Biology subjects

Vargas, C.

Publications and source records attributed to Vargas, C..

11 recordsLinked to original sources

bioRxiv2026

Tocilizumab induces significant changes in longitudinal proteomes of blood serum from patients with severe COVID-19 pneumonia

Coronavirus disease 2019 (COVID-19) shows highly variable clinical outcomes that are not fully explained by age or comorbidities, underscoring the importance of host molecular responses in determining disease severity. Proteomic and multi-omics studies have linked severe COVID-19 to profound dysregulation of immune, inflammatory, and coagulation pathways, and have shown that circulating protein signatures can predict clinical trajectories. Tocilizumab (TCZ), a monoclonal antibody targeting the interleukin-6 receptor (IL-6R), is an established therapy for IL-6-driven inflammatory diseases and can normalize aberrant molecular profiles. Here, we applied longitudinal serum proteomics to patients with severe SARS-CoV-2 pneumonia treated with TCZ to further characterize how IL-6R blockade reshapes the systemic inflammatory milieu. After TCZ administration, several clinical and inflammatory markers, including C-reactive protein (CRP), CCL5 and CXCL10, decreased. Proteomic profiling revealed that TCZ exerts a sustained effect on the serum proteome, with the most pronounced changes emerging 7 days after treatment. These changes were associated with a broad reconfiguration of the proteomic profile toward a pattern resembling a healthy physiological state, characterized by the restoration of key protein abundances to levels comparable to those observed under homeostatic conditions. Collectively, our findings support that TCZ treatment contributes to the normalization of the inflammatory state in severe COVID-19 and represents a viable therapeutic option for managing the acute inflammatory phase of the disease, while also highlighting additional pathways and biomarkers involved in this recovery process.

pharmacology and toxicology↗

bioRxiv2026

Umbilical-brain endothelial communication via TSP-1 is linked with reduced brain angiogenesis in offspring of preeclampsia

BackgroundPreeclampsia, a maternal hypertensive syndrome affect fetal brain development and cerebral angiogenesis, with potential acute and long-term consequences. Underlying mechanisms of these brain vascular alterations are unknown. This study investigates the role of thrombospondin-1 (TSP-1), an antiangiogenic glycoprotein, as a key mediator of communication between the fetoplacental and fetal brain endothelium in the context of preeclampsia. MethodsConditioned media (CM) of human umbilical vein endothelial cells (HUVECs) from normal pregnancies (NP-CM) and preeclamptic pregnancies (PE-CM), were used to treat human (hCMEC/D3) and murine brain microvascular endothelial cells (BMECs). A proteomic analysis was performed in plasma of the umbilical cord of normal pregnancy and preeclampsia. TSP-1 was identify using proteomic analysis and confirmed by Western blot. PE-CM depleted of TSP-1, using immunoprecipitation, was used to evaluate protein-protein interaction with vascular endothelial growth factor (VEGF). Antibody-mediated blockage of TSP-1 was used to investigate antiangiogenic effect and pro-angiogenic signaling pathways in brain endothelial cells exposed to PE-CM. ResultsPE-CM significantly reduced angiogenesis, migration, and invasion of brain endothelial cells and altered cytoskeletal organization. These effects were accompanied by reduced VEGFR2 and AKT signaling, indicating impaired angiogenic pathways. Proteomic analysis of umbilical cord plasma revealed elevated TSP-1 levels in preeclampsia, which was confirmed by Western blotting. TSP-1 was also increased in PE-CM, and immunoprecipitation assays suggested a protein-protein interaction with VEGF. Antibody-mediated blockade of TSP-1 restored angiogenesis, as reflected by increased total tube length, and rescued VEGFR2 and AKT signaling in brain endothelial cells exposed to PE-CM. ConclusionTSP-1-mediated endothelium-endothelium communication between placenta-brain axis in offspring of mothers with preeclampsia. This communication mediated by TSP-1 may contribute to acute and long-lasting cerebrovascular dysfunction observed in infants exposed to preeclampsia.

