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DEEP picker is a deep neural network for accurate deconvolution of complex two-dimensional NMR spectra
The analysis of nuclear magnetic resonance (NMR) spectra for the comprehensive and unambiguous identification and characterization of peaks is a difficult, but critically important step in all NMR analyses of complex biological molecular systems. Here, we introduce DEEP Picker, a deep neural network (DNN)-based approach for peak picking and spectral deconvolution which semi-automates the analysis of two-dimensional NMR spectra. DEEP Picker includes 8 hidden convolutional layers and was trained on a large number of synthetic spectra of known composition with variable degrees of crowdedness. We show that our method is able to correctly identify overlapping peaks, including ones that are challenging for expert spectroscopists and existing computational methods alike. We demonstrate the utility of DEEP Picker on NMR spectra of folded and intrinsically disordered proteins as well as a complex metabolomics mixture, and show how it provides access to valuable NMR information. DEEP Picker should facilitate the semi-automation and standardization of protocols for better consistency and sharing of results within the scientific community.
The analysis of NMR spectra of complex biochemical samples with respect to individual resonances is challenging but critically important. Here, the authors present a deep learning-based method that accelerates this process also for crowded NMR data that are non-trivial to analyze, even by expert NMR spectroscopists.
Journal Article
Genome-wide analysis of coding and non-coding RNA reveals a conserved miR164-NAC regulatory pathway for fruit ripening
Kiwifruit (Actinidia spp.) is a climacteric fruit with high sensitivity to ethylene, influenced by multiple ethylene-responsive structural genes and transcription factors. However, the roles of other post-transcriptional regulators (e.g. miRNAs) necessary for ripening remain elusive.
High-throughput sequencing sRNAome, degradome and transcriptome methods were used to identify further contributors to ripening control in the kiwifruit (A. deliciosa cv ‘Hayward’).
Two NAM/ATAF/CUC domain transcription factors (AdNAC6 and AdNAC7), both predicted targets for miR164, showed significant upregulation by exogenous ethylene. Gene expression analysis and luciferase reporter assays indicated that Ade-miR164 and one of its precursor miRNAs (Ade-MIR164b) were repressed by ethylene treatment and negatively correlated with AdNAC6/7 expression. Subsequent analysis indicated that both AdNAC6 and AdNAC7 proteins are transcriptional activators and physically bind the promoters of AdACS1 (1-aminocyclopropane-1-carboxylate synthase), AdACO1 (1-aminocyclopropane-1-carboxylic acid oxidase), AdMAN1 (endo-β-mannanase) and AaTPS1 (terpene synthase). Moreover, subcellular analysis indicated that the location of the AdNAC6/7 proteins was influenced by Ade-miR164.
Multiple omics-based approaches revealed a novel regulatory link for fruit ripening that involved ethylene-miR164-NAC. The regulatory pathway for miR164-NAC is present in various fruit (e.g. Rosaceae fruit, citrus, grape), with implications for fruit ripening regulation.
Journal Article
Occurrence of plastidial triacylglycerol synthesis and the potential regulatory role of AGPAT in the model diatom Phaeodactylum tricornutum
Background Microalgae have emerged as a potential feedstock for biofuels and bioactive components. However, lack of microalgal strains with promising triacylglycerol (TAG) content and desirable fatty acid composition have hindered its commercial feasibility. Attempts on lipid overproduction by metabolic engineering remain largely challenging in microalgae. Results In this study, a microalgal 1-acyl-sn-glycerol-3-phosphate acyltransferase designated AGPAT1 was identified in the model diatom Phaeodactylum tricornutum. AGPAT1 contained four conserved acyltransferase motifs I-IV. Subcellular localization prediction and thereafter immuno-electron microscopy revealed the localization of AGPAT1 to plastid membranes. AGPAT1 overexpression significantly altered the primary metabolism, with increased total lipid content but decreased content of total carbohydrates and soluble proteins. Intriguingly, AGPAT1 overexpression coordinated the expression of other key genes such as DGAT2 and GPAT involved in TAG synthesis, and consequently increased TAG content by 1.81-fold with a significant increase in polyunsaturated fatty acids, particularly EPA and DHA. Moreover, besides increased lipid droplets in the cytosol, ultrastructural observation showed a number of TAG-rich plastoglobuli formed in plastids. Conclusion The results suggested that AGPAT1 overexpression could elevate TAG biosynthesis and, moreover, revealed the occurrence of plastidial TAG synthesis in the diatom. Overall, our data provide a new insight into microalgal lipid metabolism and candidate target for metabolic engineering.
