Degradation of FATTY ACID EXPORT PROTEIN1 by RHOMBOID-LIKE PROTEASE11 contributes to cold tolerance in Arabidopsis.

Plants need to acclimate to different stresses to optimize growth under unfavorable conditions. In Arabidopsis (Arabidopsis thaliana), the abundance of the chloroplast envelope protein FATTY ACID EXPORT PROTEIN1 (FAX1) decreases after the onset of low temperatures. However, how FAX1 degradation occurs and whether altered FAX1 abundance contributes to cold tolerance in plants remains unclear. The rapid cold-induced increase in RHOMBOID-LIKE PROTEASE11 (RBL11) transcript levels, the physical interaction of RBL11 with FAX1, the specific FAX1 degradation after RBL11 expression, and the absence of cold-induced FAX1 degradation in rbl11 loss-of-function mutants suggest that this enzyme is responsible for FAX1 degradation. Proteomic analyses showed that rbl11 mutants have higher levels of FAX1 and other proteins involved in membrane lipid homeostasis, suggesting that RBL11 is a key element in the remodeling of membrane properties during cold conditions. Consequently, in the cold, rbl11 mutants show a shift in lipid biosynthesis toward the eukaryotic pathway, which coincides with impaired cold tolerance. To test whether cold sensitivity is due to increased FAX1 levels, we analyzed FAX1 overexpressors. The rbl11 mutants and FAX1 overexpressor lines show superimposable phenotypic defects upon exposure to cold temperatures. Our re-sults show that the cold-induced degradation of FAX1 by RBL11 is critical for Arabidop-sis to survive cold and freezing periods.

PLANTdataHUB: a collaborative platform for continuous FAIR data sharing in plant research.

In modern reproducible, hypothesis-driven plant research, scientists are increasingly relying on research data management (RDM) services and infrastructures to streamline the processes of collecting, processing, sharing, and archiving research data. FAIR (i.e., findable, accessible, interoperable, and reusable) research data play a pivotal role in enabling the integration of interdisciplinary knowledge and facilitating the comparison and synthesis of a wide range of analytical findings. The PLANTdataHUB offers a solution that realizes RDM of scientific (meta)data as evolving collections of files in a directory - yielding FAIR digital objects called ARCs - with tools that enable scientists to plan, communicate, collaborate, publish, and reuse data on the same platform while gaining continuous quality control insights. The centralized platform is scalable from personal use to global communities and provides advanced federation capabilities for institutions that prefer to host their own satellite instances. This approach borrows many concepts from software development and adapts them to fit the challenges of the field of modern plant science undergoing digital transformation. The PLANTdataHUB supports researchers in each stage of a scientific project with adaptable continuous quality control insights, from the early planning phase to data publication. The central live instance of PLANTdataHUB is accessible at (https://git.nfdi4plants.org), and it will continue to evolve as a community-driven and dynamic resource that serves the needs of contemporary plant science.

A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP-modulated NFAT signaling.

Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca2+ entry (SOCE) triggering activation of transcription factors. Here, we characterize Stim1A, a splice variant with an additional 31 amino acid domain inserted in frame within its cytosolic domain. Prominent expression of exon A is found in astrocytes, heart, kidney, and testes. Full-length Stim1A functions as a dominant-negative regulator of SOCE and ICRAC, facilitating sequence-specific fast calcium-dependent inactivation and destabilizing gating of Orai channels. Downregulation or absence of native Stim1A results in increased SOCE. Despite reducing SOCE, Stim1A leads to increased NFAT translocation. Differential proteomics revealed an interference of Stim1A with the cAMP-SOCE crosstalk by altered modulation of phosphodiesterase 8 (PDE8), resulting in reduced cAMP degradation and increased PIP5K activity, facilitating NFAT activation. Our study uncovers a hitherto unknown mechanism regulating NFAT activation and indicates that cell-type-specific splicing of Stim1 is a potent means to regulate the NFAT signalosome and cAMP-SOCE crosstalk.

