Blood. 2023 Mar 1:blood.2022017295. doi: 10.1182/blood.2022017295. Online ahead of print.

ABSTRACT

Primary resistance to tyrosine kinase inhibitors (TKI) is a significant barrier to optimal outcomes in chronic myeloid leukemia, but little is known about the factors contributing to response heterogeneity. Using scRNA-sequencing, we identified eight statistically significant features in pretreatment bone marrow mononuclear cells which correlated with either sensitivity (major molecular response or MMR) or extreme resistance to imatinib (eventual blast crisis transformation). Employing machine-learning, we also identified LSC and NK gene expression profiles predicting imatinib response with >80% accuracy, including zero false positives for predicting BC. A canonical erythroid-specifying (TAL1/KLF1/GATA1) regulon was a hallmark of LSCs from patients with MMR and was associated with erythroid progenitor (ERP) expansion in vivo (p<0.05), and a marked 2-10-fold (6.3-fold in Group A vs 1.09-fold in Group C) erythroid over myeloid bias in vitro. Notably, ERPs demonstrated exquisite TKI sensitivity compared to myeloid progenitors (p<0.001). These LSC features were lost with progressive resistance, and in patients who transformed, MYC- and IRF1-driven inflammatory regulons became evident. Patients with MMR also exhibited a 56-fold expansion (p<0.01) of a normally rare subset of hyperfunctional adaptive-like NK cells (CD57+NKG2C+) which diminished with progressive resistance, while patients destined for BC accumulated inhibitory NKG2A+ NK cells favoring NK cell tolerance (through HLA-E binding on target cells). Finally, we developed a parsimonious set of antibodies to validate our scRNA-seq findings. This panel will be useful in prospective studies of primary resistance, and assessing the contribution of predetermined versus acquired factors in TKI response heterogeneity.

PMID:36857629 | DOI:10.1182/blood.2022017295

Sci Adv. 2023 Mar;9(9):eadd5220. doi: 10.1126/sciadv.add5220. Epub 2023 Mar 1.

ABSTRACT

Cellular metabolism is important for adult neural stem/progenitor cell (NSPC) behavior. However, its role in the transition from quiescence to proliferation is not fully understood. We here show that the mitochondrial pyruvate carrier (MPC) plays a crucial and unexpected part in this process. MPC transports pyruvate into mitochondria, linking cytosolic glycolysis to mitochondrial tricarboxylic acid cycle and oxidative phosphorylation. Despite its metabolic key function, the role of MPC in NSPCs has not been addressed. We show that quiescent NSPCs have an active mitochondrial metabolism and express high levels of MPC. Pharmacological MPC inhibition increases aspartate and triggers NSPC activation. Furthermore, genetic Mpc1 ablation in vitro and in vivo also activates NSPCs, which differentiate into mature neurons, leading to overall increased hippocampal neurogenesis in adult and aged mice. These findings highlight the importance of metabolism for NSPC regulation and identify an important pathway through which mitochondrial pyruvate import controls NSPC quiescence and activation.

PMID:36857455 | DOI:10.1126/sciadv.add5220

PLoS Pathog. 2023 Mar 1;19(3):e1011155. doi: 10.1371/journal.ppat.1011155. Online ahead of print.

ABSTRACT

RNA viruses can exchange genetic material during coinfection, an interaction that creates novel strains with implications for viral evolution and public health. Influenza A viral genetic exchange can occur when genome segments from distinct strains reassort in coinfected cells. Predicting potential genomic reassortment between influenza strains has been a long-standing goal. Experimental coinfection studies have shed light on factors that limit or promote reassortment. However, determining the reassortment potential between diverse Influenza A strains has remained elusive. To address this challenge, we developed a high throughput genotyping approach to quantify reassortment among a diverse panel of human influenza virus strains encompassing two pandemics (swine and avian origin), three specific epidemics, and both circulating human subtypes A/H1N1 and A/H3N2. We found that reassortment frequency (the proportion of reassortants generated) is an emergent property of specific pairs of strains where strain identity is a predictor of reassortment frequency. We detect little evidence that antigenic subtype drives reassortment as intersubtype (H1N1xH3N2) and intrasubtype reassortment frequencies were, on average, similar. Instead, our data suggest that certain strains bias the reassortment frequency up or down, independently of the coinfecting partner. We observe that viral productivity is also an emergent property of coinfections, but uncorrelated to reassortment frequency; thus viral productivity is a separate factor affecting the total number of reassortants produced. Assortment of individual segments among progeny and pairwise segment combinations within progeny generally favored homologous combinations. These outcomes were not related to strain similarity or shared subtype but reassortment frequency was closely correlated to the proportion of both unique genotypes and of progeny with heterologous pairwise segment combinations. We provide experimental evidence that viral genetic exchange is potentially an individual social trait subject to natural selection, which implies the propensity for reassortment is not evenly shared among strains. This study highlights the need for research incorporating diverse strains to discover the traits that shift the reassortment potential to realize the goal of predicting influenza virus evolution resulting from segment exchange.

