Plant Physiol Biochem. 2024 Oct 11;216:109193. doi: 10.1016/j.plaphy.2024.109193. Online ahead of print.

ABSTRACT

Calcium plays a crucial role in regulating plant senescence. However, the specific effects of increased intranuclear calcium versus cytoplasmic calcium on aging remain unclear. Cyclic nucleotide-gated channels (CNGCs), which manage Ca2⁺ levels in plant cells, are particularly significant in this context. These channels are known to relocate between the nuclear envelope and the plasma membrane in response to stress and developmental signals. Through this movement, CNGCs help regulate the balance of cytosolic and intranuclear Ca2⁺. In this study, we categorized the 16 CNGC genes in rice into five subgroups. OsCNGCs are notably expressed in leaves, especially during the reproductive stage. Both OsCNGC6 and OsCNGC7 exhibit dual localization to the plasma membrane and the nuclear envelope. Knockdown of OsCNGC7 led to reduced levels of Ca2⁺ and K⁺ in plants. Conversely, yeast expressing the OsCNGC7 gene showed increased sensitivity to Ca2⁺. Additionally, while the [Ca2⁺]cyt was maintained at relatively low levels in both wild-type and OsCNGC7-RNAi lines, the fluorescence intensity was significantly higher in OsCNGC7-overexpressing lines, particularly in the nucleus of root tips. Overexpression of OsCNGC7 resulted in enhanced stomatal opening and accelerated leaf senescence from the tillering stage to grain filling in rice. Treatment with MeJA rapidly induced OsCNGC7 expression, while knockdown of OsCNGC7 delayed both MeJA-induced and dark-induced leaf senescence. Further analysis revealed that OsCNGC7 interacts with OsKAT2 and OsALMT2. In conclusion, our findings highlight the distinct roles of OsCNGCs in regulating senescence. This knowledge could provide new strategies for manipulating plant senescence and enhancing crop productivity.

PMID:39406002 | DOI:10.1016/j.plaphy.2024.109193

Transl Oncol. 2024 Oct 13;50:102152. doi: 10.1016/j.tranon.2024.102152. Online ahead of print.

ABSTRACT

BACKGROUND: Plasma-derived Extracellular Vesicles (EVs) have been suggested as novel biomarkers in melanoma, due to their ability to reflect the cell of origin and ease of collection. This study aimed to identify novel EV biomarkers that can discriminate between disease stages. This was achieved by characterising the plasma-derived EVs of patients with melanoma, and comparing their proteomic and metabolomic profile to those from healthy controls.

METHODS: EVs were isolated from the plasma of 36 patients with melanoma and 13 healthy controls using Size Exclusion Chromatography. Proteomic and Metabolomic Analyses were performed, and machine learning algorithms were used to identify potential proteins and metabolites to differentiate the plasma-derived EVs from melanoma patients of different disease stages.

RESULTS: The concentration and size of the EV population isolated was similar between groups. Proteins (APOC4, PRG4, PLG, TNC, VWF and SERPIND1) and metabolites (lyso PC a C18:2, PC ae C44:3) previously associated with melanoma pathogenesis were identified as relevant in differentiating between disease stages.

CONCLUSION: The results further support the continued investigation of circulating plasma-derived EVs as biomarkers in melanoma. Furthermore, the potential of combined proteo-metabolomic signatures for differentiation between disease stages may provide valuable insights into early detection, prognosis, and personalised treatment strategies.

PMID:39405606 | DOI:10.1016/j.tranon.2024.102152

ACS Nano. 2024 Oct 15. doi: 10.1021/acsnano.3c12337. Online ahead of print.

