PLOS ONE: [sortOrder=DATE_NEWEST_FIRST, sort=Date, newest first, q=subject:"Molecular biology 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:%22Molecular+biology+techniques%22&sort=Date,+newest+firstAll PLOS articles are Open Access.https://journals.plos.org/plosone/resource/img/favicon.icohttps://journals.plos.org/plosone/resource/img/favicon.ico2024-03-28T19:51:04ZDisulfide-constrained peptide scaffolds enable a robust peptide-therapeutic discovery platformLijuan ZhouFei CaiYanjie LiXinxin GaoYuehua WeiAnna FedorovaDaniel KirchhoferRami N. HannoushYingnan Zhang10.1371/journal.pone.03001352024-03-28T14:00:00Z2024-03-28T14:00:00Z<p>by Lijuan Zhou, Fei Cai, Yanjie Li, Xinxin Gao, Yuehua Wei, Anna Fedorova, Daniel Kirchhofer, Rami N. Hannoush, Yingnan Zhang</p>
Peptides present an alternative modality to immunoglobulin domains or small molecules for developing therapeutics to either agonize or antagonize cellular pathways associated with diseases. However, peptides often suffer from poor chemical and physical stability, limiting their therapeutic potential. Disulfide-constrained peptides (DCP) are naturally occurring and possess numerous desirable properties, such as high stability, that qualify them as drug-like scaffolds for peptide therapeutics. DCPs contain loop regions protruding from the core of the molecule that are amenable to peptide engineering via direct evolution by use of phage display technology. In this study, we have established a robust platform for the discovery of peptide therapeutics using various DCPs as scaffolds. We created diverse libraries comprising seven different DCP scaffolds, resulting in an overall diversity of 2 x 10<sup>11</sup>. The effectiveness of this platform for functional hit discovery has been extensively evaluated, demonstrating a hit rate comparable to that of synthetic antibody libraries. By utilizing chemically synthesized and <i>in vitro</i> folded peptides derived from selections of phage displayed DCP libraries, we have successfully generated functional inhibitors targeting the HtrA1 protease. Through affinity maturation strategies, we have transformed initially weak binders against Notch2 with micromolar Kd values to high-affinity ligands in the nanomolar range. This process highlights a viable hit-to-lead progression. Overall, our platform holds significant potential to greatly enhance the discovery of peptide therapeutics.Changes in the intestinal microbiota of individuals with non-alcoholic fatty liver disease based on sequencing: An updated systematic review and meta-analysisWenpin CaiTing QiuWeitao HuTaiyong Fang10.1371/journal.pone.02999462024-03-28T14:00:00Z2024-03-28T14:00:00Z<p>by Wenpin Cai, Ting Qiu, Weitao Hu, Taiyong Fang</p>
Background <p>Alterations in the composition and abundance of the intestinal microbiota occur in non-alcoholic fatty liver disease (NAFLD). However, the results are inconsistent because of differences in the study design, subject area, and sequencing methodology. In this study, we compared the diversity and abundance of the intestinal microbiota of patients with NAFLD and healthy individuals through a systematic review and meta-analysis.</p> Methods <p>Three databases (PubMed, EMBASE, and Cochrane Library) were searched from their inception to March 20, 2023. A meta-analysis was performed using Stata software to analyze variations in the richness and abundance of the intestinal microbiota in patients with NAFLD. The Newcastle-Ottawa Quality Assessment Scale (NOS) was used for quality assessment.</p> Results <p>A total of 28 articles were included. Shannon diversity was reduced in patients with NAFLD (SMD = -0.24 (95% CI -0.43–0.05, I<sup>2</sup> = 71.7%). The relative abundance of <i>Ruminococcus</i>, <i>Faecalibacterium</i>, and <i>Coprococcus</i> all decreased, with total SMDs of -0.96 (95% CI -1.29 to -0.63, I<sup>2</sup> = 4.8%), -1.13 (95% CI -2.07 to -0.19, I<sup>2</sup> = 80.5%), and -1.66 (95% CI -3.04 to -0.28, I<sup>2</sup> = 91.5%). <i>Escherichia</i> was increased in individuals with NAFLD (SMD = 1.78, 95% CI 0.12 to 3.45, I<sup>2</sup> = 94.4%).</p> Conclusion <p>Increasing the species diversity and altering the abundance of specific gut microbiota, including <i>Coprococcus</i>, <i>Faecalibacterium</i>, <i>Ruminococcus</i>, and <i>Escherichia</i>, may be beneficial for improving NAFLD.</p>A phage-displayed disulfide constrained peptide discovery platform yields novel human plasma protein bindersXinxin GaoHarini KaluarachchiYingnan ZhangSunhee HwangRami N. Hannoush10.1371/journal.pone.02998042024-03-28T14:00:00Z2024-03-28T14:00:00Z<p>by Xinxin Gao, Harini Kaluarachchi, Yingnan Zhang, Sunhee Hwang, Rami N. Hannoush</p>
