iamshanova, Anne-Flore Hämmerli, Elise Ramaye, Arbresh Seljmani, Daniela Ross-Kaschitza, Noëlia Schärz, Maria Essers, Sabrina Guichard, Jean-Sébastien Rougier, Hugues Abriel</p>
Background <p>14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Na<sub>v</sub>1.5. Given the many cardiac arrhythmias associated with Na<sub>v</sub>1.5 dysfunction, understanding its regulation by the protein partners is crucial.</p> Aims <p>In this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Na<sub>v</sub>1.5.</p> Methods and results <p>Amongst the seven 14-3-3 isoforms, only 14-3-3η (encoded by <i>YWHAH</i> gene) weakly co-immunoprecipitated with Na<sub>v</sub>1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Na<sub>v</sub>1.5 was however not modified by 14-3-3η overexpression or inhibition with difopein, and 14-3-3η did not affect physical interaction between Na<sub>v</sub>1.5 α-α subunits. The current-voltage relationship and the amplitude of Na<sub>v</sub>1.5-mediated sodium peak current density were also not changed.</p> Conclusions <p>Our findings illustrate that the direct implication of 14-3-3 proteins in regulating Na<sub>v</sub>1.5 is not evident in a transformed human kidney cell line tsA201.</p>Src-NADH dehydrogenase subunit 2 complex and recognition memory of imprinting in domestic chicksLela ChitadzeMaia MeparishviliVincenzo LaganiZaza KhuchuaBrian J. McCabeRevaz Solomonia10.1371/journal.pone.02971662024-01-29T14:00:00Z2024-01-29T14:00:00Z<p>by Lela Chitadze, Maia Meparishvili, Vincenzo Lagani, Zaza Khuchua, Brian J. McCabe, Revaz Solomonia</p>
Src is a non-receptor tyrosine kinase participating in a range of neuronal processes, including synaptic plasticity. We have recently shown that the amounts of total Src and its two phosphorylated forms, at tyrosine-416 (activated) and tyrosine-527 (inhibited), undergoes time-dependent, region-specific learning-related changes in the domestic chick forebrain after visual imprinting. These changes occur in the intermediate medial mesopallium (IMM), a site of memory formation for visual imprinting, but not the posterior pole of the nidopallium (PPN), a control brain region not involved in imprinting. Src interacts with mitochondrial genome-coded NADH dehydrogenase subunit 2 (NADH2), a component of mitochondrial respiratory complex I. This interaction occurs at brain excitatory synapses bearing NMDA glutamate receptors. The involvement of Src-NADH2 complexes in learning and memory is not yet explored. We show for the first time that, independently of changes in total Src or total NADH2, NADH2 bound to Src immunoprecipitated from the P2 plasma membrane-mitochondrial fraction: (i) is increased in a learning-related manner in the left IMM 1 h after the end of training; (ii), is decreased in the right IMM in a learning-related way 24 h after training. These changes occurred in the IMM but not the PPN. They are attributable to learning occurring during training rather than a predisposition to learn. Learning-related changes in Src-bound NADH2 are thus time- and region-dependent.Identification of <i>MYC</i> intron 2 regions that modulate expressionVan S. TompkinsZheng XueJake M. PetersonWarren B. RouseCollin A. O’LearyWalter N. Moss10.1371/journal.pone.02968892024-01-18T14:00:00Z2024-01-18T14:00:00Z<p>by Van S. Tompkins, Zheng Xue, Jake M. Peterson, Warren B. Rouse, Collin A. O’Leary, Walter N. Moss</p>