ABSTRACT

The stimulator of interferon genes (STING) pathway is crucial for tumor immunity, leading to the exploration of STING agonists as potential immunotherapy adjuvants. However, their clinical application faces obstacles including poor pharmacokinetics, transient activation, and an immunosuppressive tumor microenvironment (TME). Addressing these limitations, our study aims to develop an injectable silk fibroin hydrogel-based in situ vaccine. It incorporates a nanoscale STING agonist, an immunogenic cell death (ICD) inducer, and an immunomodulator to ensure their controlled and sustained release. cGAMP nanoparticles (cGAMPnps) with a core-shell structure ensure optimal delivery of cGAMP to dendritic cells (DCs), thereby activating the STING pathway and fostering DC maturation. ICD-associated damage-associated molecular patterns amplify and prolong STING activation via enhanced type I IFN and other inflammatory pathways, along with delayed degradation of cGAMP and STING. Furthermore, the STING-driven vascular normalization by cGAMPnps and ICD, in conjunction with immunomodulators like antiprogrammed cell death protein 1 antibody (anti-PD-1 Ab) or OX40 ligand (OX40L), effectively remodels the immunosuppressive TME. This in situ gel vaccine, when used independently or with surgery as neoadjuvant/adjuvant immunotherapy, enhances DC and CD8+ T-cell activation, suppressing tumor progression and recurrence across various immunologically cold tumor models. It revolutionizes the application of STING agonists in cancer immunotherapy, offering substantial promise for improving outcomes across a broad spectrum of malignancies.

PMID:39405469 | DOI:10.1021/acsnano.3c12337

Elife. 2024 Oct 15;13:RP97754. doi: 10.7554/eLife.97754.

ABSTRACT

Synthetic genetic oscillators can serve as internal clocks within engineered cells to program periodic expression. However, cell-to-cell variability introduces a dispersion in the characteristics of these clocks that drives the population to complete desynchronization. Here, we introduce the optorepressilator, an optically controllable genetic clock that combines the repressilator, a three-node synthetic network in E. coli, with an optogenetic module enabling to reset, delay, or advance its phase using optical inputs. We demonstrate that a population of optorepressilators can be synchronized by transient green light exposure or entrained to oscillate indefinitely by a train of short pulses, through a mechanism reminiscent of natural circadian clocks. Furthermore, we investigate the system's response to detuned external stimuli observing multiple regimes of global synchronization. Integrating experiments and mathematical modeling, we show that the entrainment mechanism is robust and can be understood quantitatively from single cell to population level.

PMID:39405096 | DOI:10.7554/eLife.97754

Am J Physiol Gastrointest Liver Physiol. 2024 Oct 15. doi: 10.1152/ajpgi.00110.2024. Online ahead of print.

ABSTRACT

The choline-deficient L-amino acid defined-high fat diet (CDAA-HFD) mouse model is widely used in preclinical metabolic dysfunction-associated steatohepatitis (MASH) research. To validate the CDAA-HFD mouse, we evaluated disease progression and responsiveness to dietary and pharmacological interventions with semaglutide, lanifibranor, elafibranor, obeticholic acid (OCA), firsocostat and resmetirom.Disease phenotyping was performed in C57BL/6J mice fed CDAA-HFD for 3-20 weeks and ranked using the MASLD Human Proximity Score (MHPS). Semaglutide, lanifibranor, elafibranor, OCA, firsocostat or resmetirom were profiled as treatment intervention for 8 weeks, starting after 6 weeks of CDAA-HFD feeding. Semaglutide and lanifibranor were further evaluated as early (preventive) therapy for 9 weeks, starting 3 weeks after CDAA-HFD diet feeding. Additionally, benefits of dietary intervention (chow reversal) for 8 weeks were characterized following 6 weeks of CDAA-HFD feeding. CDAA-HFD mice demonstrated a non-obese phenotype with fast onset and progression of MASH and fibrosis, high similarity to human MASH-fibrosis, and tumor development after 20 weeks of diet-induction. Semaglutide and lanifibranor partially reversed fibrosis when administered as prevention, but not as treatment intervention. Elafibranor was the only interventional drug to improve fibrosis. In comparison, chow-reversal resulted in complete steatosis regression with improved liver inflammation and fibrosis in CDAA-HFD mice. CDAA-HFD mice recapitulate histological hallmarks of advanced MASH with progressive severe fibrosis, however, in the absence of a clinical translational obese dysmetabolic phenotype. CDAA-HFD mice are suitable for profiling drug candidates directly targeting hepatic lipid metabolism, inflammation, and fibrosis. The timing of pharmacological intervention is critical for determining antifibrotic drug efficacy in the model.

PMID:39404770 | DOI:10.1152/ajpgi.00110.2024

J Cell Biol. 2025 Jan 6;224(1):e202312141. doi: 10.1083/jcb.202312141. Epub 2024 Oct 15.

ABSTRACT

The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here, we show that its lysosomal degradation is dependent on ubiquitylation at lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4, and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells, and cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive nor a localization-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4 or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.

