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.

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.

Proteomics and constraint-based modelling reveal enzyme kinetic properties of Chlamydomonas reinhardtii on a genome scale.

Metabolic engineering of microalgae offers a promising solution for sustainable biofuel production, and rational design of engineering strategies can be improved by employing metabolic models that integrate enzyme turnover numbers. However, the coverage of turnover numbers for Chlamydomonas reinhardtii, a model eukaryotic microalga accessible to metabolic engineering, is 17-fold smaller compared to the heterotrophic cell factory Saccharomyces cerevisiae. Here we generate quantitative protein abundance data of Chlamydomonas covering 2337 to 3708 proteins in various growth conditions to estimate in vivo maximum apparent turnover numbers. Using constrained-based modeling we provide proxies for in vivo turnover numbers of 568 reactions, representing a 10-fold increase over the in vitro data for Chlamydomonas. Integration of the in vivo estimates instead of in vitro values in a metabolic model of Chlamydomonas improved the accuracy of enzyme usage predictions. Our results help in extending the knowledge on uncharacterized enzymes and improve biotechnological applications of Chlamydomonas.

Reconstruction After Skin Cancer Resection of the Head and Neck.

Skin cancer defects of the head and neck region can vary greatly in their complexity. Reconstructive surgeons are tasked with maintaining or restoring function as well as providing an excellent aesthetic outcome. This article provides an overview of reconstructive options following skin cancer resection broken down by different aesthetic regions and subunits. Although not meant to serve as an all-encompassing source, it provides common indications for using different sections of the reconstructive ladder based on location of defects, tissues involved, and patient factors.

Utilizing Dynamic Risk Stratification in Patients With Tall Cell Variant Papillary Thyroid Cancer.

OBJECTIVES: Tall cell variant (TCV) papillary thyroid cancer (PTC) is a subtype of PTC associated with aggressive tumor behavior, advanced stage, and higher rates of recurrence and mortality. The present study aimed to test an established dynamic risk stratification tool in the TCV population, with the goal of better predicting the postoperative course of these patients. STUDY DESIGN: Retrospective chart review. METHODS: A total of 94 patients with TCV who underwent total thyroidectomy with radioactive iodine ablation were retrospectively reviewed from 1998 through 2020. Biochemical, structural, and overall response to treatment was determined for each patient, based on postoperative thyroglobulin levels and imaging findings. Primary outcomes were locoregional and distant recurrence, presence of disease at final follow-up, need for additional intervention, and disease-specific mortality. RESULTS: Patients with TCV who were stratified as having an excellent overall response to treatment had lower rates of locoregional recurrence than indeterminate, biochemical incomplete, and structural incomplete responses (2.0%, 33.3%, 55.0%, and 85.7% at 5 years respectively, p < 0.001). The same was true for distant recurrence as well (2.0%, 9.0%, 35.1%, and 42.9%, p < 0.001). An excellent response was also associated with lower rates of presence of disease at final follow-up, need for additional intervention, and disease-specific mortality. CONCLUSIONS: Although TCV is an aggressive subtype associated with worse clinical outcomes than classical PTC, patients with an excellent overall response to treatment have significantly improved outcomes when compared to indeterminate, biochemical incomplete, and structural incomplete responses. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:2430-2438, 2023.

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.

Systems-wide analysis revealed shared and unique responses to moderate and acute high temperatures in the green alga Chlamydomonas reinhardtii.

