Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects.

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric ß-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

Higher prevalence of sacbrood virus in Apis mellifera (Hymenoptera: Apidae) colonies after pollinating highbush blueberries.

Highbush blueberry pollination depends on managed honey bees (Apis mellifera) L. for adequate fruit sets; however, beekeepers have raised concerns about the poor health of colonies after pollinating this crop. Postulated causes include agrochemical exposure, nutritional deficits, and interactions with parasites and pathogens, particularly Melisococcus plutonius [(ex. White) Bailey and Collins, Lactobacillales: Enterococcaceae], the causal agent of European foulbrood disease, but other pathogens could be involved. To broadly investigate common honey bee pathogens in relation to blueberry pollination, we sampled adult honey bees from colonies at time points corresponding to before (t1), during (t2), at the end (t3), and after (t4) highbush blueberry pollination in British Columbia, Canada, across 2 years (2020 and 2021). Nine viruses, as well as M. plutonius, Vairimorpha ceranae, and V. apis [Tokarev et al., Microsporidia: Nosematidae; formerly Nosema ceranae (Fries et al.) and N. apis (Zander)], were detected by PCR and compared among colonies located near and far from blueberry fields. We found a significant interactive effect of time and blueberry proximity on the multivariate pathogen community, mainly due to differences at t4 (corresponding to ~6 wk after the beginning of the pollination period). Post hoc comparisons of pathogens in near and far groups at t4 showed that detections of sacbrood virus (SBV), which was significantly higher in the near group, not M. plutonius, was the primary driver. Further research is needed to determine if the association of SBV with highbush blueberry pollination is contributing to the health decline that beekeepers observe after pollinating this crop.

Development of automated proteomic workflows utilizing silicon-based coupling agents.

Automated methods for enzyme immobilization via 4-triethoxysilylbutyraldehyde (TESB) derived silicone-based coupling agents were developed. TESB and its oxidized derivative, 4-triethoxysilylbutanoic acid (TESBA), were determined to be the most effective. The resulting immobilized enzyme particles (IEPs) displayed robustness, rapid digestion, and immobilization efficiency of 51 ± 8%. Furthermore, we automated the IEP procedure, allowing for multiple enzymes, and/or coupling agents to be fabricated at once, in a fraction of the time via an Agilent Bravo. The automated trypsin TESB and TESBA IEPs were shown to rival a classical in-gel digestion method. Moreover, pepsin IEPs favored cleavage at leucine (>50%) over aromatic and methionine residues. The IEP method was then adapted for an in-situ immobilized enzyme microreactor (IMER) fabrication. We determined that TESBA could functionalize the silica capillary's inner wall while simultaneously acting as an enzyme coupler. The IMER digestion of bovine serum albumin (BSA), mirroring IEP digestion conditions, yielded a 33-40% primary sequence coverage per LC-MS/MS analysis in as little as 15 min. Overall, our findings underscore the potential of both IEP and IMER methods, paving the way for automated analysis and a reduction in enzyme waste through reuse, thereby contributing to a more cost-effective and timely study of the proteome. SIGNIFICANCE: This research introduces 4-triethoxysilylbutyraldehyde (TESB) and its derivatives as silicon-based enzyme coupling agents and an automated liquid handling method for bottom-up proteomics (BUP) while streamlining sample preparation for high-throughput processing. Additionally, immobilized enzyme particle (IEP) fabrication and digestion within the 96-well plate allows for flexibility in protocol where different enzyme-coupler combinations can be employed simultaneously. By enabling the digestion of entire microplates and reducing manual labor, the proposed method enhances reproducibility and offers a more efficient alternative to classical in-gel techniques. Furthermore, pepsin IEPs were noted to favor cleavage at leucine residues which represents an interesting finding when compared to the literature that warrants further study. The capability of immobilized enzyme microreactors (IMER) for rapid digestion (in as little as 15 min) demonstrated the system's efficiency and potential for rapid proteomic analysis. This advancement in BUP not only improves efficiency, but also opens avenues for a fully automated, mass spectrometry-integrated proteomics workflow, promising to expedite research and discoveries in complex biological studies.