DeepSTABp: A Deep Learning Approach for the Prediction of Thermal Protein Stability.

Proteins are essential macromolecules that carry out a plethora of biological functions. The thermal stability of proteins is an important property that affects their function and determines their suitability for various applications. However, current experimental approaches, primarily thermal proteome profiling, are expensive, labor-intensive, and have limited proteome and species coverage. To close the gap between available experimental data and sequence information, a novel protein thermal stability predictor called DeepSTABp has been developed. DeepSTABp uses a transformer-based protein language model for sequence embedding and state-of-the-art feature extraction in combination with other deep learning techniques for end-to-end protein melting temperature prediction. DeepSTABp can predict the thermal stability of a wide range of proteins, making it a powerful and efficient tool for large-scale prediction. The model captures the structural and biological properties that impact protein stability, and it allows for the identification of the structural features that contribute to protein stability. DeepSTABp is available to the public via a user-friendly web interface, making it accessible to researchers in various fields.

iMLP, a predictor for internal matrix targeting-like sequences in mitochondrial proteins.

Matrix targeting sequences (MTSs) direct proteins from the cytosol into mitochondria. Efficient targeting often relies on internal matrix targeting-like sequences (iMTS-Ls) which share structural features with MTSs. Predicting iMTS-Ls was tedious and required multiple tools and webservices. We present iMLP, a deep learning approach for the prediction of iMTS-Ls in protein sequences. A recurrent neural network has been trained to predict iMTS-L propensity profiles for protein sequences of interest. The iMLP predictor considerably exceeds the speed of existing approaches. Expanding on our previous work on iMTS-L prediction, we now serve an intuitive iMLP webservice available at http://iMLP.bio.uni-kl.de and a stand-alone command line tool for power user in addition.

Identification of Chloroplast Envelope Proteins with Critical Importance for Cold Acclimation.

The ability of plants to withstand cold temperatures relies on their photosynthetic activity. Thus, the chloroplast is of utmost importance for cold acclimation and acquisition of freezing tolerance. During cold acclimation, the properties of the chloroplast change markedly. To provide the most comprehensive view of the protein repertoire of the chloroplast envelope, we analyzed this membrane system in Arabidopsis (Arabidopsis thaliana) using mass spectrometry-based proteomics. Profiling chloroplast envelope membranes was achieved by a cross comparison of protein intensities across the plastid and the enriched membrane fraction under both normal and cold conditions. We used multivariable logistic regression to model the probabilities for the classification of an envelope localization. In total, we identified 38 envelope membrane intrinsic or associated proteins exhibiting altered abundance after cold acclimation. These proteins comprise several solute carriers, such as the ATP/ADP antiporter nucleotide transporter2 (NTT2; substantially increased abundance) or the maltose exporter MEX1 (substantially decreased abundance). Remarkably, analysis of the frost recovery of ntt loss-of-function and mex1 overexpressor mutants confirmed that the comparative proteome is well suited to identify key factors involved in cold acclimation and acquisition of freezing tolerance. Moreover, for proteins with known physiological function, we propose scenarios explaining their possible roles in cold acclimation. Furthermore, spatial proteomics introduces an additional layer of complexity and enables the identification of proteins differentially localized at the envelope membrane under the changing environmental regime.

Differential degradation of RNA species by autophagy-related pathways in Arabidopsis.

