Unveiling the TRAPP: The role of plant TRAPPII in adaptive growth decisions.

The regulation of intracellular membrane traffic is coupled with the cell's need to respond to environmental stimuli, which ultimately is critical for different processes such as cell growth and development. In this issue, Wiese et al. (https://www.doi.org/10.1083/jcb.202311125) explore the role of the trans-Golgi network (TGN) in stress response, exposing its role in mediating adaptive growth decisions.

Stability, Content of Bioactive Compounds and Antioxidant Activity of Emulsions with Propolis Extracts during Simulated In Vitro Digestion.

The objective in this work was the evaluation of the stability and content of bioactive compounds (total phenols and total flavonoids) and antioxidant activity of emulsions of ethanolic extracts of propolis obtained by ultrasound, during simulated in vitro digestion. The emulsions prepared with propolis extracts were evaluated on certain properties: their emulsion efficiency, stability (zeta potential, particle size, electrical conductivity), content of bioactive compound (total phenolics and total flavonoids), antioxidant activity and their behavior during simulated in vitro digestion. Based on the total phenol content, an emulsification efficiency of 87.8 ± 1.9% to 97.8 ± 3.8% was obtained. The particle size of the emulsions was 322.5 ± 15.33 nm to 463.9 ± 33.65 nm, with a zeta potential of -31.5 ± 0.66 mV to -28.2 ± 1.0 mV and electrical conductivity of 22.7 ± 1.96 µS/cm to 30.6 ± 0.91 µS/cm. These results indicate good emulsion stability. During simulated in vitro digestion, the content of bioactive compounds (total phenolics, total flavonoids) and antioxidant activity were affected during 77 days of storage at 4 °C. It was concluded that the emulsion process fulfills the function of protecting the bioactive compounds and therefore their biological activity.

The Uso1 globular head interacts with SNAREs to maintain viability even in the absence of the coiled-coil domain.

Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering, and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Genetic studies placed Uso1 paradoxically acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S in the GHD that rescued lethality of rab1-deficient Aspergillus nidulans. The mutations are phenotypically additive, their combination suppressing the complete absence of RAB1, which emphasizes the key physiological role of the GHD. In living hyphae Uso1 recurs on puncta (60 s half-life) colocalizing partially with the Golgi markers RAB1, Sed5, and GeaA/Gea1/Gea2, and totally with the retrograde cargo receptor Rer1, consistent with Uso1 dwelling in a very early Golgi compartment from which ER residents reaching the Golgi recycle back to the ER. Localization of Uso1, but not of Uso1E6K/G540S, to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. That Uso1 delocalization correlates with a decrease in the number of Gea1 cisternae supports that Uso1-and-Rer1-containing puncta are where the protein exerts its physiological role. In S-tag-coprecipitation experiments, Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing a stronger association. Using purified proteins, we show that Bos1 and Bet1 bind the Uso1 GHD directly. However, Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. G540S alone markedly increases GHD binding to Bos1, whereas E6K causes a weaker effect, correlating with their phenotypic contributions. AlphaFold2 predicts that G540S increases the binding of the GHD to the Bos1 Habc domain. In contrast, E6K lies in an N-terminal, potentially alpha-helical, region that sensitive genetic tests indicate as required for full Uso1 function. Remarkably, this region is at the end of the GHD basket opposite to the end predicted to interact with Bos1. We show that, unlike dimeric full-length and CTR∆ Uso1 proteins, the GHD lacking the CC/CTR dimerization domain, whether originating from bacteria or Aspergillus extracts and irrespective of whether it carries or not E6K/G540S, would appear to be monomeric. With the finding that overexpression of E6K/G540S and wild-type GHD complement uso1∆, our data indicate that the GHD monomer is capable of providing, at least partially, the essential Uso1 functions, and that long-range tethering activity is dispensable. Rather, these findings strongly suggest that the essential role of Uso1 involves the regulation of SNAREs.

SLC25A46 promotes mitochondrial fission and mediates resistance to lipotoxic stress in INS-1E insulin-secreting cells.