Disulfide constrained peptides (DCPs) show great potential as templates for drug discovery. They are characterized by conserved cysteine residues that form intramolecular disulfide bonds. Taking advantage of phage display technology, we designed and generated twenty-six DCP phage libraries with enriched molecular diversity to enable the discovery of ligands against disease-causing proteins of interest. The libraries were designed based on five DCP scaffolds, namely <i>Momordica charantia</i> 1 (Mch1), gurmarin, Asteropsin-A, antimicrobial peptide-1 (AMP-1), and potato carboxypeptidase inhibitor (CPI). We also report optimized workflows for screening and producing synthetic and recombinant DCPs. Examples of novel DCP binders identified against various protein targets are presented, including human IgG Fc, serum albumin, vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF). We identified DCPs against human IgG Fc and serum albumin with sub-micromolar affinity from primary panning campaigns, providing alternative tools for potential half-life extension of peptides and small protein therapeutics. Overall, the molecular diversity of the DCP scaffolds included in the designed libraries, coupled with their distinct biochemical and biophysical properties, enables efficient and robust identification of <i>de novo</i> binders to drug targets of therapeutic relevance.17β-Estradiol promotes metastasis in triple-negative breast cancer through the Calpain/YAP/β-catenin signaling axisXuemei NiuJianan WangJinguang LiuQinglong YuMingwei Ci10.1371/journal.pone.02981842024-03-28T14:00:00Z2024-03-28T14:00:00Z<p>by Xuemei Niu, Jianan Wang, Jinguang Liu, Qinglong Yu, Mingwei Ci</p>
β-catenin is an important regulator of malignant progression. 17β-Estradiol (E2), an important sex hormone in women, promotes the growth and metastasis of triple-negative breast cancer (TNBC). However, whether β-catenin is involved in E2-induced metastasis of TNBC remains unknown. In this study, we show that E2 induces the proliferation, migration, invasion, and metastasis of TNBC cells. E2 induces β-catenin protein expression and nuclear translocation, thereby regulating the expression of target genes such as Cyclin D1 and MMP-9. The inhibition of β-catenin reversed the E2-induced cell malignant behaviors. Additionally, E2 activated Calpain by increasing intracellular Ca<sup>2+</sup> levels and reducing calpastatin levels. When Calpain was inhibited, E2 did not induce the proliferation, migration, invasion, or metastasis of TNBC cells. In addition, E2 promoted translocation of YAP into the nucleus by inhibiting its phosphorylation. Calpain inhibition reversed the E2-induced YAP dephosphorylation. Inhibition of YAP transcriptional activity reversed the effects of E2 on the proliferation, migration, invasion, and β-catenin of TNBC cells. In conclusion, we demonstrated that E2 induced metastasis-related behaviors in TNBC cells and this effect was mediated through the Calpain/YAP/β-catenin signaling pathway.Diagnostic efficacy of hand-held digital refractometer for determining total serum protein in indigenous sheep of PakistanMadiha SharifMushtaq Hussain LashariUmer FarooqMusadiq IdrisMuhammad Abrar Afzal10.1371/journal.pone.02951072024-03-28T14:00:00Z2024-03-28T14:00:00Z<p>by Madiha Sharif, Mushtaq Hussain Lashari, Umer Farooq, Musadiq Idris, Muhammad Abrar Afzal</p>