The plant vacuole recycles proteins and RNA delivered to it by autophagy. In this study, by isolating intact vacuoles from Arabidopsis plants, followed by subsequent RNA purification, and deep sequencing, we provide a comprehensive characterization of Arabidopsis vacuolar RNAome. In the vacuolar RNAome, we detected ribosomal RNAs, transfer RNAs, including those of chloroplast origin, and in addition small RNA types. As autophagy is a main mechanism for the transport of RNA to the vacuole, atg5-1 mutants deficient in autophagy were included in our analysis. We observed severely reduced amounts of most chloroplast-derived RNA species in these mutants. Comparisons with cellular RNA composition provided an indication of possible up-regulation of alternative RNA breakdown pathways. By contrast, vacuolar RNA processing and composition in plants lacking vacuolar ribonuclease 2, involved in cellular RNA homeostasis, only showed minor alterations, possibly because of the presence of further so far unknown vacuolar RNase species. Among the small RNA types, we detected mature miRNAs in all vacuolar preparations but at much lower frequency in atg5-1, raising the possibility of a biological role for vacuolar miRNAs.

Did the ACA's "guaranteed issue" provision cause adverse selection into nongroup insurance? Analysis using a copula-based hurdle model.

Prior to the Affordable Care Act (ACA), insurance companies could charge higher premiums, or outright deny coverage, to people with preexisting health problems. But the ACA's "guaranteed issue" provision forbids such price discrimination and denials of coverage. This paper seeks to determine whether, after implementation of the ACA, nongroup private insurance plans have experienced adverse selection. Our empirical approach employs a copula-based hurdle regression model, with dependence modeled as a function of dimensions along which adverse selection might occur. Our main finding is that, after implementation of the ACA, nongroup insurance enrollees with preexisting health problems do not appear to exhibit adverse selection. This finding suggests that the ACA's mandate that everyone acquire coverage might have attracted enough healthy enrollees to offset any adverse selection.

Management of a Pediatric Type 3C Open Femoral Fracture Following a High-Velocity Gunshot Wound at an Adult Level II Trauma Center.

ABSTRACT: We present a case of a 10-year-old girl shot in the thigh by a stray bullet who had a favorable outcome when treated with a multidisciplinary approach at the nearest nonpediatric level II trauma center. Point-of-care thromboelastography facilitated effective resuscitation based on her coagulation profile, minimized blood product use, and allowed for damage-control surgery to stabilize and revascularize her complex femur fracture.

The Chlamydomonas deg1c Mutant Accumulates Proteins Involved in High Light Acclimation.

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent Ser endopeptidases that perform key aspects of protein quality control in all domains of life. Here, we characterized Chlamydomonas reinhardtii DEG1C, which together with DEG1A and DEG1B is orthologous to Arabidopsis (Arabidopsis thaliana) Deg1 in the thylakoid lumen. We show that DEG1C is localized to the stroma and the periphery of thylakoid membranes. Purified DEG1C exhibited high proteolytic activity against unfolded model substrates and its activity increased with temperature and pH. DEG1C forms monomers, trimers, and hexamers that are in dynamic equilibrium. DEG1C protein levels increased upon nitrogen, sulfur, and phosphorus starvation; under heat, oxidative, and high light stress; and when Sec-mediated protein translocation was impaired. DEG1C depletion was not associated with any obvious aberrant phenotypes under nonstress conditions, high light exposure, or heat stress. However, quantitative shotgun proteomics revealed differences in the abundance of 307 proteins between a deg1c knock-out mutant and the wild type under nonstress conditions. Among the 115 upregulated proteins are PSII biogenesis factors, FtsH proteases, and proteins normally involved in high light responses, including the carbon dioxide concentrating mechanism, photorespiration, antioxidant defense, and photoprotection. We propose that the lack of DEG1C activity leads to a physiological state of the cells resembling that induced by high light intensities and therefore triggers high light protection responses.

The Role of Plastidic Trigger Factor Serving Protein Biogenesis in Green Algae and Land Plants.