physiology↗

bioRxiv2025

Mouse pups lacking a cerebral cortex develop abnormal vocal behavior

Vocal learning is an essential component of spoken language and critically depends on the cerebral cortex. The evolutionary origins of cortical/pallial control over vocal learning abilities in mammals and songbirds remains largely unknown. For instance, reports conflict on whether the cerebral cortex contributes, in any way, to vocal communication in vocal non-learning mice. Physiological studies in adult mice have shown that regions of the motor cortex have roles in modulation of vocalizations in mice, yet, genetic ablation of the cerebral cortex reportedly has minimal, if any, impact on mouse vocal behavior. Re-analysis of adult acortical mice revealed that deep learning machine classifiers could distinguish mutant ultrasonic vocalizations from wildtypes. However, the specific acoustic features underlying these differences were not identified. Here, we investigated isolation calls of acortical mouse pups using statistical analysis of acoustic features and playback experiments to determine whether mutants lacking a cerebral cortex have altered vocal development. We find that a subset of acoustic features differ between acortical and wildtype pup vocalizations and that these differences are indicative of distress. Moreover, call bouts of acortical pups have lower informational complexity that are more comparable to random probability sampling. Playbacks indicate that dams preferentially approach vocalizations of acortical pups. Our analyses provide evidence that the murine cerebral cortex influences development of complex vocal behaviors, suggesting mice can be used to gain useful insights into the foundations of vocal learning.

neuroscience↗

bioRxiv2025

Clear Native Gel Electrophoresis for the Purification of Fluorescently Labeled Membrane Proteins in Native Nanodiscs

Native gel electrophoresis techniques, such as blue or clear native gel electrophoresis (BNE or CNE), are widely used to separate and characterize proteins. However, in high-resolution CNE, mild anionic or neutral detergents are often used at concentrations too low to prevent membrane-protein aggregation. Additionally, the identification of proteins is hampered by the lack of suitable molecular-weight markers, like those used in SDS-PAGE. Here, we introduce a novel approach that combines charged polymer-encapsulated nanodiscs and fluorescence correlation spectroscopy (FCS) to address both challenges. Membrane proteins are first extracted using Glyco-DIBMA, a negatively charged amphiphilic copolymer. This enables the spontaneous formation of nanodiscs harboring the fluorescently labeled target protein within a native-like lipid-bilayer environment, which is confirmed by FCS. The nanodiscs are then subjected to detergent-free CNE. As the number of protomers increases, the nanodiscs grow larger resulting in increased migration distances in CNE due to higher charge densities. Crucially, the nanodiscs remain intact throughout CNE, as demonstrated by FCS analysis of resolubilized bands excised from the gels. Moreover, the membrane proteins used in this study: a potassium channel (KvAP), a sodium channel (NavMs), a water channel (GlpF), and a urea channel (HpUreI) do not aggregate, as shown by the fluorescent brightnesses and diffusion times of individual nanodiscs. Moreover, the oligomeric states of membrane proteins can be deduced from the brightness per nanodisc. Since purified membrane proteins remain within a native-like lipid-bilayer environment and avoid detergent exposure, they are immediately suitable for downstream structural and functional studies.

biophysics↗

bioRxiv2024

An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins

FtsH, a AAA protease, associates with HflK/C subunits to form a megadalton complex that spans the inner membrane and extends into the periplasm of E. coli. How this complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following overproduction of protein components, recent cryo-EM structures reveal symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit degradation of membrane-embedded substrates. Here, we present structures of native complexes in which HflK/C instead forms an asymmetric nautilus-like assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. The membrane curvature in our FtsH{middle dot}HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid-scramblase activity and possibly with FtsHs function in the degradation of membrane-embedded proteins.

biochemistry↗

bioRxiv2024

Phylogeny, morphology, and behavior of the new ciliate species Stentor stipatus

BackgroundStentor, the genus of large trumpet-shaped ciliates, is well-known for its complex morphology and striking behaviors. Members of this genus are distributed throughout the world in a wide and diverse pool of freshwater ecosystems. Recently, the molecular phylogeny of Stentor has been explored through comparison of 18S small subunit (SSU) ribosomal DNA (rDNA) sequences, clarifying several previously mischaracterized species and species complexes. However, despite their wide distribution, to-date, only about a dozen species of Stentor have been described and verified by phylogenetic means. ResultsHere, we introduce the discovery of a new species within genus Stentor: Stentor stipatus spec. nov., so named for their distinctive cytosolic dark pigmented granules which surround the macronucleus and are also present cortically alongside cortically-distributed green microalgae. We present morphological, phylogenetic, ecological, and behavioral characterizations of these cells. Phylogenetic analysis of S. stipatus spec. nov. by comparison of SSU rDNA sequence suggests it is a distinct species from its closest relative, S. amethystinus. We demonstrate that S. stipatus spec. nov. is capable of habituation in response to repeated mechanical stimulation. Further, S. stipatus spec. nov. exhibits strongly directed positive phototaxis, like its relative S. pyriformis, but with a distinct action spectrum from both S. coeruleus and S. pyriformis. Finally, S. stipatus phototaxis response strength varies in a consistent pattern throughout the day, providing evidence of potential circadian regulation. ConclusionsThis work expands the current understanding of the ecological distribution of and behavioral features present within genus Stentor.