<i>MYC</i> pre-mRNA is spliced with high fidelity to produce the transcription factor known to regulate cellular differentiation, proliferation, apoptosis, and alternative splicing. The mechanisms underpinning the pre-mRNA splicing of <i>MYC</i>, however, remain mostly unexplored. In this study, we examined the interaction of heterogeneous nuclear ribonucleoprotein C (HNRNPC) with <i>MYC</i> intron 2. Building off published eCLIP studies, we confirmed this interaction with poly(U) regions in intron 2 of <i>MYC</i> and found that full binding is correlated with optimal protein production. The interaction appears to be compensatory, as mutational disruption of all three poly(U) regions was required to reduce both HNRNPC binding capacity and fidelity of either splicing or translation. Poly(U) sequences in <i>MYC</i> intron 2 were relatively conserved across sequences from several different species. Lastly, we identified a short sequence just upstream of an HNRNPC binding region that when removed enhances <i>MYC</i> translation.Restored forested wetland surprisingly resistant to experimental salinizationEmily A. UryMarcelo ArdónJustin P. WrightEmily S. Bernhardt10.1371/journal.pone.02961282023-12-21T14:00:00Z2023-12-21T14:00:00Z<p>by Emily A. Ury, Marcelo Ardón, Justin P. Wright, Emily S. Bernhardt</p>
Salinization of coastal freshwater wetlands is an increasingly common and widespread phenomenon resulting from climate change. The ecosystem consequences of added salinity are poorly constrained and highly variable across prior observational and experimental studies. We added 1.8 metric tons of marine salts to replicated 200 m<sup>2</sup> plots within a restored forested wetland in Eastern North Carolina over the course of four years. Based on prior small-scale experiments at this site, we predicted that salinization would lead to slower tree growth and suppressed soil carbon cycling. Results from this large-scale field experiment were subtle and inconsistent over space and time. By the fourth year of the experiment, we observed the predicted suppression of soil respiration and a reduction of water extractable carbon from soils receiving salt treatments. However, we found no cumulative effects of four years of salinization on total soil carbon stocks, tree growth, or root biomass. We observed substantial variation in soil solution chemistry (notably, pH and base saturation) across replicated treatment blocks; the effective salt levels, ionic composition, and pH varied following treatment depending upon pre-existing differences in edaphic factors. Our multi-year monitoring also revealed an underlying trend of wetland acidification across the entire site, a suspected effect of ecosystem recovery following wetland restoration on former agricultural land. The overwhelming resistance to our salt treatments could be attributed to the vigor of a relatively young, healthy wetland ecosystem. The heterogeneous responses to salt that we observed over space and time merits further investigation into the environmental factors that control carbon cycling in wetlands. This work highlights the importance of multi-year, large-scale field experiments for investigating ecosystem responses to global environmental change.ATXN2 is a target of N-terminal proteolysisMonika ChitrePatrick Emery10.1371/journal.pone.02960852023-12-21T14:00:00Z2023-12-21T14:00:00Z<p>by Monika Chitre, Patrick Emery</p>
Spinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder caused by the expansion of the poly-glutamine (polyQ) tract of Ataxin-2 (ATXN2). Other polyQ-containing proteins such as ATXN7 and huntingtin are associated with the development of neurodegenerative diseases when their N-terminal polyQ domains are expanded. Furthermore, they undergo proteolytic processing events that produce N-terminal fragments that include the polyQ stretch, which are implicated in pathogenesis. Interestingly, N-terminal ATXN2 fragments were reported in a brain extract from a SCA2 patient, but it is currently unknown whether an expanded polyQ domain contributes to ATXN2 proteolytic susceptibility. Here, we used transient expression in HEK293 cells to determine whether ATXN2 is a target for specific N-terminal proteolysis. We found that ATXN2 proteins with either normal or expanded polyQ stretches undergo proteolytic cleavage releasing an N-terminal polyQ-containing fragment. We identified a short amino acid sequence downstream of the polyQ domain that is necessary for N-terminal cleavage of full-length ATXN2 and sufficient to induce proteolysis of a heterologous