Biochemical processes in chloroplasts are important for virtually all life forms. Tight regulation of protein homeostasis and the coordinated assembly of protein complexes, composed of both imported and locally synthesized subunits, are vital to plastid functionality. Protein biogenesis requires the action of cotranslationally acting molecular chaperones. One such chaperone is trigger factor (TF), which is known to cotranslationally bind most newly synthesized proteins in bacteria, thereby assisting their correct folding and maturation. However, how these processes are regulated in chloroplasts remains poorly understood. We report here functional investigation of chloroplast-localized TF (TIG1) in the green alga (Chlamydomonas reinhardtii) and the vascular land plant Arabidopsis (Arabidopsis thaliana). We show that chloroplastic TIG1 evolved as a specialized chaperone. Unlike other plastidic chaperones that are functionally interchangeable with their prokaryotic counterpart, TIG1 was not able to complement the broadly acting ortholog in Escherichia coli. Whereas general chaperone properties such as the prevention of aggregates or substrate recognition seems to be conserved between bacterial and plastidic TFs, plant TIG1s differed by associating with only a relatively small population of translating ribosomes. Furthermore, a reduction of plastidic TIG1 levels leads to deregulated protein biogenesis at the expense of increased translation, thereby disrupting the chloroplast energy household. This suggests a central role of TIG1 in protein biogenesis in the chloroplast.

Characterization of filament-forming CTP synthases from Arabidopsis thaliana.

Cytidine triphosphate (CTP) is essential for DNA, RNA and phospholipid biosynthesis. De novo synthesis is catalyzed by CTP synthases (CTPS). Arabidopsis encodes five CTPS isoforms that unanimously share conserved motifs found across kingdoms, suggesting all five are functional enzymes. Whereas CTPS1-4 are expressed throughout Arabidopsis tissues, CTPS5 reveals exclusive expression in developing embryos. CTPS activity and substrates affinities were determined for a representative plant enzyme on purified recombinant CTPS3 protein. As demonstrated in model organisms such as yeast, fruit fly and mammals, CTPS show the capacity to assemble into large filaments called cytoophidia. Transient expression of N- and C-terminal YFP-CTPS fusion proteins in Nicotiana benthamiana allowed to monitor such filament formation. Interestingly, CTPS1 and 2 always appeared as soluble proteins, whereas filaments were observed for CTPS3, 4 and 5 independent of the YFP-tag location. However, when similar constructs were expressed in Saccharomyces cerevisiae, no filaments were observed, pointing to a requirement for organism-specific factors in vivo. Indications for filament assembly were also obtained in vitro when recombinant CTPS3 protein was incubated in the presence of CTP. T-DNA-insertion mutants in four CTPS loci revealed no apparent phenotypical alteration. In contrast, CTPS2 T-DNA-insertion mutants did not produce homozygous progenies. An initial characterization of the CTPS protein family members from Arabidopsis is presented. We provide evidence for their involvement in nucleotide de novo synthesis and show that only three of the five CTPS isoforms were able to form filamentous structures in the transient tobacco expression system. This represents a striking difference from previous observations in prokaryotes, yeast, Drosophila and mammalian cells. This finding will be highly valuable to further understand the role of filament formation to regulate CTPS activity.

Use of Viscoelastography in Malignancy-Associated Coagulopathy and Thrombosis: A Review.

The relationship between malignancy and coagulopathy is one that is well documented yet incompletely understood. Clinicians have attempted to quantify the hypercoagulable state produced in various malignancies using common coagulation tests such as prothrombin time, activated partial thromboplastin time, and platelet count; however, due to these tests' focus on individual aspects of coagulation during one specific time point, they have failed to provide clinicians the complete picture of malignancy-associated coagulopathy (MAC). Viscoelastic tests (VETs), such as thromboelastography (TEG) and rotational thromboelastometry (ROTEM), are whole blood analyses that have the advantage of providing information related to the cumulative effects of plasma clotting factors, platelets, leukocytes, and red cells during all stages of the coagulation and fibrinolytic processes. VETs have gained popularity in the care of trauma patients to objectively measure trauma-induced coagulopathy (TIC), but the utility of VETs remains yet unrealized in many other medical specialties. The authors discuss the similarities and differences between TIC and MAC, and propose a mechanism for the hypercoagulable state of MAC that revolves around the thrombomodulin-thrombin complex as it switches between activating the protein C anticoagulation pathway or the thrombin activatable fibrinolysis inhibitor coagulation pathway. Additionally, they review the current literature on the use of TEG and ROTEM in patients with various malignancies. Although limited research is currently available, early results demonstrate the utility of both TEG and ROTEM in the prediction of hypercoagulable states and thromboembolic complications in oncologic patients.