What proteomics has taught us about honey bee (Apis mellifera) health and disease.

The Western honey bee, Apis mellifera, is currently navigating a gauntlet of environmental pressures, including the persistent threat of parasites, pathogens, and climate change - all of which compromise the vitality of honey bee colonies. The repercussions of their declining health extend beyond the immediate concerns of apiarists, potentially imposing economic burdens on society through diminished agricultural productivity. Hence, there is an imperative to devise innovative monitoring techniques for assessing the health of honey bee populations. Proteomics, recognized for its proficiency in biomarker identification and protein-protein interactions, is poised to play a pivotal role in this regard. It offers a promising avenue for monitoring and enhancing the resilience of honey bee colonies, thereby contributing to the stability of global food supplies. This review delves into the recent proteomic studies of A. mellifera, highlighting specific proteins of interest and envisioning the potential of proteomics to improve sustainable beekeeping practices amidst the challenges of a changing planet.

Sex-Based Mechanisms of Cardiac Development and Function: Applications for Induced-Pluripotent Stem Cell Derived-Cardiomyocytes.

Males and females exhibit intrinsic differences in the structure and function of the heart, while the prevalence and severity of cardiovascular disease vary in the two sexes. However, the mechanisms of this sex-based dimorphism are yet to be elucidated. Sex chromosomes and sex hormones are the main contributors to sex-based differences in cardiac physiology and pathophysiology. In recent years, the advances in induced pluripotent stem cell-derived cardiac models and multi-omic approaches have enabled a more comprehensive understanding of the sex-specific differences in the human heart. Here, we provide an overview of the roles of these two factors throughout cardiac development and explore the sex hormone signaling pathways involved. We will also discuss how the employment of stem cell-based cardiac models and single-cell RNA sequencing help us further investigate sex differences in healthy and diseased hearts.

Reversible Phase Transformations in a Double-Walled Diamondoid Coordination Network with a Stepped Isotherm for Methane.

Flexible metal-organic materials (FMOMs) with stepped isotherms can offer enhanced working capacity in storage applications such as adsorbed natural gas (ANG) storage. Unfortunately, whereas >1000 FMOMs are known, only a handful exhibit methane uptake of >150 cm3/cm3 at 65 atm and 298 K, conditions relevant to ANG. Here, we report a double-walled 2-fold interpenetrated diamondoid (dia) network, X-dia-6-Ni, [Ni2L4(µ-H2O)]n, comprising a new azo linker ligand, L- (L- = (E)-3-(pyridin-4-yldiazenyl)benzoate) and 8-connected dinuclear molecular building blocks. X-dia-6-Ni exhibited gas (CO2, N2, CH4) and liquid (C8 hydrocarbons)-induced reversible transformations between its activated narrow-pore ß phase and γ, a large-pore phase with ca. 33% increase in unit cell volume. Single-crystal X-ray diffraction (SCXRD) studies of the as-synthesized phase α, ß, and γ revealed that structural transformations were enabled by twisting of the azo moiety and/or deformation of the MBB. Further insight into these transformations was gained from variable temperature powder XRD and in situ variable pressure powder XRD. Low-temperature N2 and CO2 sorption revealed stepped Type F-II isotherms with saturation uptakes of 422 and 401 cm3/g, respectively. X-dia-6-Ni exhibited uptake of 200 cm3/cm3 (65 atm, 298 K) and a high CH4 working capacity of 166 cm3/cm3 (5-65 bar, 298 K, 33 cycles), the third highest value yet reported for an FMOM and the highest value for an FMOM with a Type F-II isotherm.

Local Self-Assembly of Dissipative Structures Sustained by Substrate Diffusion.