Journal Article
Molecular characterization of a glycerol-3-phosphate acyltransferase reveals key features essential for triacylglycerol production in Phaeodactylum tricornutum
Background The marine diatom, Phaeodactylum tricornutum, has become a model for studying lipid metabolism and its triacylglycerol (TAG) synthesis pathway makes it an ideal target for metabolic engineering to improve lipid productivity. However, the genetic background and metabolic networks of fatty acid biosynthesis in diatoms are not well understood. Glycerol-3-phosphate acyltransferase (GPAT) is the critical enzyme that catalyzes the first step of TAG formation. So far, characterization of GPAT in marine microalgae has not been reported, especially at the level of comprehensive sequence-structure and functional analysis. Results A GPAT was cloned from P. tricornutum and overexpressed in P. tricornutum. Volumes of oil bodies were produced and the neutral lipid content was increased by twofold determined by Nile red fluorescence staining. Fatty acid composition was analyzed by GC-MS, which showed significantly higher proportion of unsaturated fatty acids compared to wild type. Conclusion These results suggested that the identified GPAT could upregulate TAG biosynthesis in P. tricornutum. Moreover, this study offers insight into the lipid metabolism of diatoms and supports the role of microalgal strains for biofuels production.
Journal Article
Association of p53 with Neurodegeneration in Parkinson’s Disease
p53 is a vital transcriptional protein implicated in regulating diverse cellular processes, including cell cycle arrest, DNA repair, mitochondrial metabolism, redox homeostasis, autophagy, senescence, and apoptosis. Recent studies have revealed that p53 levels and activity are substantially increased in affected neurons in cellular and animal models of Parkinson’s disease (PD) as well as in the brains of PD patients. p53 activation in response to neurodegenerative stress is closely associated with the degeneration of dopaminergic neurons accompanied by mitochondrial dysfunction, reactive oxygen species (ROS) production, abnormal protein aggregation, and impairment of autophagy, and these pathogenic events have been implicated in the pathogenesis of PD. Pathogenic p53 integrates diverse cellular stresses and activate these downstream events to induce the degeneration of dopaminergic neurons; thus, it plays a crucial role in the pathogenesis of PD and appears to be a potential target for the treatment of the disease. We reviewed the current knowledge concerning p53-dependent neurodegeneration to better understand the underlying mechanisms and provide possible strategies for PD treatment by targeting p53.
Journal Article
Neovascularization and tissue regeneration by endothelial progenitor cells in ischemic stroke
Abstract Endothelial progenitor cells (EPCs) are immature endothelial cells (ECs) capable of proliferating and differentiating into mature ECs. These progenitor cells migrate from bone marrow (BM) after vascular injury to ischemic areas, where they participate in the repair of injured endothelium and new blood vessel formation. EPCs also secrete a series of protective cytokines and growth factors that support cell survival and tissue regeneration. Thus, EPCs provide novel and promising potential therapies to treat vascular disease, including ischemic stroke. However, EPCs are tightly regulated during the process of vascular repair and regeneration by numerous endogenous cytokines that are associated closely with the therapeutic efficacy of the progenitor cells. The regenerative capacity of EPCs also is affected by a range of exogenous factors and drugs as well as vascular risk factors. Understanding the functional properties of EPCs and the factors related to their regenerative capacity will facilitate better use of these progenitor cells in treating vascular disease. Here, we review the current knowledge of EPCs in cerebral neovascularization and tissue regeneration after cerebral ischemia and the factors associated with their regenerative function to better understand the underlying mechanisms and provide more effective strategies for the use of EPCs in treating ischemic stroke.