Different intensities of high temperatures affect the growth of photosynthetic cells in nature. To elucidate the underlying mechanisms, we cultivated the unicellular green alga Chlamydomonas reinhardtii under highly controlled photobioreactor conditions and revealed systems-wide shared and unique responses to 24-hour moderate (35°C) and acute (40°C) high temperatures and subsequent recovery at 25°C. We identified previously overlooked unique elements in response to moderate high temperature. Heat at 35°C transiently arrested the cell cycle followed by partial synchronization, up-regulated transcripts/proteins involved in gluconeogenesis/glyoxylate-cycle for carbon uptake and promoted growth. But 40°C disrupted cell division and growth. Both high temperatures induced photoprotection, while 40°C distorted thylakoid/pyrenoid ultrastructure, affected the carbon concentrating mechanism, and decreased photosynthetic efficiency. We demonstrated increased transcript/protein correlation during both heat treatments and hypothesize reduced post-transcriptional regulation during heat may help efficiently coordinate thermotolerance mechanisms. During recovery after both heat treatments, especially 40°C, transcripts/proteins related to DNA synthesis increased while those involved in photosynthetic light reactions decreased. We propose down-regulating photosynthetic light reactions during DNA replication benefits cell cycle resumption by reducing ROS production. Our results provide potential targets to increase thermotolerance in algae and crops.

Use of Thromboelastography and Rotational Thromboelastometry in Otolaryngology: A Narrative Review.

In the field of otolaryngology-head and neck surgery (ENT), coagulopathies present unique diagnostic and therapeutic challenges. In both hyper- and hypocoagulable patients, management of coagulopathies requires intricate attention to the nature of hemostatic competence. Common coagulation tests (CCTs) offer only a snapshot of hemostatic competence and do not provide a clear insight into the patient's real-time hemostatic condition. Viscoelastic tests (VETs) offer a holistic and concurrent picture of the coagulation process. Although VETs have found prominent utilization in hepatic transplants, obstetrics, and emergent surgical settings, they have not been fully adopted in the realm of otolaryngology. The objective of this manuscript is to provide an overview of the literature evaluating the current utilization and possible future uses of VETs in the field of otolaryngology. The authors performed a comprehensive literature search of the utilization of VETs in otolaryngology and identified applicable studies that included descriptions of viscoelastic testing. Twenty-five studies were identified in this search, spanning topics from head and neck oncology, microvascular free flap reconstruction, obstructive sleep apnea, adenotonsillectomy, facial trauma, and epistaxis. The applicability of VETs has been demonstrated in head and neck oncology and microvascular free flap management, although their pervasiveness in practice is limited. Underutilization of VETs in the field of otolaryngology may be due to a lack of familiarity of the tests amongst practitioners. Instead, most otolaryngologists continue to rely on CCTs, including PT, PTT, INR, CBC, fibrinogen levels, and thrombin time. Learning to perform, interpret, and skillfully employ VETs in clinical and operative practice can greatly improve the management of coagulopathic patients who are at increased risk of bleeding or thrombosis.

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.

Topology of the redox network during induction of photosynthesis as revealed by time-resolved proteomics in tobacco.

Photosynthetically produced electrons provide energy for various metabolic pathways, including carbon reduction. Four Calvin-Benson cycle enzymes and several other plastid proteins are activated in the light by reduction of specific cysteines via thioredoxins, a family of electron transporters operating in redox regulation networks. How does this network link the photosynthetic chain with cellular metabolism? Using a time-resolved redox proteomic method, we have investigated the redox network in vivo during the dark­to­low light transition. We show that redox states of some thioredoxins follow the photosynthetic linear electron transport rate. While some redox targets have kinetics compatible with an equilibrium with one thioredoxin (TRXf), reduction of other proteins shows specific kinetic limitations, allowing fine-tuning of each redox-regulated step of chloroplast metabolism. We identified five new redox-regulated proteins, including proteins involved in Mg2+ transport and 1O2 signaling. Our results provide a system-level functional view of the photosynthetic redox regulation network.

Data Management and Modeling in Plant Biology.