Glucose sensing in pancreatic ß-cells depends on oxidative phosphorylation and mitochondria-derived signals that promote insulin secretion. Using mass spectrometry-based phosphoproteomics to search for downstream effectors of glucose-dependent signal transduction in INS-1E insulinoma cells, we identified the outer mitochondrial membrane protein SLC25A46. Under resting glucose concentrations, SLC25A46 was phosphorylated on a pair of threonine residues (T44/T45) and was dephosphorylated in response to glucose-induced Ca2+ signals. Overexpression of SLC25A46 in INS-1E cells caused complete mitochondrial fragmentation, resulting in a mild mitochondrial defect associated with lowered glucose-induced insulin secretion. In contrast, inactivation of the Slc25a46 gene resulted in dramatic mitochondrial hyperfusion, without affecting respiratory activity or insulin secretion. Consequently, SLC25A46 is not essential for metabolism-secretion coupling under normal nutrient conditions. Importantly, insulin-secreting cells lacking SLC25A46 had an exacerbated sensitivity to lipotoxic conditions, undergoing massive apoptosis when exposed to palmitate. Therefore, in addition to its role in mitochondrial dynamics, SLC25A46 plays a role in preventing mitochondria-induced apoptosis in INS-E cells exposed to nutrient stress. By protecting mitochondria, SLC25A46 might help to maintain ß-cell mass essential for blood glucose control.

The TRAPP complexes: oligomeric exchange factors that activate the small GTPases Rab1 and Rab11.

The Transport Protein Particle (TRAPP) complexes are highly conserved multisubunit complexes that act as nucleotide exchange factors (GEFs) for Rab GTPases. They act in both protein secretion and autophagy and have also been proposed to have a role in other processes such as cytokinesis and ciliogenesis. There are two TRAPP complexes in metazoans: TRAPPII, which activates Rab11; and TRAPPIII, which activates Rab1. Both complexes share a core of small subunits that form the active site for the exchange of GDP for GTP. In addition, each TRAPP complex has distinct large subunits that determine the specificity of each complex towards its substrate Rab and are essential for activity in vivo. Crystal structures have revealed the organisation of the TRAPP core and the mechanism of Rab1 activation, whilst recent cryo-EM structures have unveiled the arrangement of the specific subunits around the core to form each complex. Combining these findings with functional experiments has allowed the proposal of mechanisms for how the specificity of each complex towards their cognate Rab is determined and for the arrangement of these large complexes on the membrane.

Criobiopsia mediastínica guiada por ecobroncoscopia. Un nuevo enfoque, a propósito de tres casos / Transbronchial mediastinal cryobiopsy guided by endobronchial ultrasound. A case series

La punción aspiración transbronquial con aguja guiada por ultrasonido endobronquial (EBUS-TBNA) es a técnica de elección para el estudio de ganglios linfáticos malignos y estratificación del cáncer de pulmón; sin embargo, cada vez se necesitan muestras de mayor tamaño para el estudio molecular. La combinación de esta técnica con la criobiopsia puede ser una forma novedosa para poder obtener un número de muestras mayores y más adecuadas evitando la necesidad de procedimientos repetidos o más invasivos. Presentamos una serie de tres casos donde se realizó la combinación de ambas técnicas para el estudio de lesiones/adenopatías mediastínicas.(AU)

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) ins the technique of choice for study of malignant lymph nodes and lung cancer stratification; however, larger samples are increasingly needs for molecular study. Combining this technique with cryobiopsy may be a novel way to obtain a larger and more adequate number of samples, avoiding the need for repeated or more invasive procedures. We present a series of three cases where the combination of both techniques was performed for study of mediastinal lesions/adenopathy.(AU)

The Application of Ultrasound in Honey: Antioxidant Activity, Inhibitory Effect on α-amylase and α-glucosidase, and In Vitro Digestibility Assessment.

In the present study, the effects of ultrasound (10, 20, and 30 min) on the bioactive compounds, antioxidant capacity, enzymatic inhibition, and in vitro digestion of six honey extracts from the Oaxaca state, Mexico, were analyzed. Significant differences were found in each honey extract with respect to the ultrasonic treatment applied (p < 0.05). In the honey extract P-A1 treated with 20 min of ultrasound, the phenols reached a maximum concentration of 29.91 ± 1.56 mg EQ/100 g, and the flavonoids of 1.92 ± 0.01 mg EQ/100 g; in addition, an inhibition of α-amylase of 37.14 ± 0.09% was noted. There were also differences in the phases of intestinal and gastric digestion, presenting a decrease in phenols (3.92 ± 0.042 mg EQ/100 g), flavonoids (0.61 ± 0.17 mg EAG/100 mg), antioxidant capacity (8.89 ± 0.56 mg EAG/100 mg), and amylase inhibition (9.59 ± 1.38%). The results obtained from this study indicate that, in some honeys, the processing method could increase the concentration of bioactive compounds, the antioxidant capacity, and the enzymatic inhibition; however, when subjected to in vitro digestion, the properties of honey are modified. The results obtained could aid in the development of these compounds for use in traditional medicine as a natural source of bioactive compounds.