protein. However, this sequence is not required for cleavage of a short ATXN2 isoform produced from an alternative start codon located just upstream of the CAG repeats encoding the polyQ domain. Our study extends our understanding of ATXN2 posttranslational regulation by revealing that this protein can be the target of specific proteolytic cleavage events releasing polyQ-containing products that are modulated by the N-terminal domain of ATXN2. N-terminal ATXN2 proteolysis of expanded polyQ domains might contribute to SCA2 pathology, as observed in other neurodegenerative disorders caused by polyQ domain expansion.Polyethylene glycol precipitation is an efficient method to obtain extracellular vesicle-depleted fetal bovine serumPeng WangOnno J. ArntzJohanna F. A. HuschVan der Kraan P. M.Jeroen J. J. P. van den BeuckenFons A. J. van de Loo10.1371/journal.pone.02950762023-12-05T14:00:00Z2023-12-05T14:00:00Z<p>by Peng Wang, Onno J. Arntz, Johanna F. A. Husch, Van der Kraan P. M., Jeroen J. J. P. van den Beucken, Fons A. J. van de Loo</p>
Mesenchymal stromal/stem cell derived-extracellular vesicles (MSC-EVs) have gained interest as drug delivery nanoparticles, having immunoregulatory and potentiating tissue repair property. To maintain growth of MSCs and obtain pure MSC-derived EVs, the culture media should contain fetal bovine serum (FBS) devoid of EVs, as the presence of FBS EVs confounds the properties of MSC-EVs. Therefore, we tested three methods: 18h ultracentrifugation (UC) and ultrafiltration (UF), which are common FBS EV depletion methods in current MSC-EV research, and polyethylene glycol (PEG) precipitation to obtain three EV depleted FBS (EVdFBS) batches, and compared them to FBS and commercial (Com) EVdFBS on human adipose stem cell (hADSC) growth, differentiation, enrichment of EVs in hADSC supernatant and their biological function on collagen metabolism. Our comparative study showed UC and UF vary in terms of depletion efficiency and do not completely deplete EVs and affects the growth-promoting quality of FBS. Specifically, FBS EV depletion was comparable between PEG (95.6%) and UF (96.6%) but less by UC (82%), as compared to FBS. FBS protein loss was markedly different among PEG (47%), UF (87%), and UC (51%), implying the ratio of EV depletion over protein loss was PEG (2.03), UF (1.11), and UC (1.61). A significant decrease of TGFβ/Smad signaling, involving in MSC growth and physiology, was observed by UF. After 96 hours of exposure to 5% FBS or 5% four different EVdFBS cell growth media, the osteogenesis ability of hADSCs was not impaired but slightly lower mRNA expression level of Col2a observed in EVdFBS media during chondrogenesis. In consistent with low confluency of hADSCs observed by optical microscope, cell proliferation in response to 5% UF EVdFBS media was inhibited significantly. Importantly, more and purer ADSCs EVs were obtained from ADSCs cultured in 5% PEG EVdFBS media, and they retained bioactive as they upregulated the expression of Col1a1, TIMP1 of human knee synovial fibroblast. Taken together, this study showed that PEG precipitation is the most efficient method to obtain EV depleted FBS for growth of MSCs, and to obtain MSC EVs with minimal FBS EV contamination.Golgi ELMO1 binds QUA1, QUA2, GAUT9, and ELMO4 and is required for pectin accumulation in ArabidopsisBruce D. KohornNuoya YangMargaret WeinstockGarrison AsperIsabel BallDevaki Rajiv10.1371/journal.pone.02939612023-11-08T14:00:00Z2023-11-08T14:00:00Z<p>by Bruce D. Kohorn, Nuoya Yang, Margaret Weinstock, Garrison Asper, Isabel Ball, Devaki Rajiv</p>