PMID:36857394 | DOI:10.1371/journal.ppat.1011155

PLoS One. 2023 Mar 1;18(3):e0280075. doi: 10.1371/journal.pone.0280075. eCollection 2023.

ABSTRACT

Stingless bees play a crucial role in the environment and agriculture as they are effective pollinators. Furthermore, they can produce various products that can be exploited economically, such as propolis and honey. Despite their economic value, the knowledge of microbial community of stingless bees, and their roles on the bees' health, especially in Thailand, are in its infancy. This study aimed to investigate the composition and the functions of bacterial community associated with Tetragonula pagdeni stingless bees using culture-independent and culture-dependent approaches with emphasis on lactic acid bacteria. The culture-independent results showed that the dominant bacterial phyla were Firmicutes, Proteobacteria and Actinobacteria. The most abundant families were Lactobacillaceae and Halomonadaceae. Functional prediction indicated that the prevalent functions of bacterial communities were chemoheterotrophy and fermentation. In addition, the bacterial community might be able to biosynthesize amino acid and antimicrobial compounds. Further isolation and characterization resulted in isolates that belonged to the dominant taxa of the community and possessed potentially beneficial metabolic activity. This suggested that they are parts of the nutrient acquisition and host defense bacterial functional groups in Thai commercial stingless bees.

PMID:36857385 | DOI:10.1371/journal.pone.0280075

Proc Natl Acad Sci U S A. 2023 Mar 7;120(10):e2210891120. doi: 10.1073/pnas.2210891120. Epub 2023 Mar 1.

ABSTRACT

SMAD-mediated signaling regulates apoptosis, cell cycle arrest, and epithelial-to-mesenchymal transition to safeguard tissue homeostasis. However, it remains elusive how the relatively simple pathway can determine such a broad range of cell fate decisions and how it differentiates between varying ligands. Here, we systematically investigate how SMAD-mediated responses are modulated by various ligands of the transforming growth factor β (TGFβ) family and compare these ligand responses in quiescent and proliferating MCF10A cells. We find that the nature of the phenotypic response is mainly determined by the proliferation status, with migration and cell cycle arrest being dominant in proliferating cells for all tested TGFβ family ligands, whereas cell death is the major outcome in quiescent cells. In both quiescent and proliferating cells, the identity of the ligand modulates the strength of the phenotypic response proportional to the dynamics of induced SMAD nuclear-to-cytoplasmic translocation and, as a consequence, the corresponding gene expression changes. Interestingly, the proliferation state of a cell has little impact on the set of genes induced by SMAD signaling; instead, it modulates the relative cellular sensitivity to TGFβ superfamily members. Taken together, diversity of SMAD-mediated responses is mediated by differing cellular states, which determine ligand sensitivity and phenotypic effects, while the pathway itself merely serves as a quantitative relay from the cell membrane to the nucleus.

PMID:36857347 | DOI:10.1073/pnas.2210891120

STAR Protoc. 2023 Jan 20;4(1):101964. doi: 10.1016/j.xpro.2022.101964. Online ahead of print.

ABSTRACT

Genotyping single-nucleotide polymorphisms (SNPs) in microbiomes enables strain-level quantification. In this protocol, we describe a computational pipeline that performs fast and accurate SNP genotyping using metagenomic data. We first demonstrate how to use Maast to catalog SNPs from microbial genomes. Then we use GT-Pro to extract unique SNP-covering k-mers, optimize a data structure for storing these k-mers, and finally perform metagenotyping. For proof of concept, the protocol leverages public whole-genome sequences to metagenotype a synthetic community. For complete details on the use and execution of this protocol, please refer to Shi et al. (2022a)1 and Shi et al. (2022b).2.