The type V myosin-containing complex HUM is a RAB11 effector powering movement of secretory vesicles.

In the apex-directed RAB11 exocytic pathway of Aspergillus nidulans, kinesin-1/KinA conveys secretory vesicles (SVs) to the hyphal tip, where they are transferred to the type V myosin MyoE. MyoE concentrates SVs at an apical store located underneath the PM resembling the presynaptic active zone. A rod-shaped RAB11 effector, UDS1, and the intrinsically disordered and coiled-coil HMSV associate with MyoE in a stable HUM (HMSV-UDS1-MyoE) complex recruited by RAB11 to SVs through an interaction network involving RAB11 and HUM components, with the MyoE globular tail domain (GTD) binding both HMSV and RAB11-GTP and RAB11-GTP binding both the MyoE-GTD and UDS1. UDS1 bridges RAB11-GTP to HMSV, an avid interactor of the MyoE-GTD. The interaction between the UDS1-HMSV sub-complex and RAB11-GTP can be reconstituted in vitro. Ablating UDS1 or HMSV impairs actomyosin-mediated transport of SVs to the apex, resulting in spreading of RAB11 SVs across the apical dome as KinA/microtubule-dependent transport gains prominence.

Proteomics reveals unique plasma signatures in constitutional thinness.

PURPOSE: Studying the plasma proteome of control versus constitutionally thin (CT) individuals, exposed to overfeeding, may give insights into weight-gain management, providing relevant information to the clinical entity of weight-gain resistant CT, and discovering new markers for the condition. EXPERIMENTAL DESIGN: Untargeted protein relative quantification of 63 CT and normal-weight individuals was obtained in blood plasma at baseline, during and after an overfeeding challenge using mass spectrometry-based proteomics. RESULTS: The plasma proteome of CT subjects presented limited specificity with respect to controls at baseline. Yet, CT showed lower levels of inflammatory C-reactive protein and larger levels of protective insulin-like growth factor-binding protein 2. Differences were more marked during and after overfeeding. CT plasma proteome showed larger magnitude and significance in response, suggesting enhanced "resilience" and more rapid adaptation to changes. Four proteins behaved similarly between CT and controls, while five were regulated in opposite fashion. Ten proteins were differential during overfeeding in CT only (including increased fatty acid-binding protein and glyceraldehyde-3-phosphate dehydrogenase, and decreased apolipoprotein C-II and transferrin receptor protein 1). CONCLUSIONS AND CLINICAL RELEVANCE: This first proteomic profiling of a CT cohort reveals different plasma proteomes between CT subjects and controls in a longitudinal clinical trial. Our molecular observations further support that the resistance to weight gain in CT subjects appears predominantly biological. CLINICALTRIALS: gov Identifier: NCT02004821.

Assessing normalization methods in mass spectrometry-based proteome profiling of clinical samples.

BACKGROUND: Large-scale proteomic studies have to deal with unwanted variability, especially when samples originate from different centers and multiple analytical batches are needed. Such variability is typically added throughout all the steps of a clinical research study, from human biological sample collection and storage, sample preparation, spectral data acquisition, to peptide and protein quantification. In order to remove such diverse and unwanted variability, normalization of the protein data is performed. There have been already several published reviews comparing normalization methods in the -omics field, but reports focusing on proteomic data generated with mass spectrometry (MS) are much fewer. Additionally, most of these reports have only dealt with small datasets. RESULTS: As a case study, here we focused on the normalization of a large MS-based proteomic dataset obtained from an overweight and obese pan-European cohort, where different normalization methods were evaluated, namely: center standardize, quantile protein, quantile sample, global standardization, ComBat, median centering, mean centering, single standard and removal of unwanted variation (RUV); some of these are generic normalization methods while others have been specifically created to deal with genomic or metabolomic data. We checked how relationships between proteins and clinical variables (e.g., gender, levels of triglycerides or cholesterol) were improved after normalizing the data with the different methods. CONCLUSIONS: Some normalization methods were better adapted for this particular large-scale shotgun proteomic dataset of human plasma samples labeled with isobaric tags and analyzed with liquid chromatography-tandem MS. In particular, quantile sample normalization, RUV, mean and median centering showed very good performances, while quantile protein normalization provided worse results than those obtained with unnormalized data.

Crosstalk between Drp1 phosphorylation sites during mitochondrial remodeling and their impact on metabolic adaptation.