The coupling between energy-consuming molecular processes and the macroscopic dimension plays an important role in nature and in the development of active matter. Here, we study the temporal evolution of a macroscopic system upon the local activation of a dissipative self-assembly process. Injection of surfactant molecules in a substrate-containing hydrogel results in the local substrate-templated formation of assemblies, which are catalysts for the conversion of substrate into waste. We show that the system develops into a macroscopic (pseudo-)non-equilibrium steady state (NESS) characterized by the local presence of energy-dissipating assemblies and persistent substrate and waste concentration gradients. For elevated substrate concentrations, this state can be maintained for more than 4 days. The studies reveal an interdependence between the dissipative assemblies and the concentration gradients: catalytic activity by the assemblies results in sustained concentration gradients and, vice versa, continuous diffusion of substrate to the assemblies stabilizes their size. The possibility to activate dissipative processes with spatial control and create long lasting non-equilibrium steady states enables dissipative structures to be studied in the space-time domain, which is of relevance for understanding biological systems and for the development of active matter.

Characterization of Argonaute-containing protein complexes in Leishmania-infected human macrophages.

The intracellular protozoan parasite Leishmania causes leishmaniasis in humans, leading to serious illness and death in tropical and subtropical areas worldwide. Unfortunately, due to the unavailability of approved vaccines for humans and the limited efficacy of available drugs, leishmaniasis is on the rise. A comprehensive understanding of host-pathogen interactions at the molecular level could pave the way to counter leishmaniasis. There is growing evidence that several intracellular pathogens target RNA interference (RNAi) pathways in host cells to facilitate their persistence. The core elements of the RNAi system are complexes of Argonaute (Ago) proteins with small non-coding RNAs, also known as RNA-induced silencing complexes (RISCs). Recently, we have shown that Leishmania modulates Ago1 protein of host macrophages for its survival. In this study, we biochemically characterize the Ago proteins' interactome in Leishmania-infected macrophages compared to non-infected cells. For this, a quantitative proteomic approach using stable isotope labelling by amino acids in cell culture (SILAC) was employed, followed by purification of host Ago-complexes using a short TNRC6 protein-derived peptide fused to glutathione S-transferase beads as an affinity matrix. Proteomic-based detailed biochemical analysis revealed Leishmania modulated host macrophage RISC composition during infection. This analysis identified 51 Ago-interacting proteins with a broad range of biological activities. Strikingly, Leishmania proteins were detected as part of host Ago-containing complexes in infected cells. Our results present the first report of comprehensive quantitative proteomics of Ago-containing complexes isolated from Leishmania-infected macrophages and suggest targeting the effector complex of host RNAi machinery. Additionally, these results expand knowledge of RISC in the context of host-pathogen interactions in parasitology in general.

Pivotal functions and impact of long con-coding RNAs on cellular processes and genome integrity.

Recent advances in uncovering the mysteries of the human genome suggest that long non-coding RNAs (lncRNAs) are important regulatory components. Although lncRNAs are known to affect gene transcription, their mechanisms and biological implications are still unclear. Experimental research has shown that lncRNA synthesis, subcellular localization, and interactions with macromolecules like DNA, other RNAs, or proteins can all have an impact on gene expression in various biological processes. In this review, we highlight and discuss the major mechanisms through which lncRNAs function as master regulators of the human genome. Specifically, the objective of our review is to examine how lncRNAs regulate different processes like cell division, cell cycle, and immune responses, and unravel their roles in maintaining genomic architecture and integrity.

Control Compounds for Preclinical Drug-Induced Liver Injury Assessment: Consensus-driven systematic review by the ProEuroDILI Network.