PMID:36856771 | DOI:10.1016/j.xpro.2022.101964

Microbiol Spectr. 2023 Mar 1:e0383522. doi: 10.1128/spectrum.03835-22. Online ahead of print.

ABSTRACT

Burkholderia pseudomallei, a soil and water saprophyte, is responsible for the tropical human disease melioidosis. A hundred years since its discovery, there is still much to learn about B. pseudomallei proteins that are essential for the bacterium's survival in and interaction with the infected host, as well as their roles within the bacterium's natural soil habitat. To address this gap, bacteria grown under conditions mimicking the soil environment were subjected to transcriptome sequencing (RNA-seq) analysis. A dual RNA-seq approach was used on total RNA from spleens isolated from a B. pseudomallei mouse infection model at 5 days postinfection. Under these conditions, a total of 1,434 bacterial genes were induced, with 959 induced in the soil environment and 475 induced in bacteria residing within the host. Genes encoding metabolism and transporter proteins were induced when the bacteria were present in soil, while virulence factors, metabolism, and bacterial defense mechanisms were upregulated during active infection of mice. On the other hand, capsular polysaccharide and quorum-sensing pathways were inhibited during infection. In addition to virulence factors, reactive oxygen species, heat shock proteins, siderophores, and secondary metabolites were also induced to assist bacterial adaptation and survival in the host. Overall, this study provides crucial insights into the transcriptome-level adaptations which facilitate infection by soil-dwelling B. pseudomallei. Targeting novel therapeutics toward B. pseudomallei proteins required for adaptation provides an alternative treatment strategy given its intrinsic antimicrobial resistance and the absence of a vaccine. IMPORTANCE Burkholderia pseudomallei, a soil-dwelling bacterium, is the causative agent of melioidosis, a fatal infectious disease of humans and animals. The bacterium has a large genome consisting of two chromosomes carrying genes that encode proteins with important roles for survival in diverse environments as well as in the infected host. While a general mechanism of pathogenesis has been proposed, it is not clear which proteins have major roles when the bacteria are in the soil and whether the same proteins are key to successful infection and spread. To address this question, we grew the bacteria in soil medium and then in infected mice. At 5 days postinfection, bacteria were recovered from infected mouse organs and their gene expression was compared against that of bacteria grown in soil medium. The analysis revealed a list of genes expressed under soil growth conditions and a different set of genes encoding proteins which may be important for survival, replication, and dissemination in an infected host. These proteins are a potential resource for understanding the full adaptation mechanism of this pathogen. In the absence of a vaccine for melioidosis and with treatment being reliant on combinatorial antibiotic therapy, these proteins may be ideal targets for designing antimicrobials to treat melioidosis.

PMID:36856434 | DOI:10.1128/spectrum.03835-22

J Transl Med. 2023 Feb 28;21(1):158. doi: 10.1186/s12967-023-04014-9.

ABSTRACT

BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapies for the treatment of hematological malignancies experienced tremendous progress in the last decade. However, essential limitations need to be addressed to further improve efficacy and reduce toxicity to assure CAR-T cell persistence, trafficking to the tumor site, resistance to an hostile tumor microenvironment (TME), and containment of toxicity restricting production of powerful but potentially toxic bioproducts to the TME; the last could be achieved through contextual release upon tumor antigen encounter of factors capable of converting an immune suppressive TME into one conducive to immune rejection.

METHODS: We created an HER2-targeting CAR-T (RB-312) using a clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system, which induces the expression of the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. This circuit includes two lentiviral constructs. The first one (HER2-TEV) expresses an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3z co-stimulatory domains linked to the tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the interleukin (IL)-12A and IL12B transcription start site (TSS), respectively. The second construct (LdCV) encodes linker for activation of T cells (LAT) fused to nuclease-deactivated Streptococcus Pyogenes Cas9 (dCas9)-VP64-p65-Rta (VPR) via a TEV-cleavable sequence (TCS). Activation of the CAR brings HER2-TEV in close proximity to LdCV releasing dCas9 for nuclear localization. This conditional circuit leads to conditional and reversible induction of the IL-12/p70 heterodimer. RB-312 was compared in vitro to controls (cRB-312), lacking the IL-12 sgRNAs and conventional HER2 CAR (convCAR).