Pectin and its modification influence the plasticity and strength of the plant cell wall controlling cell adhesion, size, shape, and pathogen resistance. The Golgi membrane anchored QUA1, QUA2, and GAUT9 Golgi enzymes synthesize and esterify pectin, which is then secreted and selectively de-esterified to potentiate structure influencing crosslinks in the cell wall. Mutations in members of the family of non-enzymatic ELMO Golgi membrane proteins lead to a reduction of pectin levels, cell adhesion, and hypocotyl tensile strength. Results from immunoprecipitation of Golgi protein complexes reveal that ELMO1-GFP is associated with pectin biosynthesis and modifying enzymes QUA1, QUA2, and GAUT9. In a yeast two and three hybrid assay, ELMO1 can bind directly to QUA1, GAUT9 or ELMO4, but QUA1, QUA2 or GAUT9 do not bind to each other. A yeast 3 hybrid assay provides evidence that ELMO1 can mediate the binding of QUA1 and QUA2. Taken together, these results indicate that the 20 kDa ELMO1 serves to facilitate some aspect of pectin synthesis and modification that leads to sufficient accumulation to allow cell adhesion, and we speculate that ELMOs help to scaffold key enzymes in this process.USP7 attenuates endoplasmic reticulum stress-induced apoptotic cell death through deubiquitination and stabilization of FBXO7Su Hyoun LeeKwang Chul Chung10.1371/journal.pone.02903712023-10-24T14:00:00Z2023-10-24T14:00:00Z<p>by Su Hyoun Lee, Kwang Chul Chung</p>
Parkinson’s disease (PD) is a common neurodegenerative disease (NDD) characterized by the loss of dopaminergic neurons in the substantia nigra. Similar to other NDDs, the buildup of toxic protein aggregates in PD leads to progressive neuronal loss, culminating in neurodegeneration. Accumulating evidence indicates that alterations in subcellular organelles, particularly the endoplasmic reticulum (ER), are critically involved in pathological neurodegenerative events in NDDs, including PD. Mutations in the F-box only protein 7 (<i>FBXO7</i> or <i>PARK15</i>) gene have been found to cause early onset autosomal recessive familiar PD. FBXO7 functions as an adaptor protein in the Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex, which promotes substrate ubiquitination. Although FBXO7 is involved in the ubiquitination of various target proteins, little is known about the upstream regulatory mechanism of FBXO7 and/or its modulator(s). Ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme that regulates the balance between protein synthesis and degradation by removing ubiquitin from target substrates. The role of USP7 in various types of cancer is well-established; however, its role in NDDs has not been elucidated to date. In this study, we identified that USP7 acts as a novel regulator of FBXO7, positively regulating the stability of FBXO7 through Lys48-linked deubiquitination. Moreover, USP7 was found to mitigate ER stress-induced cytotoxicity and apoptosis by preventing the proteasomal degradation of FBXO7. Taken together, our study suggests that the functional relationship between FBXO7 and USP7 may play a crucial role in ER stress-induced apoptosis and the pathogenesis of PD.Hunting for the elusive target antigen in gestational alloimmune liver disease (GALD)Klaus RieneckKaren Koefoed RasmussenErwin M. SchoofFrederik Banch ClausenHenrietta HolzeThomas BergholtMarianne Hørby JørgensenVibeke Brix ChristensenRunar AlmaasPeter Lüttge JordalMarie Locard-PauletKasper RunagerLeif Kofoed NielsenBalthasar Clemens SchlotmannJoachim Lütken WeischenfeldtLars Juhl JensenMorten Hanefeld Dziegiel10.1371/journal.pone.02864322023-10-20T14:00:00Z2023-10-20T14:00:00Z<p>by Klaus Rieneck, Karen Koefoed Rasmussen, Erwin M. Schoof, Frederik Banch Clausen, Henrietta Holze, Thomas Bergholt, Marianne Hørby Jørgensen, Vibeke Brix Christensen, Runar Almaas, Peter Lüttge Jordal, Marie Locard-Paulet, Kasper Runager, Leif Kofoed Nielsen, Balthasar Clemens Schlotmann, Joachim Lütken Weischenfeldt, Lars Juhl Jensen, Morten Hanefeld Dziegiel</p>
