Biology subjects

Pacheco, D.

Publications and source records attributed to Pacheco, D..

9 recordsLinked to original sources

bioRxiv2025

Mucus-associated microbiota consortia: Insights from the advanced TIM-2muc model

The mucus layer lining the epithelium of the colon contains a microbiome that is distinct from the luminal microbiome; this is referred to as the mucus-associated microbiome (MAM). Recently, research on the MAM has gained momentum due to its significant role in immune and metabolic health. However, the study of the MAM is limited by conventional experimental techniques, as sampling this microbiome often requires invasive procedures, making in vivo research challenging. Additionally, obtaining samples from the MAM can compromise the lumen, complicating simultaneous studies of both the MAM and the lumen. To address these limitations, we present the TIM-2muc, an advanced version of the colonic in vitro model TIM-2, which includes a mucus compartment comprising both colonic mucus beads - Gut3Beads and an electrospun sheet coated with Gut3Gel. To validate whether the integrated mucus compartment behaves like the in vivo MAM, we examined the bacterial composition, alpha and beta diversity, and the MAMs composition compared to the lumen compartment of both the TIM-2muc and the conventional TIM-2 model. Our findings suggest that the MAM in the TIM-2muc model closely resembles its in vivo counterpart, thereby enhancing the physiological relevance of the luminal microbiome. TIM-2muc, an advanced TIM-2 model, presents a novel, precise, standardized, and non-invasive method for studying the MAM and LM simultaneously.

microbiology↗

bioRxiv2025

Gut3Gel as an in vitro model to investigate dietary modulation of the intestinal microbiota: An inulin supplementation case study

The intestinal microbiota plays a key role in human health, influencing digestion, immunity, and metabolism. While factors such as genetics and medications shape its composition, diet remains a primary driver of microbial modulation. Despite growing interest in using dietary interventions to beneficially alter the intestinal microbiota, assessing their effects in humans is challenging due to individual variability and the complexity of in vivo systems. This study explores Gut3Gel gradient colonic (G3GG) preclinical model as a tool to assess its representativeness on studying the effect of inulin supplementation on the intestinal microbiota of five healthy individuals compared to clinical evaluation studies. Significant inter-individual variability in baseline microbiota composition was observed, which strongly influenced microbiota response to inulin. While inulin supplementation led to a general decrease in alpha diversity, it significantly increased the abundance of health-associated genera such as Bifidobacterium and Lacticaseibacillus, along with enhanced microbial metabolic activity. Despite the intrinsic selectivity of G3GG for beneficial microbes, the model successfully captured inulins prebiotic effects and inter-individual differences, underscoring its relevance as a physiologically relevant high throughput tool for evaluating microbiota-targeting compounds and supporting personalized nutrition strategies.

microbiology↗

bioRxiv2024

Distinct representational properties of cues and contexts shape fear learning and extinction

When we learn that something is dangerous, a fear memory is formed. However, this memory is not fixed and can be updated through new experiences, such as learning that the threat is no longer present. This process of updating, known as extinction or reversal learning, is highly dependent on the context in which it occurs. How the brain represents cues, contexts, and their changing threat value remains a major question. Here, we used functional magnetic resonance imaging and a novel fear learning paradigm to track the neural representations of stimuli across fear acquisition, reversal, and test phases. We found that initial fear learning creates generalized neural representations for all threatening cues in the brains fear network. During reversal learning, when threat contingencies switched for some of the cues, two distinct representational strategies were observed. On the one hand, we still identified generalized patterns for currently threatening cues, whereas on the other hand, we observed highly stable representations of individual cues (i.e., item-specific) that changed their valence, particularly in the precuneus and prefrontal cortex. Furthermore, we observed that the brain represents contexts more distinctly during reversal learning. Furthermore, additional exploratory analyses showed that the degree of this context specificity in the prefrontal cortex predicted the subsequent return of fear, providing a potential neural mechanism for fear renewal. Our findings reveal that the brain uses a flexible combination of generalized and specific representations to adapt to a changing world, shedding new light on the mechanisms that support cognitive flexibility and the treatment of anxiety disorders via exposure therapy.

neuroscience↗

bioRxiv2024

Retrieval of human aversive memories involves reactivation of gamma activity patterns in the hippocampus that originate in the amygdala during encoding