The study was designed to ascertain the diagnostic efficacy of hand-held digital refractometer in determining total protein (TP). The Sipli sheep (n = 128) were grouped as per gender (females = 99, males = 29) and age (G1 = up till 1 year, n = 35; G2 = from 1 to 2 years, n = 63; G3 = above 2 years, n = 30). The results regarding the overall mean (±SE) values for the TPs attained through serum chemistry analyzer (TP1) and hand-held digital refractometer (TP2) were non-significantly (P≥0.05) different (59.2±1.6g/L and 59.8±0.5g/L, respectively). However, the reference intervals (RIs) were quite different for the two TPs being 45.1–95.7g/L and 57.0–67.0g/L for TP1 and TP2, respectively. Similar results were seen for gender-wise and group-wise results. On the contrary, the results regarding correlation coefficient and logilinear regression showed a negative correlation between the two TPs (r = -0.0244) with an adjusted r-square of 0.059 (5.9% probability). Furthermore, the three tests implied to assess the level of agreement between the two methods (Cronbach alpha, Intraclass correlation coefficient, and Bland & Altman test) revealed least agreement between the two methods. In a nutshell, the results of TP through digital refractometer were not in concordance with those attained through serum chemistry analyzer. However, it can cautiously be used if these results are compared with relevant corrected RIs.A natural bacterial pathogen of <i>C</i>. <i>elegans</i> uses a small RNA to induce transgenerational inheritance of learned avoidanceTitas SenguptaJonathan St. AngeRachel KaletskyRebecca S. MooreRenee J. SetoJacob MarogiCameron MyhrvoldZemer GitaiColeen T. Murphy10.1371/journal.pgen.10111782024-03-28T14:00:00Z2024-03-28T14:00:00Z<p>by Titas Sengupta, Jonathan St. Ange, Rachel Kaletsky, Rebecca S. Moore, Renee J. Seto, Jacob Marogi, Cameron Myhrvold, Zemer Gitai, Coleen T. Murphy</p>
<i>C</i>. <i>elegans</i> can learn to avoid pathogenic bacteria through several mechanisms, including bacterial small RNA-induced learned avoidance behavior, which can be inherited transgenerationally. Previously, we discovered that a small RNA from a clinical isolate of <i>Pseudomonas aeruginosa</i>, PA14, induces learned avoidance and transgenerational inheritance of that avoidance in <i>C</i>. <i>elegans</i>. <i>Pseudomonas aeruginosa</i> is an important human pathogen, and there are other <i>Pseudomonads</i> in <i>C</i>. <i>elegans’</i> natural habitat, but it is unclear whether <i>C</i>. <i>elegans</i> ever encounters PA14-like bacteria in the wild. Thus, it is not known if small RNAs from bacteria found in <i>C</i>. <i>elegans’</i> natural habitat can also regulate host behavior and produce heritable behavioral effects. Here we screened a set of wild habitat bacteria, and found that a pathogenic <i>Pseudomonas vranovensis</i> strain isolated from the <i>C</i>. <i>elegans</i> microbiota, GRb0427, regulates worm behavior: worms learn to avoid this pathogenic bacterium following exposure, and this learned avoidance is inherited for four generations. The learned response is entirely mediated by bacterially-produced small RNAs, which induce avoidance and transgenerational inheritance, providing further support that such mechanisms of learning and inheritance exist in the wild. We identified Pv1, a small RNA expressed in <i>P</i>. <i>vranovensis</i>, that has a 16-nucleotide match to an exon of the <i>C</i>. <i>elegans</i> gene <i>maco-1</i>. Pv1 is both necessary and sufficient to induce learned avoidance of Grb0427. However, Pv1 also results in avoidance of a beneficial microbiome strain, <i>P</i>. <i>mendocina</i>. Our findings suggest that bacterial small RNA-mediated regulation of host behavior and its transgenerational inheritance may be functional in <i>C</i>. <i>elegans’</i> natural environment, and that this potentially maladaptive response may favor reversal of the transgenerational memory after a few generations. Our data also suggest that different bacterial small RNA-mediated regulation systems evolved independently, but define shared molecular features of bacterial small RNAs that produce transgenerationally-inherited effects.MYCN drives oncogenesis by cooperating with the histone methyltransferase G9a and the WDR5 adaptor to orchestrate global gene transcriptionZhihui LiuXiyuan ZhangMan XuJason J. HongAmanda CiardielloHaiyan LeiJack F. ShernCarol J. Thiele10.1371/journal.pbio.30022402024-03-28T14:00:00Z2024-03-28T14:00:00Z<p>by Zhihui Liu, Xiyuan Zhang, Man Xu, Jason J. Hong, Amanda Ciardiello, Haiyan Lei, Jack F. Shern, Carol J. Thiele</p>