PMID:39404738 | DOI:10.1083/jcb.202312141

Adv Ecol Res. 2017;57:201-281. doi: 10.1016/bs.aecr.2016.11.002.

ABSTRACT

The study of biological invasions of ecological systems has much to offer research on within-host systems, particularly for understanding infections and developing therapies using biological agents. Thanks to the ground-work established in other fields, such as community ecology and evolutionary biology, and to modern methods of measurement and quantification, the study of microbiomes has quickly become a field at the forefront of modern systems biology. Investigations of host-associated microbiomes (e.g., for studying human health) are often centered on measuring and explaining the structure, functions and stability of these communities. This momentum promises to rapidly advance our understanding of ecological networks and their stability, resilience and resistance to invasions. However, intrinsic properties of host-associated microbiomes that differ from those of free-living systems present challenges to the development of a within-host invasion ecology framework. The elucidation of principles underlying the invasibility of within-host networks will ultimately help in the development of medical applications and help shape our understanding of human health and disease.

PMID:39404686 | PMC:PMC7616576 | DOI:10.1016/bs.aecr.2016.11.002

Mol Biol Evol. 2024 Oct 15:msae210. doi: 10.1093/molbev/msae210. Online ahead of print.

ABSTRACT

Comparative genomics approaches seek to associate molecular evolution with the evolution of phenotypes across a phylogeny. Many of these methods lack the ability to analyze non-ordinal categorical traits with more than two categories. To address this limitation, we introduce an expansion to RERconverge that associates shifts in evolutionary rates with the convergent evolution of categorical traits. The categorical RERconverge expansion includes methods for performing categorical ancestral state reconstruction, statistical tests for associating relative evolutionary rates with categorical variables, and a new method for performing phylogeny-aware permutations, "permulations", on categorical traits. We demonstrate our new method on a three-category diet phenotype and we compare its performance to binary RERconverge analyses and two existing methods for comparative genomic analyses of categorical traits: phylogenetic simulations and a phylogenetic signal based method. We present an analysis of how the categorical permulations scale with the number of species and the number of categories included in the analysis. Our results show that our new categorical method outperforms phylogenetic simulations at identifying genes and enriched pathways significantly associated with the diet phenotypes and that the categorical ancestral state reconstruction drives an improvement in our ability to capture diet-related enriched pathways compared to binary RERconverge when implemented without user input on phenotype evolution. The categorical expansion to RERconverge will provide a strong foundation for applying the comparative method to categorical traits on larger data sets with more species and more complex trait evolution than have previously been analyzed.

PMID:39404101 | DOI:10.1093/molbev/msae210

J Clin Invest. 2024 Oct 15;134(20):e172886. doi: 10.1172/JCI172886.

ABSTRACT

Opioid misuse, addiction, and associated overdose deaths remain global public health crises. Despite the tremendous need for pharmacological treatments, current options are limited in number, use, and effectiveness. Fundamental leaps forward in our understanding of the biology driving opioid addiction are needed to guide development of more effective medication-assisted therapies. This Review focuses on the omics-identified biological features associated with opioid addiction. Recent GWAS have begun to identify robust genetic associations, including variants in OPRM1, FURIN, and the gene cluster SCAI/PPP6C/RABEPK. An increasing number of omics studies of postmortem human brain tissue examining biological features (e.g., histone modification and gene expression) across different brain regions have identified broad gene dysregulation associated with overdose death among opioid misusers. Drawn together by meta-analysis and multi-omic systems biology, and informed by model organism studies, key biological pathways enriched for opioid addiction-associated genes are emerging, which include specific receptors (e.g., GABAB receptors, GPCR, and Trk) linked to signaling pathways (e.g., Trk, ERK/MAPK, orexin) that are associated with synaptic plasticity and neuronal signaling. Studies leveraging the agnostic discovery power of omics and placing it within the context of functional neurobiology will propel us toward much-needed, field-changing breakthroughs, including identification of actionable targets for drug development to treat this devastating brain disease.

PMID:39403933 | DOI:10.1172/JCI172886

F1000Res. 2024 Sep 5;13:781. doi: 10.12688/f1000research.153243.2. eCollection 2024.