cell biology↗

bioRxiv2024

Capturing G protein-coupled receptors into native lipid-bilayer nanodiscs using new diisobutylene/maleic acid (DIBMA) copolymers

Many membrane proteins, including G-protein-coupled receptors (GPCRs), are susceptible to denaturation when extracted from their native membrane by detergents. Therefore, alternative methods have been developed, including amphiphilic copolymers that enable the direct extraction of functional membrane proteins along with their surrounding lipids, leading to the formation of native lipid-bilayer nanodiscs. Among these amphiphilic copolymers, styrene/maleic acid (SMA) and diisobutylene/maleic acid (DIBMA) polymers have been extensively studied and successfully utilized to isolate various types of membrane proteins, including GPCRs. Despite their many benefits, SMA and DIBMA polymers also have significant drawbacks that limit their application. Most notably, both SMA and DIBMA carry high negative charge densities, which can interfere with protein-protein and protein-lipid interactions through unspecific Coulombic attraction or repulsion. Herein, we describe a series of new amphiphilic copolymers derived from DIBMA via partial amidation of the carboxylate pendant groups with various biocompatible amines. The nanodisc-forming properties of the new polymers were assessed using model membranes as well as in the context of extracting the melanocortin 4 receptor (MC4R), a prototypical class A GPCR. While each new DIBMA variant displays distinct features that may be favorable for selected applications, we identified a new PEGylated DIBMA variant called mPEG4-DIBMA as particularly promising for the studied purpose. On the one hand, mPEG4-DIBMA abolishes unspecific interactions with the tested peptide ligand, a prerequisite for reliably characterizing GPCR-ligand interactions. On the other hand, mPEG4-DIBMA outperforms other polymers such as SMA and DIBMA by achieving higher extraction efficiencies of MC4R from Sf9 insect cell membranes. Thus, this new nanodisc-forming polymer combines two key advantages that are crucial for investigating GPCRs in a well-defined but still native lipid-bilayer environment, thus paving the way for manifold future applications.

biochemistry↗

bioRxiv2023

Effects of Polyphosphate metabolism Mutations on Biofilm, Capsule Formation and Virulence Traits in Hypervirulent ST23 Klebsiella pneumoniae SGH10

The emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains poses a significant threat to public health due to their high mortality rates and propensity to cause severe community-acquired infections in otherwise healthy individuals. The ability of hvKP to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Therefore, understanding the molecular mechanisms underlying hvKP virulence and biofilm formation is crucial for developing new therapeutic strategies. Polyphosphate Kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using Dictyostelium discoideum amoeba as a model host. We found that the PPK1 null-mutant was impaired in biofilm and capsule formation and showed attenuated virulence in D. discoideum compared to the wild-type strain. We performed a shotgun proteomic analysis of the PPK1 mutant and wild-type strain to gain further insight into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins (DEP) involved in capsule synthesis (Wzi - Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB -LpxC - PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hypervirulent K. pneumoniae. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in K. pneumoniae and provides insights into potential therapeutic targets for treating K. pneumoniae infections.

microbiology↗

bioRxiv2023

The intrinsically disordered region of the E3 ubiquitin ligase TRIP12 induces the formation of chromatin condensates and interferes with DNA damage response.

Chromatin compaction is crucial for the faithful expression and integrity of the genome. Although largely studied, proteins and mechanisms that control the chromatin compaction are not entirely discovered. We previously showed that the nuclear HECT-type E3 ubiquitin ligase Thyroid hormone Receptor Interacting Protein 12 (TRIP12) is tightly associated to chromatin. As TRIP12 is overexpressed in several types of cancers, we explored herein the consequences of a TRIP12 overexpression on chromatin homeostasis. First, we established the TRIP12 proxisome and unveiled its pleiotropic role in chromatin regulation. Second, we demonstrated that TRIP12 overexpression leads to the formation of chromatin condensates enriched in heterochromatin marks via its intrinsically disordered region (IDR). We further discovered that the formation of TRIP12-mediated chromatin condensates is highly dynamic and driven by a mechanism of phase separation. Chromatin condensate formation depends on the TRIP12 concentration, the length of the TRIP12-IDR and relies on electrostatic interactions. We found that the formation of TRIP12 mediated-condensates alters cell cycle progression, genome accessibility, transcription as well as DNA damage response by inhibiting the accumulation of Mediator of DNA Damage Checkpoint 1 (MDC1). Altogether, this study reveals a novel dynamic role for TRIP12 in chromatin compaction independently of its ubiquitin ligase activity with important consequences on cellular homeostasis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/556486v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@13b3d90org.highwire.dtl.DTLVardef@46b991org.highwire.dtl.DTLVardef@1410bdeorg.highwire.dtl.DTLVardef@1722197_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