Journal Article
Improvement of Neutral Lipid and Polyunsaturated Fatty Acid Biosynthesis by Overexpressing a Type 2 Diacylglycerol Acyltransferase in Marine Diatom Phaeodactylum tricornutum
Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the last step of triacylglyceride (TAG) biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.
Journal Article
Systematic dissection of genomic features determining the vast diversity of conotoxins
Background
Conus
, a highly diverse species of venomous predators, has attracted significant attention in neuroscience and new drug development due to their rich collection of neuroactive peptides called conotoxins. Recent advancements in transcriptome, proteome, and genome analyses have facilitated the identification of conotoxins within
Conus
’ venom glands, providing insights into the genetic features and evolutionary patterns of conotoxin genes. However, the underlying mechanism behind the extraordinary hypervariability of conotoxins remains largely unknown.
Results
We analyzed the transcriptomes of 34
Conus
species, examining various tissues such as the venom duct, venom bulb, and salivary gland, leading to the identification of conotoxin genes. Genetic variation analysis revealed that a subset of these genes (15.78% of the total) in
Conus
species underwent positive selection (Ka/Ks > 1,
p
< 0.01). Additionally, we reassembled and annotated the genome of
C. betulinus
, uncovering 221 conotoxin-encoding genes. These genes primarily consisted of three exons, with a significant portion showing high transcriptional activity in the venom ducts. Importantly, the flanking regions and adjacent introns of conotoxin genes exhibited a higher prevalence of transposon elements, suggesting their potential contribution to the extensive variability observed in conotoxins. Furthermore, we detected genome duplication in
C. betulinus
, which likely contributed to the expansion of conotoxin gene numbers. Interestingly, our study also provided evidence of introgression among
Conus
species, indicating that interspecies hybridization may have played a role in shaping the evolution of diverse conotoxin genes.
Conclusions
This study highlights the impact of adaptive evolution and introgressive hybridization on the genetic diversity of conotoxin genes and the evolution of
Conus
. We also propose a hypothesis suggesting that transposable elements might significantly contribute to the remarkable diversity observed in conotoxins. These findings not only enhance our understanding of peptide genetic diversity but also present a novel approach for peptide bioengineering.
Journal Article
Heat shock transcription factor-mediated thermal tolerance and cell size plasticity in marine diatoms
Diatoms are a crucial component of marine ecosystems, recognized for their broad environmental adaptability and wide temperature tolerance. However, the molecular mechanisms underlying their adaptability to diverse temperatures are unknown. In this study, we discover that heat shock transcription factors (HSFs) are potentially important for thermal tolerance in diatoms. Our study focuses on PtHSF2, annotated as HSF2 in
Phaeodactylum tricornutum
’s genome, which is ubiquitous in diatoms. Overexpression of PtHSF2 markedly enhances thermal tolerance and increases cell size; causes significant differential expression of several genes, including cell division cycle protein 45-like (
PtCdc45-like
),
ATM
(ataxia telangiectasia mutated),
ATR
(ataxia telangiectasia and Rad3-related), light-harvesting complex protein 2 (
Lhcx2
), and fatty acid desaturase. Cleavage Under Targets and Tagmentation (CUT&Tag) and CUT&Tag-qPCR analyses demonstrate that PtHSF2 directly targets and upregulates
PtCdc45-like
and
Lhcx2
while downregulating ATP-binding cassette transporter. Functional validation of
PtCdc45-like
shows that its overexpression results in larger cell size, enhances antioxidant capacity, and improves cell survival at elevated temperatures. Collectively, our findings elucidate the molecular mechanism by which PtHSF2 mediates high-temperature tolerance in diatoms and validate the functions of its target gene
PtCdc45-like
. These results highlight the importance of HSFs in diatom temperature adaptation and provide insights into temperature acclimation in microalgae.
Diatoms are crucial marine microorganisms, but the molecular mechanisms underlying their temperature tolerance remain unclear. This study identifies the heat shock transcription factor PtHSF2 as a key regulator of thermal tolerance and cell size plasticity in Phaeodactylum tricornutum, revealing its role in gene regulation and stress response.
Journal Article