BACKGROUND & AIMS: Idiosyncratic drug-induced liver injury (DILI) is a complex and unpredictable event caused by drugs, herbal or dietary supplements. Early identification of human hepatotoxicity at preclinical stages remains a major challenge, in which the selection of validated in vitro systems and test drugs has a significant impact. This systematic review analyzed the compounds used in hepatotoxicity assays and established a list of DILI positive and negative control drugs for validation of in vitro models of DILI, supported by literature and clinical evidence and endorsed by an expert committee from COST Action ProEuroDILI Network (CA17112). METHODS: Following 2020 PRISMA guidelines, original research articles focusing on DILI which used in vitro human models and performed at least one hepatotoxicity assay with positive and negative control compounds, were included. Bias of the studies was assessed by a modified 'Toxicological Data Reliability Assessment Tool'. RESULTS: 51 studies (out of 2,936) met the inclusion criteria, with 30 categorized as reliable without restrictions. Although there was a broad consensus on positive compounds, the selection of negative compounds lacked clarity. 2D monoculture, short exposure times and cytotoxicity endpoints were the most tested, although there was no consensus on the drug concentrations. CONCLUSIONS: The extensive analysis highlighted the lack of agreement on control compounds for in vitro DILI assessment. Following comprehensive in vitro and clinical data analysis together with input from the expert committee, an evidence-based consensus-driven list of 10 positive and negative drugs is proposed for validating in vitro models for improving preclinical drug safety testing regimes. IMPACT AND IMPLICATIONS: Prediction of human toxicity early in the drug development process remains a major challenge. For this, human in vitro models are becoming increasingly important, however, the development of more physiologically relevant liver models and careful selection of control DILI+ and DILI- drugs are requisites to better predict DILI liability of new drug candidates. Thus, this systematic study holds critical implications for standardizing validation of new in vitro models for studying drug-induced liver injury (DILI). By establishing a consensus-driven list of positive and negative control drugs, the study provides a scientifically justified framework for enhancing the consistency of preclinical testing, thereby addressing a significant challenge in early hepatotoxicity identification. The results are of paramount importance to all the actors involved in the drug development process, offering a standardized approach to assess hepatotoxic risks. Practically, these findings can guide researchers in evaluating safety profiles of new drugs, refining in vitro models, and informing regulatory agencies on potential improvements to regulatory guidelines, ensuring a more systematic and efficient approach to drug safety assessment.

AI and crisis leadership: Using the POP-DOC Loop to explore potential implications and opportunities for leaders.

In the evolving landscape of crisis leadership and emergency management, artificial intelligence (AI) emerges as a potentially transformative force with far-reaching implications. Utilizing the POP-DOC Loop, a comprehensive framework for crisis leadership analysis and decision-making, this paper delves into the diverse roles that AI is poised to play in shaping the future of crisis planning and response. The POP-DOC Loop serves as a structured methodology, encompassing key elements such as information gathering, contextual analysis informed by social determinants, enhanced predictive modeling, guided decision-making, strategic action implementation, and appropriate communication. Rather than offer definitive predictions, this review aims to catalyze exploration and discussion, equipping researchers and practitioners to anticipate future contingencies. The paper concludes by examining the limitations and challenges posed by AI within this specialized context.

Evaluation of an ultrasound-guided freeze-core biopsy system for canine and feline brain tumors.

Objective: To determine if a single brain biopsy utilizing a freeze-core needle harvest system Cassi II under ultrasound guidance provides a diagnostic sample; to evaluate the technique's efficacy in procuring diagnostic samples in comparison with "open" surgical biopsies; and to describe intraoperative complications associated with the technique. Study design: Experimental clinical study. Animals: Seventeen dogs and four cats with magnetic resonance imaging (MRI) diagnoses of readily surgically accessible intracranial masses. Methods: Immediately prior to surgical biopsy (SB), freeze-core biopsy (FCB) sample was obtained from each patient under ultrasound guidance. Results: Histopathology results from single FCB samples were found to be in 100% agreement with the SB samples. Freezing artifact was minimal and did not interfere with histopathologic interpretation. There were no intraoperative complications specifically attributable to the use of the FCB system. Conclusion: Based on the results of this small experimental study, the FCB system is expected to safely yield diagnostic quality intracranial masses biopsy specimens. Clinical significance: This system has the potential of obtaining diagnostic biopsies of more deeply seated brain lesions (i.e., intra-axial tumors considered inaccessible or with large risks/difficulties by standard surgical means) which would provide a definitive diagnosis to guide appropriate therapy.