Mitochondria constantly undergo fusion and fission events, referred as mitochondrial dynamics, which determine mitochondrial architecture and bioenergetics. Cultured cell studies demonstrate that mitochondrial dynamics are acutely regulated by phosphorylation of the mitochondrial fission orchestrator dynamin-related protein 1 (Drp1) at S579 or S600. However, the physiological impact and crosstalk of these phosphorylation sites is poorly understood. Here, we describe the functional interrelation between S579 and S600 phosphorylation sites in vivo and their role on mitochondrial remodeling. Mice carrying a homozygous Drp1 S600A knockin (Drp1 KI) mutation display larger mitochondria and enhanced lipid oxidation and respiratory capacities, granting improved glucose tolerance and thermogenic response upon high-fat feeding. Housing mice at thermoneutrality blunts these differences, suggesting a role for the brown adipose tissue in the protection of Drp1 KI mice against metabolic damage. Overall, we demonstrate crosstalk between Drp1 phosphorylation sites and provide evidence that their modulation could be used in the treatment and prevention of metabolic diseases.

Cryo-EM structure of metazoan TRAPPIII, the multi-subunit complex that activates the GTPase Rab1.

The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo-EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur-TRAPP.

The Addition of Microencapsulated or Nanoemulsified Bioactive Compounds Influences the Antioxidant and Antimicrobial Activities of a Fresh Cheese.

The objective of this study was to compare the effects of the incorporation of microcapsules or nanoemulsions with Opuntiaoligacantha on the quality of fresh cheese. Three treatments were established: Control, cheese with microcapsules (Micro), and cheese with nanoemulsion (Nano). The parameters evaluated were physicochemical (moisture, ash, fat, proteins, and pH), microbiological (mesophilic aerobic bacteria, mold-yeast, and total coliforms), functional (total phenols, flavonoids, and antioxidant capacity), and texture (hardness, elasticity, cohesion, and chewiness) during storage for 45 days at 4 °C. The results showed that adding microcapsules and nanoemulsion did not affect the physicochemical parameters of the cheese. Total coliforms decreased in all samples from the first days of storage (Control: 4.23 ± 0.12, Micro: 3.27 ± 0.02, and Nano: 2.68 ± 0.08 Log10 CFU), as well as aerobic mesophiles and mold-yeast counts. Regarding the functional properties, an increase in total phenols was observed in all treatments. The texture profile analysis showed that the addition of microcapsules and nanoemulsion influenced hardness (Control: 8.60 ± 1.12, Micro: 1.61 ± 0.31, and Nano: 3.27 ± 0.37 N). The antimicrobial effect was greater when nanoemulsions were added, while adding microcapsules influenced the antioxidant activity more positively.

Proteomics of Human Milk: Definition of a Discovery Workflow for Clinical Research Studies.

Milk is a complex biological fluid composed mainly of water, carbohydrates, lipids, proteins, and diverse bioactive factors. Human milk represents a unique tailored source of nutrients that adapts during lactation to the specific needs of the developing infant. Proteins in milk have been studied for decades, and proteomics, peptidomics, and glycoproteomics are the main approaches previously deployed to decipher the proteome of human milk. In the present work, we aimed at implementing a highly automated pipeline for the proteomic analysis of human milk with liquid chromatography mass spectrometry (MS). Commercial human milk samples were used to evaluate and optimize workflows. Centrifugation for defatting milk samples was assessed before and after reduction, alkylation, and enzymatic digestion of proteins, without and with presence of surfactants. Skimmed milk samples were analyzed using isobaric labeling-based quantitative MS on an Orbitrap Tribrid mass spectrometer. Sample fractionation using isoelectric focusing was also evaluated to more deeply profile the human milk proteome. Finally, the most appropriate workflow was transferred to a liquid handling workstation for automated sample preparation. In conclusion, we have defined and describe herein an efficient highly automated proteomic workflow for human milk sample analysis. It is compatible with clinical research, possibly allowing the analysis of sufficiently large cohorts of samples.

Exploration of human cerebrospinal fluid: A large proteome dataset revealed by trapped ion mobility time-of-flight mass spectrometry.

Cerebrospinal fluid (CSF) is a biofluid in direct contact with the brain and as such constitutes a sample of choice in neurological disorder research, including neurodegenerative diseases such as Alzheimer or Parkinson. Human CSF has still been less studied using proteomic technologies compared to other biological fluids such as blood plasma or serum. In this work, a pool of "normal" human CSF samples was analysed using a shotgun proteomic workflow that combined removal of highly abundant proteins by immunoaffinity depletion and isoelectric focussing fractionation of tryptic peptides to alleviate the complexity of the biofluid. The resulting 24 fractions were analysed using liquid chromatography coupled to a high-resolution and high-accuracy timsTOF Pro mass spectrometer. This state-of-the-art mass spectrometry-based proteomic workflow allowed the identification of 3'174 proteins in CSF. The dataset reported herein completes the pool of the most comprehensive human CSF proteomes obtained so far. An overview of the identified proteins is provided based on gene ontology annotation. Mass and tandem mass spectra are made available as a possible starting point for further studies exploring the human CSF proteome.