Investigations on VELVET regulatory mutants confirm the role of host tissue acidification and secretion of proteins in the pathogenesis of Botrytis cinerea.

The Botrytis cinerea VELVET complex regulates light-dependent development and virulence. The goal of this study was to identify common virulence defects of several VELVET mutants and to reveal their molecular basis. Growth, differentiation, physiology, gene expression and infection of fungal strains were analyzed, and quantitative comparisons of in planta transcriptomes and secretomes were performed. VELVET mutants showed reduced release of citric acid, the major acid secreted by the wild-type, whereas no significant role for oxalic acid was observed. Furthermore, a common set of infection-related and secreted proteins was strongly underexpressed in the mutants. Quantitative secretome analysis with 15 N metabolic labeling revealed a correlation of changes in protein and mRNA levels between wild-type and mutants, indicating that transcript levels determine the abundance of secreted proteins. Infection sites kept at low pH partially restored lesion expansion and expression of virulence genes by the mutants. Drastic downregulation of proteases in the mutants was correlated with incomplete degradation of cellular host proteins at the infection site, but no evidence was obtained that aspartyl proteases are required for lesion formation. The B. cinerea VELVET complex controls pathogenic differentiation by regulating organic acid secretion, host tissue acidification, gene expression and protein secretion.

Using copulas to estimate the coefficient of a binary endogenous regressor in a Poisson regression: Application to the effect of insurance on doctor visits.

This paper presents a copula-based method for identifying and estimating the coefficient of a binary endogenous regressor in a Poisson regression. The method offers advantages over existing approaches. Most importantly, it relies upon standard maximum likelihood approaches, and it does not require numerical integration. Further, as part of its implementation, the method provides a convenient test for the presence of endogeneity. The empirical application investigates the effect of insurance status (a binary measure) on doctor visits (a count measure).

The Hancock Alkaloids (-)-Cuspareine, (-)-Galipinine, (-)-Galipeine, and (-)-Angustureine: Asymmetric Syntheses and Corrected 1H and 13C NMR Data.

The asymmetric syntheses of all members of the Hancock alkaloid family based upon a 2-substituted N-methyl-1,2,3,4-tetrahydroquinoline core are delineated. The conjugate addition of enantiopure lithium N-benzyl- N-(α-methyl- p-methoxybenzyl)amide to 5-( o-bromophenyl)- N-methoxy- N-methylpent-2-enamide is used to generate the requisite C-2 stereogenic center of the targets, while an intramolecular Buchwald-Hartwig coupling is used to form the 1,2,3,4-tetrahydroquinoline ring. Late-stage diversification completes construction of the C-2 side chains. Thus, (-)-cuspareine, (-)-galipinine, (-)-galipeine, and (-)-angustureine were prepared in overall yields of 30%, 28%, 15%, and 39%, respectively, in nine steps from commercially available 3-( o-bromophenyl)propanoic acid in all cases. Unambiguously corrected 1H and 13C NMR data for the originally isolated samples of (-)-cuspareine, (-)-galipinine, and (-)-angustureine are also reported, representing a valuable reference resource for these popular synthetic targets.

Cost-offsets of prescription drug expenditures: data analysis via a copula-based bivariate dynamic hurdle model.