The Loss of the E3 ubiquitin ligase TRIP12 inhibits Pancreatic Acinar Cell Plasticity and Tumor Cell Metastatic Capacity

Background & AimsAlthough specialized and dedicated to the production of digestive enzymes, pancreatic acinar cells harbor a high plasticity and are able to modify their identity. They undergo reversible acinar-to-ductal cell metaplasia (ADM) through epigenetic silencing of the acinar lineage gene program mainly controlled by PTF1a (Pancreas Transcription Factor 1a). ADM becomes irreversible in the presence of oncogenic Kras mutations and leads to the formation of preneoplastic lesions. We investigated the role of the E3 ubiquitin ligase Thyroid hormone Receptor Interacting Protein 12 (TRIP12), involved in PTF1a degradation, in pancreatic carcinogenesis. MethodsWe used genetically engineered mouse models of pancreas-selective Trip12 deletion, mutant Kras (G12D) and mutant Trp53 (R172H). We performed RNA sequencing analysis from acinar cells and cell lines derived from mice models tumors. We investigated the impact of TRIP12 deficiency on acute pancreatitis, tumor formation and metastasis development. ResultsTRIP12 is overexpressed in human pancreatic preneoplastic lesions and tumors. We show that a conditional deletion of TRIP12 in the pancreas during murine embryogenesis alters pancreas homeostasis and acinar cell genes expression patterns in adults. EGF induced-ADM is suppressed in TRIP12-depleted pancreatic acini. In vivo, a loss of TRIP12 prevents acini to develop ADM in response to pancreatic injury, the formation of Kras-induced pancreatic preneoplastic lesions, and impairs tumors and metastasis formation in the presence of mutated Trp53. TRIP12 is required for Claudin18.2 isoform expression in pancreatic tumors cells. ConclusionsOur study identifies TRIP12 as a novel regulator of acinar fate in the adult pancreas with an important dual role in pancreatic carcinogenesis, in initiation steps and in metastatic behavior of tumor cells. SynopsisThis study shows that Thyroid hormone Receptor Interacting Protein 12 plays an important dual role in the initiation steps and invasion of pancreatic carcinogenesis. Moreover, expression of TRIP12 switches on the expression of Claudin-18, a targetable biomarker of pancreatic tumors.

cancer biology↗

bioRxiv2021

A Bioinspired Glycopolymer for Capturing Membrane Proteins in Native-Like Lipid-Bilayer Nanodiscs

Amphiphilic copolymers that directly extract membrane proteins and lipids from cellular membranes to form nanodiscs combine the advantages of harsher membrane mimics with those of a native-like membrane environment. Among the few commercial polymers that are capable of forming nanodiscs, alternating diisobutylene/maleic acid (DIBMA) copolymers have gained considerable popularity as gentle and UV-transparent alternatives to aromatic polymers. However, their moderate hydrophobicities and high electric charge densities render all existing aliphatic copolymers rather inefficient under near-physiological conditions. Here, we introduce Glyco-DIBMA, a bioinspired glycopolymer that possesses increased hydrophobicity and reduced charge density but nevertheless retains excellent solubility in aqueous solutions. Glyco-DIBMA outperforms established aliphatic copolymers in that it solubilizes lipid vesicles of various compositions much more efficiently, thereby furnishing smaller, more narrowly distributed nanodiscs that preserve a bilayer architecture and exhibit rapid lipid exchange. We demonstrate the superior performance of Glyco-DIBMA in preparative and analytical applications by extracting a broad range of integral membrane proteins from cellular membranes and further by purifying a membrane-embedded voltage-gated K+ channel, which was fluorescently labeled and analyzed with the aid of microfluidic diffusional sizing (MDS) directly within native-like lipid-bilayer nano-discs.

biochemistry↗