Revisiting distinct nerve excitability patterns in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease, characterized by loss of central and peripheral motor neurones. Although the disease is clinically and genetically heterogeneous, axonal hyperexcitability is a commonly observed feature that has been suggested to reflect an early pathophysiological step linked to the neurodegenerative cascade. Therefore, it is important to clarify the mechanisms causing axonal hyperexcitability and how these relate to the clinical characteristics of patients. Measures derived directly from a nerve excitability recording are frequently used as study endpoints, even though their biophysical basis is difficult to deduce. Mathematical models can aid in the interpretation, but are only reliable when applied to group-averaged recordings. Consequently, model estimates of membrane properties cannot be compared to clinical characteristics or treatment effects in individual patients, posing a considerable limitation in heterogeneous diseases such as amyotrophic lateral sclerosis. To address these challenges, we revisited nerve excitability using a novel pattern-analysis-based approach (principal component analysis). We evaluated disease-specific patterns of excitability changes and established their biophysical origins. Based on the observed patterns, we developed novel compound measures of excitability that facilitate the implementation of this approach in clinical settings We found that excitability changes in amyotrophic lateral sclerosis patients (n = 161, median disease duration = 11 months) were characterized by four unique patterns compared to controls (n = 50, age-gender matched). These four patterns were best explained by changes in resting membrane potential (modulated by Na+/K + -currents), slow potassium- and sodium-currents (modulated by their gating kinetics) and refractory properties of the nerve. Consequently, we were able to show that altered gating of slow potassium-channels was associated with, and predictive of, the disease's progression rate on the amyotrophic lateral sclerosis functional rating scale. Based on these findings, we designed four composite measures that capture these properties to facilitate implementation outside of this study. Our findings demonstrate that nerve excitability changes in patients with amyotrophic lateral sclerosis are dominated by four distinct patterns, each with a distinct biophysical origin. Based on this new approach, we provide evidence that altered slow potassium-channel function may play a role in the rate of disease progression. The magnitudes of these patterns, quantified using either a similar approach or our novel composite measures, have potential as efficient measures to study membrane properties directly in amyotrophic lateral sclerosis patients, and thus aid prognostic stratification and trial design.

KRAS: Biology, Inhibition, and Mechanisms of Inhibitor Resistance.

KRAS is a small GTPase that is among the most commonly mutated oncogenes in cancer. Here, we discuss KRAS biology, therapeutic avenues to target it, and mechanisms of resistance that tumors employ in response to KRAS inhibition. Several strategies are under investigation for inhibiting oncogenic KRAS, including small molecule compounds targeting specific KRAS mutations, pan-KRAS inhibitors, PROTACs, siRNAs, PNAs, and mutant KRAS-specific immunostimulatory strategies. A central challenge to therapeutic effectiveness is the frequent development of resistance to these treatments. Direct resistance mechanisms can involve KRAS mutations that reduce drug efficacy or copy number alterations that increase the expression of mutant KRAS. Indirect resistance mechanisms arise from mutations that can rescue mutant KRAS-dependent cells either by reactivating the same signaling or via alternative pathways. Further, non-mutational forms of resistance can take the form of epigenetic marks, transcriptional reprogramming, or alterations within the tumor microenvironment. As the possible strategies to inhibit KRAS expand, understanding the nuances of resistance mechanisms is paramount to the development of both enhanced therapeutics and innovative drug combinations.

Honey bee stressor networks are complex and dependent on crop and region.