Emotional memories require coordinated activity of the amygdala and hippocampus. Human intracranial recordings have shown that formation of aversive memories involves an amygdala theta-hippocampal gamma phase code. Yet, the mechanisms engaged during translation of aversive experiences into memories and subsequent retrieval remain unclear. Directly recording from human amygdala and hippocampus, here we show that hippocampal gamma activity increases for correctly remembered aversive scenes, while exerting unidirectional oscillatory influence within the theta/beta frequency range on the amygdala for previously seen aversive scenes. Crucially, patterns of amygdala high amplitude gamma activity at encoding are reactivated in the hippocampus, but not amygdala, during both aversive encoding and retrieval. Trial-specific hippocampal gamma patterns showing highest representational similarity with amygdala activity at encoding are replayed in the hippocampus during aversive retrieval. This reactivation process occurs against a background of gamma activity that is otherwise decorrelated between encoding and retrieval. Thus, retrieval of aversive memories is hippocampal-centered, with hippocampal activity patterns apparently entrained by the amygdala during encoding.

neuroscience↗

bioRxiv2022

Ventral Hippocampal Infusions of C20:0 Ceramide Stimulate Microglia and Induce Anhedonia-like Behavior in Female and Male Rats

Increased long-chain C20:0 ceramides have been found in the serum of patients with depression. Moreover, ceramides are linked with increased microglia reactivity and inflammatory cytokine production, which are associated with depression. Since ceramides can readily cross the blood brain barrier, peripheral C20:0 ceramides could enter the brain, activate microglia, and induce depressive-like behavior. In this study, we determined whether localized infusion of C20:0 ceramides into the ventral hippocampus (VH) of rats is sufficient to activate microglia and induce depressive-like behaviors. Adult male and female rats received infusions of C20:0 ceramides or vehicle solution every other day for 2 weeks. After the third infusion, C20:0-infused animals showed reduced sucrose preference suggesting anhedonia-like behavior. However, 4-6 days after the last infusion, animals infused with C20:0 ceramides did not show anhedonia-like or despair-like behavior suggesting the behavioral effect was reversible. After the final behavioral test, C20:0-infused animals had more Iba-1+ microglia in the VH. Despite the increased number of VH microglia, the expression of inflammatory genes was not increased. In conclusion, our data suggest that localized increases in C20:0 ceramides in the VH are sufficient to induce microglia activation and reversible anhedonia-like behavior.

neuroscience↗

bioRxiv2019

Mediator subunit Med15 dictates the conserved 'fuzzy' binding mechanism of yeast transcription activators Gal4 and Gcn4

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in both transcription repression and activation through sequence-dependent binding to Gal80 repressor and sequence-independent binding to Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a "fuzzy" protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two ADs of different sequence, interact nearly identically with Med15. The findings that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts with Gal80 via a tight structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein interaction.

molecular biology↗

bioRxiv2019

Auditory Activity is Diverse and Widespread Throughout the Central Brain of Drosophila

Sensory pathways are typically studied by starting at receptor neurons and following postsynaptic neurons into the brain. However, this leads to a bias in analysis of activity towards the earliest layers of processing. Here, we present new methods for volumetric neural imaging with precise across-brain registration, to characterize auditory activity throughout the entire central brain of Drosophila and make comparisons across trials, individuals, and sexes. We discover that auditory activity is present in most central brain regions and in neurons known to carry responses to other modalities. Auditory responses are temporally diverse, but the majority of activity, regardless of brain region, is tuned to aspects of courtship song. We find that auditory responses are stereotyped across trials and animals in early mechanosensory regions, becoming more variable at higher layers of the putative pathway, and that this variability is largely independent of spontaneous movements. This study highlights the power of using an unbiased, brain-wide approach for mapping the functional organization of sensory activity.

neuroscience↗

bioRxiv2017

Transcription activation domains of the yeast factors Met4 and Ino2: tandem activation domains with properties similar to the yeast Gcn4 activator

Eukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation and chromatin modifications. Here we examine the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies. While AD function from both factors depends on hydrophobic residues, Ino2 further requires key conserved acidic and polar residues for optimal function. Binding studies show that the ADs bind multiple Med15 activator binding domains (ABDs) with a similar order of micromolar affinity, and similar but distinct thermodynamic properties. Protein crosslinking shows that no unique complex is formed upon Met4-Med15 binding. Rather, we observed heterogeneous AD-ABD contacts with nearly every possible AD-ABD combination. Many of these properties are similar to those observed with the yeast activator Gcn4, which forms a large heterogeneous, dynamic, and fuzzy complex with Med15. We suggest that this molecular behavior is common among eukaryotic activators.

molecular biology↗

bioRxiv2017

Transcription activator-coactivator specificity is mediated by a large and dynamic fuzzy protein-protein complex

Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient activation by yeast Gcn4 requires tandem Gcn4 ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous, containing nearly all possible AD-ABD interactions. This complex forms using a dynamic fuzzy protein-protein interface where ADs use hydrophobic residues to bind hydrophobic surfaces of the ABDs in multiple orientations. This combinatorial mechanism allows individual interactions of low affinity and specificity to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators and allows great flexibility in combinations of activators that synergize to regulate genes with variable coactivator requirements.

molecular biology↗