The study of plant-environment interactions is a multidisciplinary research field. With the emergence of quantitative large-scale and high-throughput techniques, amount and dimensionality of experimental data have strongly increased. Appropriate strategies for data storage, management, and evaluation are needed to make efficient use of experimental findings. Computational approaches of data mining are essential for deriving statistical trends and signatures contained in data matrices. Although, current biology is challenged by high data dimensionality in general, this is particularly true for plant biology. Plants as sessile organisms have to cope with environmental fluctuations. This typically results in strong dynamics of metabolite and protein concentrations which are often challenging to quantify. Summarizing experimental output results in complex data arrays, which need computational statistics and numerical methods for building quantitative models. Experimental findings need to be combined by computational models to gain a mechanistic understanding of plant metabolism. For this, bioinformatics and mathematics need to be combined with experimental setups in physiology, biochemistry, and molecular biology. This review presents and discusses concepts at the interface of experiment and computation, which are likely to shape current and future plant biology. Finally, this interface is discussed with regard to its capabilities and limitations to develop a quantitative model of plant-environment interactions.

In Vivo Structure-Function Analysis and Redox Interactomes of Leishmania tarentolae Erv.

Import and oxidative folding of proteins in the mitochondrial intermembrane space differ among eukaryotic lineages. While opisthokonts such as yeast rely on the receptor and oxidoreductase Mia40 in combination with the Mia40:cytochrome c oxidoreductase Erv, kinetoplastid parasites and other Excavata/Discoba lack Mia40 but have a functional Erv homologue. Whether excavate Erv homologues rely on a Mia40 replacement or directly interact with imported protein substrates remains controversial. Here, we used the CRISPR-Cas9 system to generate a set of tagged and untagged homozygous mutants of LTERV from the kinetoplastid model parasite Leishmania tarentolae. Modifications of the shuttle cysteine motif of LtErv were lethal, whereas replacement of clamp residue Cys17 or removal of the kinetoplastida-specific second (KISS) domain had no impact on parasite viability under standard growth conditions. However, removal of the KISS domain rendered parasites sensitive to heat stress and led to the accumulation of homodimeric and mixed LtErv disulfides. We therefore determined and compared the redox interactomes of tagged wild-type LtErv and LtErvΔKISS using stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry. While the Mia40-replacement candidate Mic20 and all but one typical substrate with twin Cx3/9C-motifs were absent in both redox interactomes, we identified a small set of alternative potential interaction partners with putative redox-active cysteine residues. In summary, our study reveals parasite-specific intracellular structure-function relationships and redox interactomes of LtErv with implications for current hypotheses on mitochondrial protein import in nonopisthokonts. IMPORTANCE The discovery of the redox proteins Mia40/CHCHD4 and Erv1/ALR, as well as the elucidation of their relevance for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals, founded a new research topic in redox biology and mitochondrial protein import. The lack of Mia40/CHCHD4 in protist lineages raises fundamental and controversial questions regarding the conservation and evolution of this essential pathway. Do protist Erv homologues act alone, or do they use the candidate Mic20 or another protein as a Mia40 replacement? Furthermore, we previously showed that Erv homologues in L. tarentolae and the human pathogen L. infantum are not only essential but also differ structurally and mechanistically from yeast and human Erv1/ALR. Here, we analyzed the relevance of such structural differences in vivo and determined the first redox interactomes of a nonopisthokont Erv homologue. Our data challenge recent hypotheses on mitochondrial protein import in nonopisthokonts.

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.

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.

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.

The effect of marrow secretome and culture environment on the rate of metastatic breast cancer cell migration in two and three dimensions.

Metastasis is responsible for over 90% of cancer-related deaths, and bone is the most common site for breast cancer metastasis. Metastatic breast cancer cells home to trabecular bone, which contains hematopoietic and stromal lineage cells in the marrow. As such, it is crucial to understand whether bone or marrow cells enhance breast cancer cell migration toward the tissue. To this end, we quantified the migration of MDA-MB-231 cells toward human bone in two- and three-dimensional (3D) environments. First, we found that the cancer cells cultured on tissue culture plastic migrated toward intact trabecular bone explants at a higher rate than toward marrow-deficient bone or devitalized bone. Leptin was more abundant in conditioned media from the cocultures with intact explants, while higher levels of IL-1ß, IL-6, and TNFα were detected in cultures with both intact bone and cancer cells. We further verified that the cancer cells migrated into bone marrow using a bioreactor culture system. Finally, we studied migration toward bone in 3D gelatin. Migration speed did not depend on stiffness of this homogeneous gel, but many more dendritic-shaped cancer cells oriented and migrated toward bone in stiffer gels than softer gels, suggesting a coupling between matrix mechanics and chemotactic signals.