RESULTS: The inducible CRISPRa system activated endogenous IL-12 expression resulting in enhanced secondary interferon (FN)-γ production, cytotoxicity, and CAR-T proliferation in vitro, prolonged in vivo persistence and greater suppression of HER2+ FaDu oropharyngeal cancer cell growth compared to the conventional CAR-T cell product. No systemic IL-12 was detected in the peripheral circulation. Moreover, the combination with programmed death ligand (PD-L1) blockade demonstrated robust synergistic effects.

CONCLUSIONS: RB-312, the first clinically relevant product incorporating a CRISPRa system with non-gene editing and reversible upregulation of endogenous gene expression that promotes CAR-T cells persistence and effectiveness against HER2-expressing tumors. The autocrine effects of reversible, nanoscale IL-12 production limits the risk of off-tumor leakage and systemic toxicity.

PMID:36855120 | DOI:10.1186/s12967-023-04014-9

BMC Bioinformatics. 2023 Feb 28;24(1):71. doi: 10.1186/s12859-023-05201-7.

ABSTRACT

Allosteric transcription factor (aTF) based biosensors can be used to engineer genetic circuits for a wide range of applications. The literature and online databases contain hundreds of experimentally validated molecule-TF pairs; however, the knowledge is scattered and often incomplete. Additionally, compared to the number of compounds that can be produced in living systems, those with known associated TF-compound interactions are low. For these reasons, new tools that help researchers find new possible TF-ligand pairs are called for. In this work, we present Sensbio, a computational tool that through similarity comparison against a TF-ligand reference database, is able to identify putative transcription factors that can be activated by a given input molecule. In addition to the collection of algorithms, an online application has also been developed, together with a predictive model created to find new possible matches based on machine learning.

PMID:36855083 | DOI:10.1186/s12859-023-05201-7

Sci Rep. 2023 Feb 28;13(1):3413. doi: 10.1038/s41598-023-29344-y.

ABSTRACT

In recent studies, the void of evaluation and in-depth understanding of unknown clinically relevant potential molecular biomarkers involved in colorectal cancer (CRC) from the inflammatory stage of ulcerative colitis (UC) to CRC metastasis, which can be suitable therapeutic targets, is deeply felt. The regulation and interaction among different cancer-promoting molecules, including messenger RNAs (mRNAs) and micro RNAs (miRNAs) in CRC and its progression, were the aim we pursued in this study. Using microarray data, we investigated the differential expression for five datasets, including mRNA and microRNA samples related to UC, tumor/normal. Then, using robust data analysis, separate lists of differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRNAs) were identified, which were used for robust rank aggregation (RRA) and co-expression network analysis. Then, comprehensive computational systems biology analyses, including gene ontology and Kyoto encyclopedia of genes and genomic pathway enrichment analyses, mRNA-miRNA regulatory network, and survival analysis, were employed to achieve the aim of this study. Finally, we used clinical samples to validate this potential and new target. According to this systems biology approach, a total of 98 DEGs and 8 DEmiRNAs with common differential expression were identified. By combining the distinct results of RRA and network, several potential therapeutic targets, and predictive and prognostic biomarkers for UC and CRC were identified. These targets include six common hub genes, CXCL1, CXCL8, MMP7, SLCA16A9, PLAU, and TIMP1, which are upregulated. Among these, the important and new biomarker SLC16A9 is negatively regulated by hsa-mir-194-5p, and hsa-miR-378a-5p take. The findings of the present study provide new insight into the pathogenesis of CRC in UC. Our study suggests future evaluation of the functional role of SLC16A9 and hsa-mir-194-5p and hsa-miR-378a-5p in CRC development.

PMID:36854781 | DOI:10.1038/s41598-023-29344-y

Nat Commun. 2023 Feb 28;14(1):1143. doi: 10.1038/s41467-023-36693-9.

