Biology subjects

Lombardi, L.

Publications and source records attributed to Lombardi, L..

9 recordsLinked to original sources

bioRxiv2024

Development and evaluation of a non-invasive brain-spine interface using transcutaneous spinal cord stimulation

Motor rehabilitation is a therapeutic process to facilitate functional recovery in people with spinal cord injury (SCI). However, its efficacy is limited to areas with remaining sensorimotor function. Spinal cord stimulation (SCS) creates a temporary prosthetic effect that may allow further rehabilitation-induced recovery in individuals without remaining sensorimotor function, thereby extending the therapeutic reach of motor rehabilitation to individuals with more severe injuries. In this work, we report our first steps in developing a non-invasive brain-spine interface (BSI) based on electroencephalography (EEG) and transcutaneous spinal cord stimulation (tSCS). The objective of this study was to identify EEG-based neural correlates of lower limb movement in the sensorimotor cortex of unimpaired individuals and to quantify the performance of a linear discriminant analysis (LDA) decoder in detecting movement onset from these neural correlates. Our results show that initiation of knee extension was associated with event-related desynchronization in the central-medial cortical regions at frequency bands between 4-44 Hz. Our neural decoder using {micro} (8-12 Hz), low {beta} (16-20 Hz), and high {beta} (24-28 Hz) frequency bands achieved an average area under the curve (AUC) of 0.83 {+/-} 0.06 s.d. (n = 7) during a cued movement task offline. Generalization to imagery and uncued movement tasks served as positive controls to verify robustness against movement artifacts and cue-related confounds, respectively. With the addition of real-time decoder-modulated tSCS, the neural decoder performed with an average AUC of 0.81 {+/-} 0.05 s.d. (n = 9) on cued movement and 0.68 {+/-} 0.12 s.d. (n = 9) on uncued movement. Our results suggest that the decrease in decoder performance in uncued movement may be due to differences in underlying cortical strategies between conditions. Furthermore, we explore alternative applications of the BSI system by testing neural decoders trained on uncued movement and imagery tasks. By developing a non-invasive BSI, tSCS can be timed to be delivered only during voluntary effort, which may have implications for improving rehabilitation.

neuroscience↗

bioRxiv2024

Confinement and Catalysis Within De Novo Designed Peptide Barrels

De novo protein design has advanced such that many peptide assemblies and protein structures can be generated predictably and quickly. The drive now is to bring functions to these structures, for example, small-molecule binding and catalysis. The formidable challenge of binding and orienting multiple small molecules to direct chemistry is particularly important for paving the way to new functionalities. To address this, here we describe the design, characterization, and application of small-molecule:peptide ternary complexes in aqueous solution. This uses -helical barrel (HB) peptide assemblies, which comprise 5 or more -helices arranged around central channels. These channels are solvent accessible, and their internal dimensions and chemistries can be altered predictably. Thus, HBs are analogous to molecular flasks made in supramolecular, polymer, and materials chemistry. Using Forster resonance energy transfer as a readout, we demonstrate that specific HBs can accept two different organic dyes, 1,6-diphenyl-1,3,5-hexatriene and Nile Red in close proximity. In addition, two anthracene molecules can be accommodated within an HB to promote photocatalytic anthracene-dimer formation. However, not all ternary complexes are productive, either in energy transfer or photocatalysis, illustrating the control that can be exerted by judicious choice and design of the HB.

synthetic biology↗

bioRxiv2024

Fundamental limitations of kilohertz-frequency carriers in afferent fiber recruitment with transcutaneous spinal cord stimulation

The use of kilohertz-frequency (KHF) waveforms has rapidly gained momentum in transcutaneous spinal cord stimulation (tSCS) to restore motor function after paralysis. However, the mechanisms by which these fast-alternating currents depolarize efferent and afferent fibers remain unknown. Our study fills this research gap by providing a hypothesis-and evidence-based investigation using peripheral nerve stimulation, lumbar tSCS, and cervical tSCS in 25 unimpaired participants together with computational modeling. Peripheral nerve stimulation experiments and computational modeling showed that KHF waveforms negatively impact the processes required to elicit action potentials, thereby increasing response thresholds and biasing the recruitment towards efferent fibers. While these results translate to tSCS, we also demonstrate that lumbar tSCS results in the preferential recruitment of afferent fibers, while cervical tSCS favors recruitment of efferent fibers. Given the assumed importance of proprioceptive afferents in motor recovery, our work suggests that the use of KHF waveforms should be reconsidered to maximize neurorehabilitation outcomes, particularly for cervical tSCS. We posit that careful analysis of the mechanisms that mediate responses elicited by novel approaches in tSCS is crucial to understanding their potential to restore motor function after paralysis.