In this paper, we estimate a copula-based bivariate dynamic hurdle model of prescription drug and nondrug expenditures to test the cost-offset hypothesis, which posits that increased expenditures on prescription drugs are offset by reductions in other nondrug expenditures. We apply the proposed methodology to data from the Medical Expenditure Panel Survey, which have the following features: (i) the observed bivariate outcomes are a mixture of zeros and continuously measured positives; (ii) both the zero and positive outcomes show state dependence and inter-temporal interdependence; and (iii) the zeros and the positives display contemporaneous association. The point mass at zero is accommodated using a hurdle or a two-part approach. The copula-based approach to generating joint distributions is appealing because the contemporaneous association involves asymmetric dependence. The paper studies samples categorized by four health conditions: arthritis, diabetes, heart disease, and mental illness. There is evidence of greater than dollar-for-dollar cost-offsets of expenditures on prescribed drugs for relatively low levels of spending on drugs and less than dollar-for-dollar cost-offsets at higher levels of drug expenditures.

Ionizable Cationic Lipids and Helper Lipids Synergistically Contribute to RNA Packing and Protection in Lipid-Based Nanomaterials.

Lipid-based nanomaterials are used as a common delivery vehicle for RNA therapeutics. They typically include a formulation containing ionizable cationic lipids, cholesterol, phospholipids, and a small molar fraction of PEGylated lipids. The ionizable cationic lipids are considered a crucial element of the formulation for the way they mediate interactions with the anionic RNA as a function of pH. Here, we show, by means of molecular dynamics simulation of lipid formulations containing two different ionizable cationic lipids (DLinDMA and DLinDAP), that the direct interactions of those lipids with RNA, taken alone, may not be sufficient to determine the level of protection and packaging of mRNA. Our simulations help and highlight how the collective behavior of the lipids in the formulation, which determines the ability to envelop the RNA, and the level of hydration of the lipid-RNA interface may also play a significant role. This allows the drawing of a hypothesis about the experimentally observed differences in the transfection efficiency of the two ionizable cationic lipids.

Lead optimisation of OXS007417: in vivo PK profile and hERG liability modulation to optimise a small molecule differentiation agent for the potential treatment of acute myeloid leukaemia.

The development of a safe, efficacious, and widely effective differentiation therapy for AML would dramatically improve the outlook for many patients worldwide. To this aim, our laboratory has discovered a class of differentiation agents that demonstrate tumour regression in murine models in vivo. Herein, we report a lead optimisation process around compound OXS007417, which led to improved potency, solubility, metabolic stability, and off-target toxicity of this compound class. A hERG liability was investigated and successfully alleviated through addition of nitrogen atoms into key positions of the compound. OXS008255 and OXS008474 demonstrated an improved murine PK profile in respect to OXS007417 and a delay in tumour growth in a subcutaneous in vivo model using HL-60 cells.

A prognostic neural epigenetic signature in high-grade glioma.

Neural-tumor interactions drive glioma growth as evidenced in preclinical models, but clinical validation is limited. We present an epigenetically defined neural signature of glioblastoma that independently predicts patients' survival. We use reference signatures of neural cells to deconvolve tumor DNA and classify samples into low- or high-neural tumors. High-neural glioblastomas exhibit hypomethylated CpG sites and upregulation of genes associated with synaptic integration. Single-cell transcriptomic analysis reveals a high abundance of malignant stemcell-like cells in high-neural glioblastoma, primarily of the neural lineage. These cells are further classified as neural-progenitor-cell-like, astrocyte-like and oligodendrocyte-progenitor-like, alongside oligodendrocytes and excitatory neurons. In line with these findings, high-neural glioblastoma cells engender neuron-to-glioma synapse formation in vitro and in vivo and show an unfavorable survival after xenografting. In patients, a high-neural signature is associated with decreased overall and progression-free survival. High-neural tumors also exhibit increased functional connectivity in magnetencephalography and resting-state magnet resonance imaging and can be detected via DNA analytes and brain-derived neurotrophic factor in patients' plasma. The prognostic importance of the neural signature was further validated in patients diagnosed with diffuse midline glioma. Our study presents an epigenetically defined malignant neural signature in high-grade gliomas that is prognostically relevant. High-neural gliomas likely require a maximized surgical resection approach for improved outcomes.