The prevailing concept is that gestational alloimmune liver disease (GALD) is caused by maternal antibodies targeting a currently unknown antigen on the liver of the fetus. This leads to deposition of complement on the fetal hepatocytes and death of the fetal hepatocytes and extensive liver injury. In many cases, the newborn dies. In subsequent pregnancies early treatment of the woman with intravenous immunoglobulin can be instituted, and the prognosis for the fetus will be excellent. Without treatment the prognosis can be severe. Crucial improvements of diagnosis require identification of the target antigen. For this identification, this work was based on two hypotheses: 1. The GALD antigen is exclusively expressed in the fetal liver during normal fetal life in all pregnancies; 2. The GALD antigen is an alloantigen expressed in the fetal liver with the woman being homozygous for the minor allele and the father being, most frequently, homozygous for the major allele. We used three different experimental approaches to identify the liver target antigen of maternal antibodies from women who had given birth to a baby with the clinical GALD diagnosis: 1. Immunoprecipitation of antigens from either a human liver cell line or human fetal livers by immunoprecipitation with maternal antibodies followed by mass spectrometry analysis of captured antigens; 2. Construction of a cDNA expression library from human fetal liver mRNA and screening about 1.3 million recombinants in <i>Escherichia coli</i> using antibodies from mothers of babies diagnosed with GALD; 3. Exome/genome sequencing of DNA from 26 presumably unrelated women who had previously given birth to a child with GALD with husband controls and supplementary HLA typing. In conclusion, using the three experimental approaches we did not identify the GALD target antigen and the exome/genome sequencing results did not support the hypothesis that the GALD antigen is an alloantigen, but the results do not yield basis for excluding that the antigen is exclusively expressed during fetal life., which is the hypothesis we favor.Effects of HSP70 chaperones Ssa1 and Ssa2 on Ste5 scaffold and the mating mitogen-activated protein kinase (MAPK) pathway in <i>Saccharomyces cerevisiae</i>Francis W. FarleyRyan R. McCullyPaul B. MasloLu YuMark A. SheffHomayoun SadeghiElaine A. Elion10.1371/journal.pone.02893392023-10-18T14:00:00Z2023-10-18T14:00:00Z<p>by Francis W. Farley, Ryan R. McCully, Paul B. Maslo, Lu Yu, Mark A. Sheff, Homayoun Sadeghi, Elaine A. Elion</p>
Ste5 is a prototype of scaffold proteins that regulate activation of mitogen-activated protein kinase (MAPK) cascades in all eukaryotes. Ste5 associates with many proteins including Gβγ (Ste4), Ste11 MAPKKK, Ste7 MAPKK, Fus3 and Kss1 MAPKs, Bem1, Cdc24. Here we show that Ste5 also associates with heat shock protein 70 chaperone (Hsp70) Ssa1 and that Ssa1 and its ortholog Ssa2 are together important for Ste5 function and efficient mating responses. The majority of purified overexpressed Ste5 associates with Ssa1. Loss of Ssa1 and Ssa2 has deleterious effects on Ste5 abundance, integrity, and localization particularly when Ste5 is expressed at native levels. The status of Ssa1 and Ssa2 influences Ste5 electrophoresis mobility and formation of high molecular weight species thought to be phosphorylated, ubiquitinylated and aggregated and lower molecular weight fragments. A Ste5 VWA domain mutant with greater propensity to form punctate foci has reduced predicted propensity to bind Ssa1 near the mutation sites and forms more punctate foci when Ssa1 Is overexpressed, supporting a dynamic protein quality control relationship between Ste5 and Ssa1. Loss of Ssa1 and Ssa2 reduces activation of Fus3 and Kss1 MAPKs and <i>FUS1</i> gene expression and impairs mating shmoo morphogenesis. Surprisingly, <i>ssa1</i>, <i>ssa2</i>, <i>ssa3</i> and <i>ssa4</i> single, double and triple mutants can still mate, suggesting compensatory mechanisms exist for folding. Additional analysis suggests Ssa1 is the major Hsp70 chaperone for the mating and invasive growth pathways and reveals several Hsp70-Hsp90 chaperone-network proteins required for mating morphogenesis.A dominant negative 14-3-3 mutant in <i>Schizosaccharomyces pombe</i> distinguishes the binding proteins involved in sexual differentiation and check pointTomohito OhshimaZhang JiajunTakuki FukamachiYuko OhnoHiroko SenooYasuhiro MatsuoMakoto Kawamukai10.1371/journal.pone.02915242023-10-03T14:00:00Z2023-10-03T14:00:00Z<p>by Tomohito Ohshima, Zhang Jiajun, Takuki Fukamachi, Yuko Ohno, Hiroko Senoo, Yasuhiro Matsuo, Makoto Kawamukai</p>