Evaluating the risks and benefits of ketorolac in transoral robotic surgery.

Transoral Robotic Surgery (TORS) is increasingly used for oropharyngeal neoplasms and obstructive sleep apnea. Post-operative pain and bleeding remain concerns. Ketorolac has proved to be a safe alternative or addition to narcotics in other operations, but has not been thoroughly evaluated in TORS. A retrospective review was carried out on all TORS cases at our institution between April 2012 and March 2019, with the vast majority of cases performed starting in 2017. Post-operative bleed rates were compared between those who received Ketorolac and those who did not. Secondary outcomes evaluated included post-operative pain scores and need for feeding tube upon discharge. A total of 81 TORS cases were evaluated, with 37 patients receiving Ketorolac. Six (7.4%) patients reported post-operative bleeding, with one major and five minor bleeds. The patient with major bleeding requiring operative intervention did not receive Ketorolac. All five patients with minor bleeding received Ketorolac, but no bleeds occurred in the immediate post-operative setting while receiving Ketorolac. The average time of bleeding was 8 days post-operative. There were no significant differences in pain scores or time to feeding tube removal. This preliminary study shows that Ketorolac use in the postoperative pain management after TORS does not increase major bleeding risk without benefits in pain management. There was increased risk of minor bleeding not requiring intervention, but this was not significant. Future prospective studies are needed to determine if it improves pain and swallowing and decreases narcotic requirements following TORS.

Clinical Versus Pathologic Laryngeal Cancer Staging and the Impact of Stage Change on Outcomes.

OBJECTIVES/HYPOTHESIS: Evaluate the impact and accuracy of clinical laryngeal cancer staging. STUDY DESIGN: Retrospective cohort study. METHODS: Two hundred sixty-five consecutive patients with laryngeal squamous cell carcinoma who underwent total laryngectomy from 2001 to 2017 were studied. Clinical versus pathologic tumor (T) and nodal (N) categories were compared. Logistic regression and Cox proportional hazards analyzed the association of stage change with perioperative factors and outcomes. RESULTS: Forty-seven patients (17.7%, accuracy = 0.969 ± 0.010 [standard error]) changed between T1-2 and T3-4. Sixty-four patients (24.1%, accuracy = 0.866 ± 0.020) had inaccurate N category. Salvage patients were less likely to have stage change (downstage: odds ratio [OR] = 0.20, 95% confidence interval [CI]: 0.08-0.50, P < .001; upstage: OR = 0.41, 95% CI: 0.23-0.74, P = .003), but more likely to have inaccurate nodal category (39.8% vs. 11.7%, P < .001). Patients with stage change tended to have greater odds of positive/close margins (upstage: OR = 1.78, 95% CI: 0.91-3.5, P = .092) and chemotherapy (downstage: OR = 2.21, 95% CI: 0.80-6.14, P = .128; upstage: OR = 1.87, 95% CI: 0.85-4.11, P = .119). Stage change was associated with recurrence (P = .047) with downstaged patients less likely to recur (hazard ratio = 0.26, 95% CI: 0.08-0.82, P = .021). Stage change was not associated with positron emission tomography scan, subsite, time to surgery, or mortality. CONCLUSIONS: A third of laryngeal cancer patients were downstaged or upstaged after laryngectomy with 18% and 24% of clinical T and N categories inaccurate, respectively. Stage change was less common for salvage patients and associated with risk of recurrence. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:559-565, 2021.

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.