Honey bees play a major role in crop pollination but have experienced declining health throughout most of the globe. Despite decades of research on key honey bee stressors (e.g., parasitic Varroa destructor mites and viruses), researchers cannot fully explain or predict colony mortality, potentially because it is caused by exposure to multiple interacting stressors in the field. Understanding which honey bee stressors co-occur and have the potential to interact is therefore of profound importance. Here, we used the emerging field of systems theory to characterize the stressor networks found in honey bee colonies after they were placed in fields containing economically valuable crops across Canada. Honey bee stressor networks were often highly complex, with hundreds of potential interactions between stressors. Their placement in crops for the pollination season generally exposed colonies to more complex stressor networks, with an average of 23 stressors and 307 interactions. We discovered that the most influential stressors in a network-those that substantively impacted network architecture-are not currently addressed by beekeepers. Finally, the stressor networks showed substantial divergence among crop systems from different regions, which is consistent with the knowledge that some crops (e.g., highbush blueberry) are traditionally riskier to honey bees than others. Our approach sheds light on the stressor networks that honey bees encounter in the field and underscores the importance of considering interactions among stressors. Clearly, addressing and managing these issues will require solutions that are tailored to specific crops and regions and their associated stressor networks.

Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects.

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SulfETF), tryptophan synthases from Salmonella typhimurium (StTS) and the dimeric ß-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

Randomized, Double-Blind Phase III Study of Pazopanib Versus Placebo in Patients With Metastatic Renal Cell Carcinoma Who Have No Evidence of Disease After Metastasectomy: ECOG-ACRIN E2810.

PURPOSE: Patients with no evidence of disease (NED) after metastasectomy for renal cell carcinoma are at high risk of recurrence. Pazopanib is an inhibitor of vascular endothelial growth factor receptor and other kinases that improves progression-free survival in patients with metastatic RCC (mRCC). We conducted a randomized, double-blind, placebo-controlled multicenter study to test whether pazopanib would improve disease-free survival (DFS) in patients with mRCC rendered NED after metastasectomy. PATIENTS AND METHODS: Patients with NED after metastasectomy were randomly assigned 1:1 to receive pazopanib 800 mg once daily versus placebo for 52 weeks. The study was designed to observe an improvement in DFS from 25% to 45% with pazopanib at 3 years, corresponding to 42% reduction in the DFS event rate. RESULTS: From August 2012 to July 2017, 129 patients were enrolled. The study was unblinded after 83 DFS events (92% information). The study did not meet its primary end point. An updated analysis at 60.5-month median follow-up from random assignment (95% CI, 59.3 to 71.0) showed that the 3-year DFS was 27.4% (95% CI, 17.9 to 41.7) for pazopanib and 21.9% (95% CI, 13.3 to 36.2) for placebo. Hazard ratio (HR) for DFS was 0.90 ([95% CI, 0.60 to 1.34]; Pone-sided = .29) in favor of pazopanib. Three-year overall survival (OS) was 81.9% (95% CI, 72.7 to 92.2) for pazopanib and 91.4% (95% CI, 84.4 to 98.9) for placebo. The HR for OS was 2.55 (95% CI, 1.23 to 5.27) in favor of placebo (Ptwo-sided = .012). Health-related quality-of-life measures deteriorated in the pazopanib group during the treatment period. CONCLUSION: Pazopanib did not improve DFS as the primary end point compared with blinded placebo in patients with mRCC with NED after metastasectomy. In addition, there was a concerning trend favoring placebo in OS.

Oral infectivity through carnivorism in murine model of Trypanosoma cruzi infection.