MYCN activates canonical MYC targets involved in ribosome biogenesis, protein synthesis, and represses neuronal differentiation genes to drive oncogenesis in neuroblastoma (NB). How MYCN orchestrates global gene expression remains incompletely understood. Our study finds that MYCN binds promoters to up-regulate canonical MYC targets but binds to both enhancers and promoters to repress differentiation genes. MYCN binding also increases H3K4me3 and H3K27ac on canonical MYC target promoters and decreases H3K27ac on neuronal differentiation gene enhancers and promoters. WDR5 facilitates MYCN promoter binding to activate canonical MYC target genes, whereas MYCN recruits G9a to enhancers to repress neuronal differentiation genes. Targeting both MYCN’s active and repressive transcriptional activities using both WDR5 and G9a inhibitors synergistically suppresses NB growth. We demonstrate that MYCN cooperates with WDR5 and G9a to orchestrate global gene transcription. The targeting of both these cofactors is a novel therapeutic strategy to indirectly target the oncogenic activity of <i>MYCN</i>.Comparison of nutritional value of snakehead fish from Guangdong and Deqing varietiesZeguo ZengMiao ZhengMengxiao ZhaoJing GuoShuifa ZhuXianguo ZouQingxiang Zeng10.1371/journal.pone.03012032024-03-27T14:00:00Z2024-03-27T14:00:00Z<p>by Zeguo Zeng, Miao Zheng, Mengxiao Zhao, Jing Guo, Shuifa Zhu, Xianguo Zou, Qingxiang Zeng</p>
Environmental pollution and overfishing of wild fish resources have led to a significant decrease in snakehead fish, thus leading to the increased demand for breeding the snakehead fish. Guangdong and Deqing snakehead fish are two common consumed varieties. However, their nutritional value was unclear. Therefore, this study aimed to evaluate the nutritional value of snakehead fish from Guangdong and Deqing varieties feeding with different fodders by analyzing and comparing the proximate composition, fatty acids and amino acids. Results showed that the contents of carbohydrate, energy and fat contents in Guangdong variety were lower than that in Deqing variety feeding commercial fodder or offal. Besides, Guangdong variety contained the highest contents of polyunsaturated fatty acids (27.99 ± 1.99%) and EPA + DHA (2.70 ± 0.04%), as well as total essential amino acid content (2550.29), compared to Deqing variety feeding commercial fodder or offal. Overall, snakehead fish from Guangdong variety displayed the highest nutritional value, and thus was a reasonable choice for farmers and consumers. The findings of this study would help farmers to choose the suitable feeding variety and patterns of snakehead fish from the perspective of fish nutritional value, which is beneficial to the sustainable fish farming.Study protocol: Identifying transcriptional regulatory alterations of chronic effects of blast and disturbed sleep in United States VeteransMolly J. SullanKelly A. Stearns-YoderZhaoyu WangAndrew J. HoisingtonAdam D. BramowethWalter CarrYongchao GeHanga GalfalvyFatemah HaghighiLisa A. Brenner10.1371/journal.pone.03010262024-03-27T14:00:00Z2024-03-27T14:00:00Z<p>by Molly J. Sullan, Kelly A. Stearns-Yoder, Zhaoyu Wang, Andrew J. Hoisington, Adam D. Bramoweth, Walter Carr, Yongchao Ge, Hanga Galfalvy, Fatemah Haghighi, Lisa A. Brenner</p>
Injury related to blast exposure dramatically rose during post-911 era military conflicts in Iraq and Afghanistan. Mild traumatic brain injury (mTBI) is among the most common injuries following blast, an exposure that may not result in a definitive physiologic marker (e.g., loss of consciousness). Recent research suggests that exposure to low level blasts and, more specifically repetitive blast exposure (RBE), which may be subconcussive in nature, may also impact long term physiologic and psychological outcomes, though findings have been mixed. For military personnel, blast-related injuries often occur in chaotic settings (e.g., combat), which create challenges in the immediate assessment of related-injuries, as well as acute and post-acute sequelae. As such, alternate means of identifying blast-related injuries are needed. Results from previous work suggest that epigenetic markers, such as DNA methylation, may provide a potential