ABSTRACT

Casein kinase II subunit alpha (CSNK2A1), a serine/threonine kinase, phosphorylates multiple protein substrates and is involved in diverse cellular and biological processes. Implicated in various human diseases, high-performing antibodies would help evaluate its potential as a therapeutic target and benefit the scientific community. In this study, we have characterized ten CSNK2A1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

PMID:39403680 | PMC:PMC11472280 | DOI:10.12688/f1000research.153243.2

Anal Chem. 2024 Oct 14. doi: 10.1021/acs.analchem.4c04607. Online ahead of print.

ABSTRACT

Extracellular vesicles (EVs), biomimetics, and other biological nanoparticles (BNs) produced from human cells are gaining increasing attention in the fields of molecular diagnostics and nanomedicine for the delivery of therapeutic cargo. In particular, BNs are considered prospective delivery vehicles for different biologics, including protein and RNA therapeutics. Moreover, EVs are widely used in molecular diagnostics for early detection of disease-associated proteins and RNA. Technical approaches for measuring biologics mostly originated from the field of EVs and were later adopted for other BNs, such as extracellular vesicle-mimetic nanovesicles, membrane nanoparticles (nanoghosts), and hybrid nanoparticles, with minimal modifications. Here, we demonstrate that BNs are highly resistant to protocols that severely underestimate the protein and RNA content of BNs, and provide the relevance of these data both for general BNs characterization and practical applications of CRISPR/Cas-based therapies. We demonstrate that the addition of saponin leads to an ∼2- to 7-fold enhancement in protein isolation and an ∼2- to 242-fold improvement in RNA recovery rates and detection efficiency. Differences in the proteolipid contents of BNs, measured by Raman and surface-enhanced Raman spectroscopy, correlate with their susceptibility to saponin treatment for cargo extraction. Finally, we develop a unified protocol using saponin to efficiently isolate proteins and RNA from the BNs. These data demonstrate that previously utilized protocols underestimate BN cargo contents and offer gold standard protocols that can be broadly adopted into the field of nanobiologics, molecular diagnostics, and analytical chemistry.

PMID:39402710 | DOI:10.1021/acs.analchem.4c04607

BMC Biol. 2024 Oct 14;22(1):235. doi: 10.1186/s12915-024-02019-4.

ABSTRACT

BACKGROUND: The transition from explanative modeling of fitted data to the predictive modeling of unseen data for systems biology endeavors necessitates the effective recovery of reaction parameters. Yet, the relative efficacy of optimization algorithms in doing so remains under-studied, as to the specific reaction kinetics and the effect of measurement noises. To this end, we simulate the reactions of an artificial pathway using 4 kinetic formulations: generalized mass action (GMA), Michaelis-Menten, linear-logarithmic, and convenience kinetics. We then compare the effectiveness of 5 evolutionary algorithms (CMAES, DE, SRES, ISRES, G3PCX) for objective function optimization in kinetic parameter hyperspace to determine the corresponding estimated parameters.

RESULTS: We quickly dropped the DE algorithm due to its poor performance. Baring measurement noise, we find the CMAES algorithm to only require a fraction of the computational cost incurred by other EAs for both GMA and linear-logarithmic kinetics yet performing as well by other criteria. However, with increasing noise, SRES and ISRES perform more reliably for GMA kinetics, but at considerably higher computational cost. Conversely, G3PCX is among the most efficacious for estimating Michaelis-Menten parameters regardless of noise, while achieving numerous folds saving in computational cost. Cost aside, we find SRES to be versatilely applicable across GMA, Michaelis-Menten, and linear-logarithmic kinetics, with good resilience to noise. Nonetheless, we could not identify the parameters of convenience kinetics using any algorithm.

CONCLUSIONS: Altogether, we identify a protocol for predicting reaction parameters under marked measurement noise, as a step towards predictive modeling for systems biology endeavors.

PMID:39402553 | DOI:10.1186/s12915-024-02019-4

BMC Genomics. 2024 Oct 14;25(1):957. doi: 10.1186/s12864-024-10859-1.

ABSTRACT

BACKGROUND: Transcription factors (TFs) bind to different parts of the genome in different types of cells, but it is usually assumed that the inherent DNA-binding preferences of a TF are invariant to cell type. Yet, there are several known examples of TFs that switch their DNA-binding preferences in different cell types, and yet more examples of other mechanisms, such as steric hindrance or cooperative binding, that may result in a "DNA signature" of differential binding.