ABSTRACT

The protein phosphatase 2A (PP2A) heterotrimer PP2A-B56α is a human tumour suppressor. However, the molecular mechanisms inhibiting PP2A-B56α in cancer are poorly understood. Here, we report molecular level details and structural mechanisms of PP2A-B56α inhibition by an oncoprotein CIP2A. Upon direct binding to PP2A-B56α trimer, CIP2A displaces the PP2A-A subunit and thereby hijacks both the B56α, and the catalytic PP2Ac subunit to form a CIP2A-B56α-PP2Ac pseudotrimer. Further, CIP2A competes with B56α substrate binding by blocking the LxxIxE-motif substrate binding pocket on B56α. Relevant to oncogenic activity of CIP2A across human cancers, the N-terminal head domain-mediated interaction with B56α stabilizes CIP2A protein. Functionally, CRISPR/Cas9-mediated single amino acid mutagenesis of the head domain blunted MYC expression and MEK phosphorylation, and abrogated triple-negative breast cancer in vivo tumour growth. Collectively, we discover a unique multi-step hijack and mute protein complex regulation mechanism resulting in tumour suppressor PP2A-B56α inhibition. Further, the results unfold a structural determinant for the oncogenic activity of CIP2A, potentially facilitating therapeutic modulation of CIP2A in cancer and other diseases.

PMID:36854761 | DOI:10.1038/s41467-023-36693-9

Nat Commun. 2023 Feb 28;14(1):1132. doi: 10.1038/s41467-023-36638-2.

ABSTRACT

The causal variants and genes underlying thousands of cardiac GWAS signals have yet to be identified. Here, we leverage spatiotemporal information on 966 RNA-seq cardiac samples and perform an expression quantitative trait locus (eQTL) analysis detecting eQTLs considering both eGenes and eIsoforms. We identify 2,578 eQTLs associated with a specific developmental stage-, tissue- and/or cell type. Colocalization between eQTL and GWAS signals of five cardiac traits identified variants with high posterior probabilities for being causal in 210 GWAS loci. Pulse pressure GWAS loci are enriched for colocalization with fetal- and smooth muscle- eQTLs; pulse rate with adult- and cardiac muscle- eQTLs; and atrial fibrillation with cardiac muscle- eQTLs. Fine mapping identifies 79 credible sets with five or fewer SNPs, of which 15 were associated with spatiotemporal eQTLs. Our study shows that many cardiac GWAS variants impact traits and disease in a developmental stage-, tissue- and/or cell type-specific fashion.

PMID:36854752 | DOI:10.1038/s41467-023-36638-2

Nat Commun. 2023 Feb 28;14(1):914. doi: 10.1038/s41467-023-36405-3.

ABSTRACT

The systematics of Madagascar's extinct elephant birds remains controversial due to large gaps in the fossil record and poor biomolecular preservation of skeletal specimens. Here, a molecular analysis of 1000-year-old fossil eggshells provides the first description of elephant bird phylogeography and offers insight into the ecology and evolution of these flightless giants. Mitochondrial genomes from across Madagascar reveal genetic variation that is correlated with eggshell morphology, stable isotope composition, and geographic distribution. The elephant bird crown is dated to ca. 30 Mya, when Madagascar is estimated to have become less arid as it moved northward. High levels of between-clade genetic variation support reclassifying Mullerornis into a separate family. Low levels of within-clade genetic variation suggest there were only two elephant bird genera existing in southern Madagascar during the Holocene. However, we find an eggshell collection from Madagascar's far north that represents a unique lineage of Aepyornis. Furthermore, divergence within Aepyornis coincides with the aridification of Madagascar during the early Pleistocene ca. 1.5 Ma, and is consistent with the fragmentation of populations in the highlands driving diversification and the evolution of extreme gigantism over shorts timescales. We advocate for a revision of their taxonomy that integrates palaeogenomic and palaeoecological perspectives.

PMID:36854679 | DOI:10.1038/s41467-023-36405-3

Curr Opin Genet Dev. 2023 Feb 26;79:102023. doi: 10.1016/j.gde.2023.102023. Online ahead of print.

ABSTRACT

In the past two years, approaches relying on high-resolution microscopy and live-cell imaging have increasingly contributed to our understanding of the 3D genome organization and its importance for transcriptional control. Here, we describe recent progress that has highlighted how flexible and heterogeneous 3D chromatin structure is, on the length scales relevant to transcriptional control. We describe work that has investigated how robust transcriptional outcomes may be derived from such flexible organization without the need for clearly distinct structures in active and silent cells. We survey the latest state of the art in directly observing the dynamics of chromatin interactions, and suggest how some recent, apparently contradictory conclusions may be reconciled.