Introduction: Oral transmission of T. cruzi is probably the most frequent transmission mechanism in wild animals. This observation led to the hypothesis that consuming raw or undercooked meat from animals infected with T. cruzi may be responsible for transmitting the infection. Therefore, the general objective of this study was to investigate host-pathogen interactions between the parasite and gastric mucosa and the role of meat consumption from infected animals in the oral transmission of T. cruzi. Methods: Cell infectivity assays were performed on AGS cells in the presence or absence of mucin, and the roles of pepsin and acidic pH were determined. Moreover, groups of five female Balb/c mice were fed with muscle tissue obtained from mice in the acute phase of infection by the clone H510 C8C3hvir of T. cruzi, and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of T. cruzi trypomastigotes and amastigotes by infecting groups of five mice Balb/c females, which were infected orally using a nasogastric probe, and the infection was monitored by a parasitemia curve. Finally, different trypomastigote and amastigote inoculums were used to determine their infective capacities. Adhesion assays of T. cruzi proteins to AGS stomach cells were performed, and the adhered proteins were detected by western blotting using monoclonal or polyclonal antibodies and by LC-MS/MS and bioinformatics analysis. Results: Trypomastigote migration in the presence of mucin was reduced by approximately 30%, whereas in the presence of mucin and pepsin at pH 3.5, only a small proportion of parasites were able to migrate (∼6%). Similarly, the ability of TCTs to infect AGS cells in the presence of mucin is reduced by approximately 20%. In all cases, 60-100% of the animals were fed meat from mice infected in the acute phase or infected with trypomastigotes or amastigotes developed high parasitemia, and 80% died around day 40 post-infection. The adhesion assay showed that cruzipain is a molecule of trypomastigotes and amastigotes that binds to AGS cells. LC-MS/MS and bioinformatics analysis, also confirmed that transialidase, cysteine proteinases, and gp63 may be involved in TCTs attachment or invasion of human stomach cells because they can potentially interact with different proteins in the human stomach mucosa. In addition, several human gastric mucins have cysteine protease cleavage sites. Discussion: Then, under our experimental conditions, consuming meat from infected animals in the acute phase allows the T. cruzi infection. Similarly, trypomastigotes and amastigotes could infect mice when administered orally, whereas cysteinyl proteinases and trans-sialidase appear to be relevant molecules in this infective process.

Non-invasive methods of monitoring Fe3O4 magnetic nanoparticle toxicity in human liver HepaRG cells using impedance biosensing and Coherent anti-Stokes Raman spectroscopic (CARS) microscopy.

Functionalized nanoparticles have been developed for use in nanomedicines for treating life threatening diseases including various cancers. To ensure safe use of these new nanoscale reagents, various assays for biocompatibility or cytotoxicity in vitro using cell lines often serve as preliminary assessments prior to in vivo animal testing. However, many of these assays were designed for soluble, colourless materials and may not be suitable for coloured, non-transparent nanoparticles. Moreover, cell lines are not always representative of mammalian organs in vivo. In this work, we use non-invasive impedance sensing methods with organotypic human liver HepaRG cells as a model to test the toxicity of PEG-Fe3O4 magnetic nanoparticles. We also use Coherent anti-Stokes Raman Spectroscopic (CARS) microscopy to monitor the formation of lipid droplets as a parameter to the adverse effect on the HepaRG cell model. The results were also compared with two commercial testing kits (PrestoBlue and ATP) for cytotoxicity. The results suggested that the HepaRG cell model can be a more realistic model than commercial cell lines while use of impedance monitoring of Fe3O4 nanoparticles circumventing the uncertainties due to colour assays. These methods can play important roles for scientists driving towards the 3Rs principle - Replacement, Reduction and Refinement.

Proteomic predictors of individualized nutrient-specific insulin secretion in health and disease.

Population level variation and molecular mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized despite ramifications for personalized nutrition. Here, we define prototypical insulin secretion dynamics in response to the three macronutrients in islets from 140 cadaveric donors, including those diagnosed with type 2 diabetes. While islets from the majority of donors exhibited the expected relative response magnitudes, with glucose being highest, amino acid moderate, and fatty acid small, 9% of islets stimulated with amino acid and 8% of islets stimulated with fatty acids had larger responses compared with high glucose. We leveraged this insulin response heterogeneity and used transcriptomics and proteomics to identify molecular correlates of specific nutrient responsiveness, as well as those proteins and mRNAs altered in type 2 diabetes. We also examine nutrient-responsiveness in stem cell-derived islet clusters and observe that they have dysregulated fuel sensitivity, which is a hallmark of functionally immature cells. Our study now represents the first comparison of dynamic responses to nutrients and multi-omics analysis in human insulin secreting cells. Responses of different people's islets to carbohydrate, protein, and fat lay the groundwork for personalized nutrition. ONE-SENTENCE SUMMARY: Deep phenotyping and multi-omics reveal individualized nutrient-specific insulin secretion propensity.