RESULTS: To survey this phenomenon systematically, we developed a deep learning method we call SigTFB (Signatures of TF Binding) to detect and quantify cell-type specificity in a TF's known genomic binding sites. We used ENCODE ChIP-seq data to conduct a wide scale investigation of 169 distinct TFs in up to 14 distinct cell types. SigTFB detected statistically significant DNA binding signatures in approximately two-thirds of TFs, far more than might have been expected from the relatively sparse evidence in prior literature. We found that the presence or absence of a cell-type specific DNA binding signature is distinct from, and indeed largely uncorrelated to, the degree of overlap between ChIP-seq peaks in different cell types, and tended to arise by two mechanisms: using established motifs in different frequencies, and by selective inclusion of motifs for distint TFs.

CONCLUSIONS: While recent results have highlighted cell state features such as chromatin accessibility and gene expression in predicting TF binding, our results emphasize that, for some TFs, the DNA sequences of the binding sites contain substantial cell-type specific motifs.

PMID:39402535 | DOI:10.1186/s12864-024-10859-1

Cell Death Dis. 2024 Oct 14;15(10):748. doi: 10.1038/s41419-024-07121-7.

ABSTRACT

After cutaneous wounds successfully heal, keratinocytes that underwent the epithelial-mesenchymal transition (EMT) regain their epithelial characteristics, while in scar tissue, epidermal cells persist in a mesenchymal state. However, the regulatory mechanisms governing this reversion are poorly understood, and the impact of persistent mesenchymal-like epidermal cells in scar tissue remains unclear. In the present study, we found that during wound healing, the regulatory factor GRHL2 is highly expressed in normal epidermal cells, downregulated in EMT epidermal cells, and upregulated again during the process of mesenchymal-epithelial transition (MET). We further demonstrated that interfering with GRHL2 expression in epidermal cells can effectively induce the EMT. Conversely, the overexpression of GRHL2 in EMT epidermal cells resulted in partial reversion of the EMT to an epithelial state. To investigate the effects of failed MET in epidermal cells on skin wound healing, we interfered with GRHL2 expression in epidermal cells surrounding the cutaneous wound. The results demonstrated that the persistence of epidermal cells in the mesenchymal state promoted fibrosis in scar tissue, manifested by increased thickness of scar tissue, deposition of collagen and fibronectin, as well as the activation of myofibroblasts. Furthermore, the miR-200s/Zeb1 axis was perturbed in GRHL2 knockdown keratinocytes, and transfection with miR-200s analogs promoted the reversion of EMT in epidermal cells, which indicates that they mediate the EMT process in keratinocytes. These results suggest that restoration of the epithelial state in epidermal cells following the EMT is essential to wound healing, providing potential therapeutic targets for preventing scar formation.

PMID:39402063 | DOI:10.1038/s41419-024-07121-7

Clin Genet. 2024 Oct 14. doi: 10.1111/cge.14632. Online ahead of print.

ABSTRACT

Malate is an important dicarboxylic acid produced from fumarate in the tricarboxylic acid cycle. Deficiencies of fumarate hydrolase (FH) and malate dehydrogenase (MDH), responsible for malate formation and metabolism, respectively, are known to cause recessive forms of neurodevelopmental disorders (NDDs). The malic enzyme isoforms, malic enzyme 1 (ME1) and 2 (ME2), are required for the conversion of malate to pyruvate. To date, there have been no reports linking deficiency of either malic enzyme isoforms to any Mendelian disease in humans. We report a patient presenting with NDD, subtle dysmorphic features, resolved dilated cardiomyopathy, and mild blood lactate elevation. Whole exome sequencing (WES) revealed a homozygous frameshift variant (c.1379_1380delTT, p.Phe460fs*22) in the malic enzyme 2 (ME2) gene resulting in truncated and unstable ME2 protein in vitro. Subsequent deletion of the yeast ortholog of human ME2 (hME2) resulted in growth arrest, which was rescued by overexpression of hME2, strongly supporting an important role of ME2 in mitochondrial function. Our results also support the pathogenicity and candidacy of the ME2 gene and variant in association with NDD. To our knowledge, this is the first report of a Mendelian human disease resulting from a biallelic variant in the ME encoding gene. Future studies are warranted to confirm ME2-associated recessive NDD.

PMID:39401966 | DOI:10.1111/cge.14632

Eur Respir J. 2024 Oct 10:2400207. doi: 10.1183/13993003.00207-2024. Online ahead of print.

