Biology subjects

Castellano, M.

Publications and source records attributed to Castellano, M..

4 recordsLinked to original sources

bioRxiv2024

Ribonuclease activity undermines immune sensing of naked extracellular RNA

The plasma membrane and the membrane of endosomal vesicles are considered physical barriers preventing extracellular RNA uptake. While naked RNA can be spontaneously internalized by certain cells types, functional delivery of naked RNA into the cytosol has been rarely observed. Here we show that extracellular ribonucleases, mainly derived from cell culture supplements, have so far hindered the study of extracellular RNA functionality. In the presence of active ribonuclease inhibitors (RI), naked bacterial RNA is pro-inflammatory when spiked in the media of dendritic cells and macrophages. In murine cells, this response mainly depends on the action of endosomal Toll-like receptors. However, we also show that naked RNA can perform endosomal escape and engage with cytosolic RNA sensors and ribosomes. For example, naked mRNAs encoding reporter proteins can be spontaneously internalized and translated by a variety of cell types, in an RI-dependent manner. In vivo, RI co-injection enhances the activation induced by naked extracellular RNA on splenic lymphocytes and myeloid-derived leukocytes. Furthermore, naked extracellular RNA is inherently pro-inflammatory in ribonuclease-poor compartments such as the peritoneal cavity. Overall, these results demonstrate that naked RNA is bioactive and does not need encapsulation inside synthetic or biological lipid vesicles for functional uptake, making a case for nonvesicular extracellular RNA-mediated intercellular communication.

molecular biology↗

bioRxiv2022

Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids

Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of up to three hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.

molecular biology↗

bioRxiv2019

Resting Eyes Closed Beta-Phase High Gamma-Amplitude Coupling Deficits in Children with Attention Deficit Hyperactivity Disorder

ObjectiveAttention-deficit hyperactivity disorder (ADHD) is the neurobehavioral disorder with the largest prevalence rate in childhood. ADHD is generally assessed based on physical examination of the child and interviews, and therefore prone to subjectivity. This fact may lead to a high risk of mis- and over-diagnosis, a problem that can be addressed through the use of objective markers.\n\nMethodsHere we propose to use phase-amplitude coupling as a digital biomarker in ADHD. We investigated the hypothesis that coupling between the phase of slow brain rhythms and the amplitude of fast rhythms is altered in the ADHD population. We tested this hypothesis measuring phase-amplitude coupling (PAC) in the 4 to 200Hz range in electroencephalographic (EEG) signals recorded in the central-frontal area in children during eyes closed resting state.\n\nResultsUsing automatic clustering, we observed statistically significant beta-gamma PAC deficits in the ADHD population in the frontal-left hemisphere.\n\nConclusionsThese findings suggests alterations in the beta-gamma coupling in the ADHD population. We discuss the hypothesis that these alterations may be indicators of working memory and attention deficits.\n\nSignificanceThe study of the coupling between the different brain rhythms can potentially contribute to the understanding and clinical diagnosis of ADHD.

neuroscience↗

bioRxiv2017

Occipital tACS bursts during a visual task impact ongoing neural oscillation power, coherence and LZW complexity

Little is known about the precise neural mechanisms by which tACS affects the human cortex. Current hypothesis suggest that transcranial current stimulation (tCS) can directly enhance ongoing brain oscillations and induce long - lasting effects through the activation of synaptic plasticity mechanisms [1]. Entrainment has been demonstrated in in - vitro studies, but its presence in non-invasive human studies is still under debate [2,3]. Here, we aim to investigate the immediate and short-term effects of tACS bursts on the occipital cortex of participants engaged in a change - of - speed detection task, a task that has previously reported to have a clear physiology - behavior relationship, where trials with faster responses also have increased power in {gamma} - oscillations (50 - 80 Hz) [4]. The dominant brain oscillations related to the visual task are modulated using multichannel tACS at 10 and 70 Hz within occipital cortex. We found that tACS stimulation at 10 Hz (tACS 10) enhanced both (8 - 13 Hz) and {gamma} oscillations, in hand with an increase in reaction time (RT) in the change - of - speed detection visual task. On the other hand, tACS at 70Hz desynchronized visual cortices, impairing both phase - locked and endogenous {gamma} - power while increasing RT. While both tACS protocols seem to revert the relationship reported in [4], we argue that tACS produces a shift in attentional resources within visual cortex while leaving unaltered the resources required to conduct the task. This theory is supported by the fact that the correlation between fast RT and high {gamma}- power trials is maintained for tACS sessions too. Finally, we measured cortical excitability by analyzing Event - Related - Potentials (ERP) Lempel - Ziv - Welch Complexity (LZW). In control sessions we observe that lower {gamma} - LZW complexity correlates to faster reaction times. Both metrics are altered by tACS stimulation, as tACS 10 decreased amplitude of the P300 peak, while increasing {gamma}- LZW complexity. To this end, our study highlights the nonlinear cross - frequency interaction between exogenous stimulation and endogenous brain dynamics, and proposes the use of complexity metrics, as LZW, to characterize excitability patterns of cortical areas in a behaviorally relevant timescale. These insights will hopefully contribute to the design of adaptive and personalized tACS protocols where cortical excitability can be characterized through complexity metrics.\n\nAdditional Title Page FootnotesO_LIWe introduce a bursting tACS protocol to study semi-concurrent tACS effects in the visual system and their impact on behavior as measured by reaction time.\nC_LIO_LIBurst 10 Hz tACS (tACS10) applied to the visual cortex entrained {gamma}-oscillations and increased RTs in a change-of-speed detection visual task more than 70 Hz tACS (tACS70) or Control conditions.\nC_LIO_LIBurst tACS10 also decreased amplitude of the P300 peak, while increasing -power and {gamma}-LZW complexity.\nC_LIO_LIPhysiological and behavioral impact of occipital tACS10 and tACS70 was frequency-specific. tACS70 reduced {gamma}-oscillations after 20min of tACS stimulation.\nC_LIO_LICognitive task may determine cortical excitation levels as measured by complexity metrics, as lower {gamma}-LZW complexity correlates to faster reaction times.\nC_LI

neuroscience↗