stable biomarker of cumulative blast exposure that can persist over time. However, more research regarding blast exposure and associations with short- and long-term sequelae is needed. Here we present the protocol for an observational study that will be completed in two phases: Phase 1 will address blast exposure among Active Duty Personnel and Phase 2 will focus on long term sequelae and biological signatures among Veterans who served in the recent conflicts and were exposed to repeated blast events as part of their military occupation. Phase 2 will be the focus of this paper. We hypothesize that Veterans will exhibit similar differentially methylated regions (DMRs) associated with changes in sleep and other psychological and physical metrics, as observed with Active Duty Personnel. Additional analyses will be conducted to compare DMRs between Phase 1 and 2 cohorts, as well as self-reported psychological and physical symptoms. This comparison between Service Members and Veterans will allow for exploration regarding the natural history of blast exposure in a quasi-longitudinal manner. Findings from this study are expected to provide additional evidence for repetitive blast-related physiologic changes associated with long-term neurobehavioral symptoms. It is expected that findings will provide foundational data for the development of effective interventions following RBE that could lead to improved long-term physical and psychological health.Comparative study on differential expression analysis methods for single-cell RNA sequencing data with small biological replicates: Based on single-cell transcriptional data of PBMCs from COVID-19 severe patientsJie XueXinfan ZhouJing YangAdan Niu10.1371/journal.pone.02993582024-03-27T14:00:00Z2024-03-27T14:00:00Z<p>by Jie Xue, Xinfan Zhou, Jing Yang, Adan Niu</p>
Single-cell RNA sequencing (scRNA-seq) is a high-throughput experimental technique for studying gene expression at the single-cell level. As a key component of single-cell data analysis, differential expression analysis (DEA) serves as the foundation for all subsequent secondary studies. Despite the fact that biological replicates are of vital importance in DEA process, small biological replication is still common in sequencing experiment now, which may impose problems to current DEA methods. Therefore, it is necessary to conduct a thorough comparison of various DEA approaches under small biological replications. Here, we compare 6 performance metrics on both simulated and real scRNA-seq datasets to assess the adaptability of 8 DEA approaches, with a particular emphasis on how well they function under small biological replications. Our findings suggest that DEA algorithms extended from bulk RNA-seq are still competitive under small biological replicate conditions, whereas the newly developed method DEF-scRNA-seq which is based on information entropy offers significant advantages. Our research not only provides appropriate suggestions for selecting DEA methods under different conditions, but also emphasizes the application value of machine learning algorithms in this field.GraphMHC: Neoantigen prediction model applying the graph neural network to molecular structureHoyeon JeongYoung-Rae ChoJungsoo GimSeung-Kuy ChaMaengsup KimDae Ryong Kang10.1371/journal.pone.02912232024-03-27T14:00:00Z2024-03-27T14:00:00Z<p>by Hoyeon Jeong, Young-Rae Cho, Jungsoo Gim, Seung-Kuy Cha, Maengsup Kim, Dae Ryong Kang</p>
Neoantigens are tumor-derived peptides and are biomarkers that can predict prognosis related to immune checkpoint inhibition by estimating their binding to major histocompatibility complex (MHC) proteins. Although deep neural networks have been primarily used for these prediction models, it is difficult to interpret the models reported thus far as accurately representing the interactions between biomolecules. In this study, we propose the GraphMHC model, which utilizes a graph neural network model applied to molecular structure to simulate the binding between MHC proteins and peptide sequences. Amino acid sequences sourced from the immune epitope database (IEDB) undergo conversion into molecular structures. Subsequently, atomic intrinsic informations and inter-atomic