ABSTRACT

RATIONALE: Lung quantitative computed tomographic (qCT) severe asthma clusters have been reported, but their replication and underlying disease mechanisms are unknown. We identified and replicated qCT clusters of severe asthma in two independent asthma cohorts and determined their association with molecular pathways.

METHODS: We used consensus clustering on qCT measurements of airway and lung CT scans, performed in 105 severe asthmatic adults from the U-BIOPRED cohort. The same qCT measurements were used to replicate qCT clusters in a subsample of the ATLANTIS asthma cohort (n=97). We performed integrated enrichment analysis using blood, sputum, bronchial biopsies, bronchial brushings and nasal brushings transcriptomics and blood and sputum proteomics to characterize radiomultiomic-associated clusters (RACs).

RESULTS: qCT clusters and clinical features in U-BIOPRED were replicated in the matched ATLANTIS cohort. In the U-BIOPRED cohort, RAC1 (n=30) was predominantly female with elevated BMI, mild airflow limitation, normal qCT parameters and upregulation of the complement pathway. RAC2 (n=34) subjects had a lower degree of airflow limitation, airway wall thickness and dilatation, with upregulation of proliferative pathways, including neurotrophic receptor tyrosine kinase 2/tyrosine kinase receptor B (NTRK2/TRKB), and down-regulation of semaphorin pathways. RAC3 (n=41) showed increased lung attenuation area and air trapping, severe airflow limitation, hyperinflation, and upregulation of cytokine signaling and signaling by interleukin pathways, and matrix metallopeptidase 1, 2 and 9.

CONCLUSIONS: U-BIOPRED severe asthma qCT clusters were replicated in a matched independent asthmatic cohort and associated with specific molecular pathways. Radiomultiomics might represent anovel strategy to identify new molecular pathways in asthma pathobiology.

PMID:39401856 | DOI:10.1183/13993003.00207-2024

Adv Sci (Weinh). 2024 Oct 14:e2402421. doi: 10.1002/advs.202402421. Online ahead of print.

ABSTRACT

Protein phosphorylation plays a crucial role in regulating disease phenotypes and serves as a key target for drug development. Mapping nanoscale-to-single-cell samples can unravel the heterogeneity of cellular signaling events. However, it remains a formidable analytical challenge due to the low detectability, abundance, and stoichiometry of phosphorylation sites. Here, we present a Chip-DIA strategy, integrating a microfluidic-based phosphoproteomic chip (iPhosChip) with data-independent acquisition mass spectrometry (DIA-MS) for ultrasensitive nanoscale-to-single-cell phosphoproteomic profiling. The iPhosChip operates as an all-in-one station that accommodates both quantifiable cell capture/imaging and the entire phosphoproteomic workflow in a highly streamlined and multiplexed manner. Coupled with a sample size-comparable library-based DIA-MS strategy, Chip-DIA achieved ultra-high sensitivity, detecting 1076±158 to 15869±1898 phosphopeptides from 10±0 to 1013±4 cells, and revealed the first single-cell phosphoproteomic landscape comprising druggable sites and basal phosphorylation-mediated networks in lung cancer. Notably, the sensitivity and coverage enabled the illumination of heterogeneous cytoskeleton remodeling and cytokeratin signatures in patient-derived cells resistant to third-generation EGFR therapy, stratifying mixed-lineage adenocarcinoma-squamous cell carcinoma subtypes, and identifying alternative targeted therapy for late-stage patients. With flexibility in module design and functionalization, Chip-DIA can be adapted to other PTM-omics to explore dysregulated PTM landscapes, thereby guiding therapeutic strategies toward precision oncology.

PMID:39401432 | DOI:10.1002/advs.202402421

Proc Natl Acad Sci U S A. 2024 Oct 22;121(43):e2405169121. doi: 10.1073/pnas.2405169121. Epub 2024 Oct 14.