Effectiveness of Hyperbaric Oxygenation Versus Normobaric Oxygenation Therapy in Carbon Monoxide Poisoning: A Systematic Review.

Carbon monoxide (CO) is a gas product of combustion, considered highly poisonous. Prolonged CO exposure is responsible for more than half of fatal poisonings and is also one of the leading causes of poisoning in Western countries. We aimed to compare the effectiveness of therapy with hyperbaric oxygen (HBO) versus normobaric oxygen (NBO) in the setting of carbon monoxide poisoning (COP). We independently searched the National Library of Medicine's Medline (PubMed™), ScienceDirect™, and Scielo™ for any relevant studies published from 1989 to 2017, using the following keywords: hyperbaric therapy, hyperbaric oxygenation, normobaric therapy, carbon monoxide poisoning, carboxyhemoglobin, Haldane effect. We analyzed the studies that suggested the effectiveness of HBO or NBO. Also, we searched for studies related to COP; including history, epidemiology (risk factors, incidence, demographics), pathophysiology, clinical manifestations, diagnosis, and treatment. Sixty-eight articles were found, sixteen of which dealt with either HBO or NBO or both. Twelve suggested HBO as the treatment of choice in COP; four studies indicated that NBO was an adequate treatment due to its cost-effectiveness and availability in the emergency department (ED). HBO has been shown in several studies to be effective in moderate to high-risk COP situations, being the therapy of choice to avoid sequelae, especially neurologically. NBO can be considered as a reasonable alternative due to its cost-effectiveness. The availability and understanding of different therapeutic interventions are critical in the management of patients with COP in ED and the Critical Care unit.

Antioxidant and antibacterial activities of a starch film with bioextracts microencapsulated from cactus fruits (Opuntia oligacantha).

The use of unconventional sources is very relevant in the food area. In the present study the development of active films with the addition of bioextract (BE) or microencapsulated bioextract (MBE) from xoconostle (Opuntia oligacantha) on chayotextle starch was investigated. The film formulations were: 4 g of chayotextle starch, 2 g of glycerol and 180 g of water, three films with BE added (0.4, 0.8 and 1.2 g) and three films with MBE added (0.4, 0.8 and 1.2 g). Total phenols, total flavonoids, antioxidant activity (ABTS and DPPH), Salmonella typhimurium inhibition, color and mechanical properties of the films were analyzed. The film with 1.2 g of MBE showed high concentration of total phenols (54.12 ± 0.77 mg EAG/100 g), total flavonoids (16.65 ± 0.10 mg QE/100 g) and antioxidant activity (29.11 ± 0.48 and 41.42 ± 1.81 mg EAA for ABTS and DPPH respectively). The addition of bioextract from xoconostle is an option for the development of active films with antioxidant properties.

Influence of Bioactive Compounds Incorporated in a Nanoemulsion as Coating on Avocado Fruits (Persea americana) during Postharvest Storage: Antioxidant Activity, Physicochemical Changes and Structural Evaluation.

The objective of the present study was to determine the effect of the application of a nanoemulsion made of orange essential oil and Opuntia oligacantha extract on avocado quality during postharvest. The nanoemulsion was applied as a coating in whole fruits, and the following treatments were assessed: concentrated nanoemulsion (CN), 50% nanoemulsion (N50), 25% nanoemulsion (N25) and control (C). Weight loss, firmness, polyphenol oxidase (PPO) activity, total soluble solids, pH, external and internal colour, total phenols, total flavonoids, antioxidant activity by 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH), while the structural evaluation of the epicarp was assessed through histological cuts. Significant differences were found (p < 0.05) among the treatments in all the response variables. The best results were with the N50 and N25 treatments for firmness and weight loss, finding that the activity of the PPO was diminished, and a delay in the darkening was observed in the coated fruits. Furthermore, the nanoemulsion treatments maintained the total phenol and total flavonoid contents and potentiated antioxidant activity at 60 days. This histological study showed that the nanoemulsion has a delaying effect on the maturation of the epicarp. The results indicate that using this nanoemulsion as a coating is an effective alternative to improve the postharvest life of avocado.