neuroscience↗

bioRxiv2023

Enhanced selectivity of transcutaneous spinal cord stimulation by multielectrode configuration

ObjectiveTranscutaneous spinal cord stimulation (tSCS) has been gaining momentum as a non-invasive rehabilitation approach to restore movement to paralyzed muscles after spinal cord injury (SCI). However, its low selectivity limits the types of movements that can be enabled and, thus, its potential applications in rehabilitation. ApproachIn this cross-over study design, we investigated whether muscle recruitment selectivity of individual muscles could be enhanced by multielectrode configurations of tSCS in 16 neurologically intact individuals. We hypothesized that due to the segmental innervation of lower limb muscles, we could identify muscle-specific optimal stimulation locations that would enable improved recruitment selectivity over conventional tSCS. We elicited leg muscle responses by delivering biphasic pulses of electrical stimulation to the lumbosacral enlargement using conventional and multielectrode tSCS. ResultsAnalysis of recruitment curve responses confirmed that multielectrode configurations could improve the rostrocaudal and lateral selectivity of tSCS. To investigate whether motor responses elicited by spatially selective tSCS were mediated by posterior root-muscle reflexes, each stimulation event was a paired pulse with a conditioning-test interval of 33.3 ms. Muscle responses to the second stimulation pulse were significantly suppressed, a characteristic of post-activation depression suggesting that spatially selective tSCS recruits proprioceptive fibers that reflexively activate muscle-specific motor neurons in the spinal cord. Moreover, the combination of leg muscle recruitment probability and segmental innervation maps revealed a stereotypical spinal activation map in congruence with each electrodes position. SignificanceImprovements in muscle recruitment selectivity could be essential for the effective translation into stimulation protocols that selectively enhance single-joint movements in neurorehabilitation.

neuroscience↗

bioRxiv2022

CRISPR-Cas9 editing induces Loss of Heterozygosity in the pathogenic yeast Candida parapsilosis

Genetic manipulation is often used to study gene function. However, unplanned genome changes (including Single Nucleotide Polymorphisms (SNPs), aneuploidy and Loss of Heterozygosity (LOH)) can affect the phenotypic traits of the engineered strains. Here, we show that CRISPR-Cas9 editing can induce LOH in the diploid human pathogenic yeast Candida parapsilosis. We sequenced the genomes of ten isolates that were edited with CRISPR-Cas9 and found that the designed changes were present in nine. However, we also observed LOH in all isolates, and aneuploidy in two isolates. LOH occurred most commonly downstream of the Cas9 cut site and extended to the telomere in three isolates. In two isolates we observed LOH on chromosomes that were not targeted by CRISPR-Cas9. Two different isolates exhibited cysteine and methionine auxotrophy caused by LOH at a heterozygous site in MET10, approximately 11 and 157 kb downstream from the Cas9 target site, respectively. C. parapsilosis isolates have relatively low levels of heterozygosity. However, our results show that mutation complementation to confirm observed phenotypes is important even when using CRISPR-Cas9, which is now the gold standard of genetic engineering. IMPORTANCECRISPR-Cas9 has greatly streamlined gene editing, and is now the gold standard and first choice for genetic engineering. However, we show that in diploid species extra care should be taken in confirming the cause of any phenotypic changes observed. We show that the Cas9-induced double strand break is often associated with loss of heterozygosity in the asexual diploid human fungal pathogen Candida parapsilosis. This can result in deleterious heterozygous variants (e.g. stop gain in one allele) becoming homozygous resulting in unplanned phenotypic changes. Our results stress the importance of mutation complementation even when using CRISPR-Cas9.

microbiology↗

bioRxiv2020

Reaching to sounds in virtual reality: A multisensory-motor approach to re-learn sound localisation