ABSTRACT

Hibernation is a widespread and highly efficient mechanism to save energy in mammals. However, one major challenge of hibernation is maintaining blood circulation at low body temperatures, which strongly depends on the viscoelastic properties of red blood cells (RBCs). Here, we examined at physiologically relevant timescales the thermomechanical properties of hundreds of thousands of individual RBCs from the hibernating common noctule bat (Nyctalus noctula), the nonhibernating Egyptian fruit bat (Rousettus aegyptiacus), and humans (Homo sapiens). We exposed RBCs to temperatures encountered during normothermia and hibernation and found a significant increase in elasticity and viscosity with decreasing temperatures. Our data demonstrate that temperature adjustment of RBCs is mainly driven by membrane properties and not the cytosol while viscous dissipation in the membrane of both bat species exceeds the one in humans by a factor of 15. Finally, our results show that RBCs from both bat species reveal a transition to a more viscous-like state when temperature decreases. This process on a minute timescale has an effect size that is comparable with fluctuations in RBC viscoelasticity over the course of the year, implying that environmental factors, such as diets, have a lower impact on the capability of RBCs to respond to different temperatures than general physical properties of the cell membrane. In summary, our findings suggest membrane viscoelasticity as a promising target for identifying mechanisms that could be manipulated to ensure blood circulation at low body temperatures in humans, which may be one first step toward safe synthetic torpor in medicine and space flight.

PMID:39401351 | DOI:10.1073/pnas.2405169121

PLoS Comput Biol. 2024 Oct 14;20(10):e1012522. doi: 10.1371/journal.pcbi.1012522. Online ahead of print.

ABSTRACT

Exquisite binding specificity is essential for many protein functions but is difficult to engineer. Many biotechnological or biomedical applications require the discrimination of very similar ligands, which poses the challenge of designing protein sequences with highly specific binding profiles. Experimental methods for generating specific binders rely on in vitro selection, which is limited in terms of library size and control over specificity profiles. Additional control was recently demonstrated through high-throughput sequencing and downstream computational analysis. Here we follow such an approach to demonstrate the design of specific antibodies beyond those probed experimentally. We do so in a context where very similar epitopes need to be discriminated, and where these epitopes cannot be experimentally dissociated from other epitopes present in the selection. Our approach involves the identification of different binding modes, each associated with a particular ligand against which the antibodies are either selected or not. Using data from phage display experiments, we show that the model successfully disentangles these modes, even when they are associated with chemically very similar ligands. Additionally, we demonstrate and validate experimentally the computational design of antibodies with customized specificity profiles, either with specific high affinity for a particular target ligand, or with cross-specificity for multiple target ligands. Overall, our results showcase the potential of leveraging a biophysical model learned from selections against multiple ligands to design proteins with tailored specificity, with applications to protein engineering extending beyond the design of antibodies.

PMID:39401247 | DOI:10.1371/journal.pcbi.1012522

PLoS One. 2024 Oct 14;19(10):e0284234. doi: 10.1371/journal.pone.0284234. eCollection 2024.

ABSTRACT

The quantity of each protein in a cell only is only partially correlated with its gene transcription rate. Independent influences on protein synthesis levels include mRNA sequence motifs, amino acyl-tRNA synthesis levels, elongation factor action, and protein susceptibility to degradation. Here we report that the amino acid composition of a protein can also influence its expression level in two distinct ways. The nutritional classification of amino acids in animals reflects their potential for scarcity-essential amino acids (EAA) are reliant on dietary supply, non-essential amino acids (NEAA) from internal biosynthesis, and conditionally essential amino acids (CEAA) from both. Accessing public proteomic datasets, we demonstrate that a protein's CEAA sequence composition is inversely correlated with expression-a correlation enhanced during rapid cellular proliferation-suggesting CEAA availability can limit translation. Similarly, proteins with the most extreme compositions of EAA are generally reduced in abundance. These latter proteins participate in biological systems such as taste and food-seeking behaviour, oxidative phosphorylation, and chemokine function, and so linking their expression to EAA availability may act as a homeostatic response to malnutrition. Protein composition can also influence general human phenotypes and disease susceptibility: stature proteins are enriched in CEAAs, and a curated dataset of over 700 cancer proteins is significantly under-represented in EAAs. We also show that individual amino acids can influence protein expression across all kingdoms of life and that this effect appears to be rooted in the unchanging structural and mRNA encoding features of each amino acid. Species-specific environmental survival pathways are shown to be enriched in proteins with individual amino acid compositions favouring higher expression. These two forms of amino acid-driven protein expression regulation promise new insights into systems biology, evolutionary studies, experimental research design, and public health intervention.

PMID:39401228 | DOI:10.1371/journal.pone.0284234