connections are extracted and structured as a graph representation. Stacked graph attention and convolution layers comprise the GraphMHC network which classifies bindings. The prediction results from the test set using the GraphMHC model showed a high performance with an area under the receiver operating characteristic curve of 92.2% (91.9-92.5%), surpassing a baseline model. Moreover, by applying the GraphMHC model to melanoma patient data from The Cancer Genome Atlas project, we found a borderline difference (0.061) in overall survival and a significant difference in stromal score between the high and low neoantigen load groups. This distinction was not present in the baseline model. This study presents the first feature-intrinsic method based on biochemical molecular structure for modeling the binding between MHC protein sequences and neoantigen candidate peptide sequences. This model can provide highly accurate responsibility information that can predict the prognosis of immune checkpoint inhibitors to cancer patients who want to apply it.A dual computational and experimental strategy to enhance TSLP antibody affinity for improved asthma treatmentYitong LvHe GongXuechao LiuJia HaoLei XuZhiwei SunChangyuan YuLida Xu10.1371/journal.pcbi.10119842024-03-27T14:00:00Z2024-03-27T14:00:00Z<p>by Yitong Lv, He Gong, Xuechao Liu, Jia Hao, Lei Xu, Zhiwei Sun, Changyuan Yu, Lida Xu</p>
Thymic stromal lymphopoietin is a key cytokine involved in the pathogenesis of asthma and other allergic diseases. Targeting TSLP and its signaling pathways is increasingly recognized as an effective strategy for asthma treatment. This study focused on enhancing the affinity of the T6 antibody, which specifically targets TSLP, by integrating computational and experimental methods. The initial affinity of the T6 antibody for TSLP was lower than the benchmark antibody AMG157. To improve this, we utilized alanine scanning, molecular docking, and computational tools including mCSM-PPI2 and GEO-PPI to identify critical amino acid residues for site-directed mutagenesis. Subsequent mutations and experimental validations resulted in an antibody with significantly enhanced blocking capacity against TSLP. Our findings demonstrate the potential of computer-assisted techniques in expediting antibody affinity maturation, thereby reducing both the time and cost of experiments. The integration of computational methods with experimental approaches holds great promise for the development of targeted therapeutic antibodies for TSLP-related diseases.Bias in the arrival of variation can dominate over natural selection in Richard Dawkins’s biomorphsNora S. MartinChico Q. CamargoArd A. Louis10.1371/journal.pcbi.10118932024-03-27T14:00:00Z2024-03-27T14:00:00Z<p>by Nora S. Martin, Chico Q. Camargo, Ard A. Louis</p>
Biomorphs, Richard Dawkins’s iconic model of morphological evolution, are traditionally used to demonstrate the power of natural selection to generate biological order from random mutations. Here we show that biomorphs can also be used to illustrate how developmental bias shapes adaptive evolutionary outcomes. In particular, we find that biomorphs exhibit phenotype bias, a type of developmental bias where certain phenotypes can be many orders of magnitude more likely than others to appear through random mutations. Moreover, this bias exhibits a strong preference for simpler phenotypes with low descriptional complexity. Such bias towards simplicity is formalised by an information-theoretic principle that can be intuitively understood from a picture of evolution randomly searching in the space of algorithms. By using population genetics simulations, we demonstrate how moderately adaptive phenotypic variation that appears more frequently upon random mutations can fix at the expense of more highly adaptive biomorph phenotypes that are less frequent. This result, as well as many other patterns found in the structure of variation for the biomorphs, such as high mutational robustness and a positive correlation between phenotype evolvability and robustness, closely resemble findings in molecular genotype-phenotype maps. Many of these patterns can be explained with an analytic model based on constrained and