The homothallic fission yeast <i>Schizosaccharomyces pombe</i> undergoes sexual differentiation when starved, but <i>sam</i> (skips the requirement of starvation for mating) mutants such as those carrying mutations in adenylate cyclase (<i>cyr1</i>) or protein kinase A (<i>pka1</i>) mate without starvation. Here, we identified <i>sam3</i>, a dominant negative allele of <i>rad24</i>, encoding one of two 14-3-3 proteins. Genetic mapping and whole-genome sequencing showed that the <i>sam3</i> mutation comprises a change in nucleotide at position 959 from guanine to adenine, which switches the amino acid at position 185 from glutamic acid to lysine (E185K). We generated the <i>rad24-</i>E185K integrated mutant and its phenotype was similar to that of the <i>sam3</i> mutant, including calcium sensitivity and UV non-sensitivity, but the phenotype is different from that of the <i>Δrad24</i> strain. While the UV-sensitive phenotype was observed in the <i>Δrad24</i> mutant, it was not observed in the <i>sam3</i> and <i>rad24-</i>E185K mutants. The expression of the <i>rad24</i>-<i>E185K</i> gene in wild type cells induced spore formation in the nutrient rich medium, confirming <i>rad24</i>-<i>E185K</i> is dominant. This dominant effect of <i>rad24</i>-<i>E185K</i> was also observed in Δ<i>ras1</i> and Δ<i>byr2</i> diploid mutants, indicating that <i>rad24</i>-<i>E185K</i> generate stronger phenotype than <i>rad24</i> null mutants. Ste11, the key transcription factor for sexual differentiation was expressed in <i>sam3</i> mutants without starvation and it predominantly localized to the nucleus. The Rad24-E185K mutant protein retained its interaction with Check point kinase1 (Chk1), whereas it reduced interaction with Ste11, an RNA binding protein Mei2, and a MAPKKK Byr2, freeing these proteins from negative regulation by Rad24, that account for the <i>sam</i> phenotype and UV non-sensitive phenotype. Glucose depletion in <i>rad24</i>-<i>E185K</i> or Δ<i>pka1</i> Δ<i>rad24</i> double mutation induced haploid meiosis, leading to the formation of spores in haploid. The position of glutamic acid 185 is conserved in all major 14-3-3s; hence, our finding of a dominant negative allele of 14-3-3 is useful for understanding 14-3-3s in other organisms.Effect of preparation conditions on the properties of nano ZnO powders during ultrasonic assisted direct precipitation processJingfeng WangHaiyang Ji10.1371/journal.pone.02867652023-08-31T14:00:00Z2023-08-31T14:00:00Z<p>by Jingfeng Wang, Haiyang Ji</p>
Transparent conductive thin films (TCO) are widely used for their excellent photoelectric properties. To prepare high-quality ZnO targets, starting with the original ZnO powder is necessary. This paper aims to explore the basic technology and method of ultrasonic-assisted direct precipitation for mass production of ZnO powder and to analyze the effects of factors such as precipitating agent, surfactant, calcination temperature, and solvent on the powder’s morphology, particle size, and crystallinity. The study found that the type and amount of precipitants and surfactants affect the powder’s morphology and dispersibility, while calcination temperature mainly affects the powder’s morphology and crystallinity. The ethanol content in the solvent mainly affects the grain size. After testing different variables, the optimal conditions for preparing spherical ZnO powder were found to be using (NH<sub>4</sub>)<sub>2</sub>·CO<sub>3</sub> as the precipitant, adding 3% wt of PEG-400 and 3% wt of TEA at a calcination temperature of 320°C and a 60% ethanol solvent. This resulted in a smooth surface, uniform particle size distribution, good dispersibility, high crystallinity, and particle sizes between 26-32nm.