PMID:36854248 | DOI:10.1016/j.gde.2023.102023

Annu Rev Anal Chem (Palo Alto Calif). 2022 Feb 28. doi: 10.1146/annurev-anchem-091622-092353. Online ahead of print.

ABSTRACT

The identification of thousands of proteins and their relative levels of expression has furthered understanding of biological processes and disease and stimulated new systems biology hypotheses. Quantitative proteomics workflows that rely on analytical assays such as mass spectrometry have facilitated high-throughput measurements of proteins partially due to multiplexing. Multiplexing allows proteome differences across multiple samples to be measured simultaneously, resulting in more accurate quantitation, increased statistical robustness, reduced analysis times, and lower experimental costs. The number of samples that can be multiplexed has evolved from as few as two to more than 50, with studies involving more than 10 samples being denoted as enhanced multiplexing or hyperplexing. In this review, we give an update on emerging multiplexing proteomics techniques and highlight advantages and limitations for enhanced multiplexing strategies. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 16 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

PMID:36854207 | DOI:10.1146/annurev-anchem-091622-092353

STAR Protoc. 2023 Feb 9;4(1):102107. doi: 10.1016/j.xpro.2023.102107. Online ahead of print.

ABSTRACT

Since changes in mitochondrial morphology regulate key functions of stem cells, it is important to assess their structure under physiological and pathophysiological conditions. Here, we present techniques optimized in rare adult muscle stem cells (MuSCs). For evaluating mitochondrial length and volume within a compact cytoplasmic area in MuSCs on intact myofibers, we describe steps for mitochondrial staining, imaging, and quantification. For evaluating mitochondrial ultrastructure in small cell numbers, we describe steps for agarose embedding and quantification by TEM. For complete details on generation and use of this protocol, please refer to Baker et al. (2022).1.

PMID:36853728 | DOI:10.1016/j.xpro.2023.102107

STAR Protoc. 2023 Jan 25;4(1):102069. doi: 10.1016/j.xpro.2023.102069. Online ahead of print.

ABSTRACT

Understanding cellular metabolism is important across biotechnology and biomedical research and has critical implications in a broad range of normal and pathological conditions. Here, we introduce the user-friendly web-based platform ImmCellFie, which allows the comprehensive analysis of metabolic functions inferred from transcriptomic or proteomic data. We explain how to set up a run using publicly available omics data and how to visualize the results. The ImmCellFie algorithm pushes beyond conventional statistical enrichment and incorporates complex biological mechanisms to quantify cell activity. For complete details on the use and execution of this protocol, please refer to Richelle et al. (2021).1.

PMID:36853701 | DOI:10.1016/j.xpro.2023.102069

STAR Protoc. 2023 Jan 24;4(1):102060. doi: 10.1016/j.xpro.2023.102060. Online ahead of print.

ABSTRACT

Mass-spectrometry-based absolute protein quantification uses labeled quantification concatamer (QconCAT) as internal standards (ISs). To calculate the amount of protein(s), the ion intensity ratio between the analyte and its cognate IS is compared in each biological sample. The present protocol describes a systematic workflow to design, produce, and purify QconCATs and to quantify soluble proteins in Pseudomonas putida KT2440. Our methodology enables the quantification of detectable peptide and serves as a versatile platform to produce ISs for different biological systems.

PMID:36853682 | DOI:10.1016/j.xpro.2023.102060

STAR Protoc. 2023 Jan 24;4(1):102059. doi: 10.1016/j.xpro.2023.102059. Online ahead of print.

ABSTRACT

Some newly translated proteins are more susceptible to misfolding and aggregation upon heat shock in comparison to other proteins. To study these newly translated thermo-sensitive proteins on a proteomic scale, we present here a protocol that combines pulse-SILAC with biochemical fractionation for mass spectrometry analysis, followed by an orthogonal validation protocol for selected candidates using the GAL promoter system in Saccharomyces cerevisiae. This approach can be further developed to study other stresses and specific post-translational modifications or adapted to mammalian cells. For complete details on the use and execution of this protocol, please refer to Zhu et al. (2022).1.

PMID:36853680 | DOI:10.1016/j.xpro.2023.102059

New Phytol. 2023 Feb 28. doi: 10.1111/nph.18803. Online ahead of print.

NO ABSTRACT

PMID:36853427 | DOI:10.1111/nph.18803