unconstrained sections of the genome. We postulate that the phenotype bias towards simplicity and other patterns biomorphs share with molecular genotype-phenotype maps may hold more widely for developmental systems.Differential carbonic anhydrase activities control EBV-induced B-cell transformation and lytic cycle reactivationSamaresh MalikJoyanta BiswasPurandar SarkarSubhadeep NagChandrima GainShatadru Ghosh RoyBireswar BhattacharyaDipanjan GhoshAbhik Saha10.1371/journal.ppat.10119982024-03-26T14:00:00Z2024-03-26T14:00:00Z<p>by Samaresh Malik, Joyanta Biswas, Purandar Sarkar, Subhadeep Nag, Chandrima Gain, Shatadru Ghosh Roy, Bireswar Bhattacharya, Dipanjan Ghosh, Abhik Saha</p>
Epstein-Barr virus (EBV) contributes to ~1% of all human cancers including several B-cell neoplasms. A characteristic feature of EBV life cycle is its ability to transform metabolically quiescent B-lymphocytes into hyperproliferating B-cell blasts with the establishment of viral latency, while intermittent lytic cycle induction is necessary for the production of progeny virus. Our RNA-Seq analyses of both latently infected naïve B-lymphocytes and transformed B-lymphocytes upon lytic cycle replication indicate a contrasting expression pattern of a membrane-associated carbonic anhydrase isoform CA9, an essential component for maintaining cell acid-base homeostasis. We show that while CA9 expression is transcriptionally activated during latent infection model, lytic cycle replication restrains its expression. Pharmacological inhibition of CA-activity using specific inhibitors retards EBV induced B-cell transformation, inhibits B-cells outgrowth and colony formation ability of transformed B-lymphocytes through lowering the intracellular pH, induction of cell apoptosis and facilitating degradation of CA9 transcripts. Reanalyses of ChIP-Seq data along with utilization of EBNA2 knockout virus, ectopic expression of EBNA2 and sh-RNA mediated knockdown of CA9 expression we further demonstrate that EBNA2 mediated CA9 transcriptional activation is essential for EBV latently infected B-cell survival. In contrast, during lytic cycle reactivation CA9 expression is transcriptionally suppressed by the key EBV lytic cycle transactivator, BZLF1 through its transactivation domain. Overall, our study highlights the dynamic alterations of CA9 expression and its activity in regulating pH homeostasis act as one of the major drivers for EBV induced B-cell transformation and subsequent B-cell lymphomagenesis.Spindle checkpoint activation by fungal orthologs of the <i>S</i>. <i>cerevisiae</i> Mps1 kinaseAmy FabritiusAnabel AlonsoAndrew WoodShaheen SulthanaMark Winey10.1371/journal.pone.03010842024-03-26T14:00:00Z2024-03-26T14:00:00Z<p>by Amy Fabritius, Anabel Alonso, Andrew Wood, Shaheen Sulthana, Mark Winey</p>
There is an ongoing need for antifungal agents to treat humans. Identification of new antifungal agents can be based on screening compounds using whole cell assays. Screening compounds that target a particular molecule is possible in budding yeast wherein sophisticated strain engineering allows for controlled expression of endogenous or heterologous genes. We have considered the yeast Mps1 protein kinase as a reasonable target for antifungal agents because mutant or druggable forms of the protein, upon inactivation, cause rapid loss of cell viability. Furthermore, extensive analysis of the Mps1 in budding yeast has offered potential tactics for identifying inhibitors of its enzymatic activity. One such tactic is based on the finding that overexpression of Mps1 leads to cell cycle arrest via activation of the spindle assembly checkpoint. We have endeavored to adapt this assay to be based on the overexpression of Mps1 orthologs from pathogenic yeast in hopes of having a whole-cell assay system to test the activity of these orthologs. Mps1 orthologous genes from seven pathogenic yeast or other pathogenic fungal species were isolated and expressed in budding yeast. Two orthologs clearly produced phenotypes similar to those produced by the overexpression of budding yeast Mps1, indicating that this system for heterologous Mps1 expression has potential as a platform for identifying prospective antifungal agents.