When localising sounds in space the brain relies on internal models that specify the correspondence between the auditory input reaching the ears and initial head-position with coordinates in external space. These models can be updated throughout life, setting the basis for re-learning spatial hearing abilities in adulthood. This is particularly important for individuals who experience long-term auditory alterations (e.g., hearing loss, hearing aids, cochlear implants) as well as individuals who have to adapt to novel auditory cues when listening in virtual auditory environments. Until now, several methodological constraints have limited our understanding of the mechanisms involved in spatial hearing re-learning. In particular, the potential role of active listening and head-movements have remained largely overlooked. Here, we overcome these limitations by using a novel methodology, based on virtual reality and real-time kinematic tracking, to study the role of active multisensory-motor interactions with sounds in the updating of sound-space correspondences. Participants were immersed in a virtual reality scenario showing 17 speakers at ear-level. From each visible speaker a free-field real sound could be generated. Two separate groups of participants localised the sound source either by reaching or naming the perceived sound source, under binaural or monaural listening. Participants were free to move their head during the task and received audio-visual feedback on their performance. Results showed that both groups compensated rapidly for the short-term auditory alteration caused by monaural listening, improving sound localisation performance across trials. Crucially, compared to naming, reaching the sounds induced faster and larger sound localisation improvements. Furthermore, more accurate sound localisation was accompanied by progressively wider head-movements. These two measures were significantly correlated selectively for the Reaching group. In conclusion, reaching to sounds in an immersive visual VR context proved most effective for updating altered spatial hearing. Head movements played an important role in this fast updating, pointing to the importance of active listening when implementing training protocols for improving spatial hearing. HIGHLIGHTS- We studied spatial hearing re-learning using virtual reality and kinematic tracking - Audio-visual feedback combined with active listening improved monaural sound localisation - Reaching to sounds improved performance more than naming sounds - Monaural listening triggered compensatory head-movement behaviour - Head-movement behaviour correlated with re-learning only when reaching to sounds

neuroscience↗

bioRxiv2020

The yeast mating-type switching endonuclease HO is a domesticated member of an unorthodox homing genetic element family

The mating-type switching endonuclease HO plays a central role in the natural life cycle of Saccharomyces cerevisiae, but its evolutionary origin is unknown. HO is a recent addition to yeast genomes, present in only a few genera. It resembles a degenerated intein fused to a zinc finger domain. Here we show that HO is structurally and phylogenetically related to a family of unorthodox homing genetic elements found in Torulaspora and Lachancea yeasts. These WHO elements integrate into the aldolase gene FBA1, replacing its 3 end each time. Their structural organization is different from all known classes of homing elements. We show that a WHO protein cleaves Torulaspora delbrueckii FBA1 efficiently and in an allele-specific manner, leading to DNA repair by gene conversion or NHEJ. The DNA rearrangement steps during WHO element homing are very similar to those during mating-type switching, and indicate that HO is a domesticated WHO-like element.

evolutionary biology↗

bioRxiv2019

Plasmid-based CRISPR-Cas9 gene editing in multiple Candida species

Many Candida species that cause infection have diploid genomes, and do not undergo classical meiosis. The application of CRISPR-Cas9 gene editing systems have therefore greatly facilitated the generation of gene disruptions, and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step, and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis and C. metapsilosis. We also show that CRISPR can easily be used to introduce molecular barcodes, and to reintroduce wild type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site, or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis. We show that editing can easily be carried out in multiple C. tropicalis isolates. NHEJ repair occurs at a high level in C. metapsilosis and C. tropicalis. IMPORTANCECandida species are a major cause of infection worldwide. The species associated with infection vary with geographical location, and patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis. We have also constructed a separate system for gene editing in C. tropicalis.

genetics↗

bioRxiv2018

Evaluation of Sb-1 bacteriophage activity in enhancing antibiotic efficacy against biofilm, degrading the exopolysaccharide matrix and targeting persister cells of Staphylococcus aureus

Most research on phage therapy focused on planktonic bacteria, whereas bacteriophage activity against biofilms is limited. We evaluated the capability of Staphylococcus aureus-specific bacteriophage Sb-1 to eradicate biofilm alone and in combination with different classes of antibiotics, to degrade the extracellular matrix and target persister cells. Biofilm of methicillin-resistant S. aureus (MRSA) ATCC 43300 was treated with Sb-1 alone or in (simultaneous or staggered) combination with either fosfomycin, rifampin, vancomycin, daptomycin or ciprofloxacin. The matrix was visualized by confocal fluorescent microscopy. Persister cells were treated with 104 and 107 PFU/mL Sb-1 for 3 hours in PBS, followed by CFU counting. Alternatively, bacteria were washed and incubated in fresh BHI medium and the bacterial growth assessed after further 24-hours. Pre-treatment with Sb-1 followed by the administration of sub-inhibitory concentrations of antibiotic exerted a considerable synergistic effect in eradicating MRSA biofilm. Sb-1 determined a dose-dependent reduction of matrix exopolysaccharide. 107 PFU/mL Sb-1 showed direct killing activity on persisters. However, even a lower titer had lytic activity when phage-treated persister cells were inoculated in fresh medium, reverting to a normal-growing phenotype. This study provides valuable data regarding the capability of Sb-1 to enhance antibiotic efficacy, exhibiting specific antibiofilm features. Its ability to degrade the MRSA polysaccharide matrix and target persister cells makes Sb-1 suitable for the therapy of biofilm-associated infections.

microbiology↗