Biology subjects

Ricci, D.

Publications and source records attributed to Ricci, D..

9 recordsLinked to original sources

bioRxiv2026

Targeting Sec61 to Silence Pathogenic Light-Chain Production in AL Amyloidosis

Unstable immunoglobulin light chains (LCs) secreted by clonal plasma cells drive organ damage in AL amyloidosis, yet no therapy exist to directly suppress their biosynthesis. Here, we targeted the Sec61 translocon to block LC biogenesis. In patient-derived plasma cells, the Sec61 blocker mycolactone redirected nascent LCs to proteasomal degradation, inducing proteotoxic stress that synergized with bortezomib. In vivo, this combination reduced plasma-cell engraftment and LC secretion but caused toxicity, underscoring the need for Sec61 client selectivity. Proteomic profiling of a model patient-derived plasma cell line treated with the synthetic Sec61 inhibitors A317 and A347 revealed intermediate- and narrow-spectrum client selectivity, respectively, while retaining activity against the LC. However, unlike A317, the more selective A347 failed to inhibit diverse amyloidogenic LCs in a signal-peptide reporter assay. In primary plasma cells from an AL cohort, A317 consistently reduced pathogenic LC production while sparing matched non-malignant bone marrow cells. Together, these findings establish Sec61 as a therapeutic target in AL amyloidosis and identify the need to balance client selectivity with coverage of diverse amyloidogenic LC signal peptides.

cell biology↗

bioRxiv2024

Creating overlapping genes by alternate-frame insertion

Overlapping genes-wherein two different proteins are translated from alternative frames of the same DNA sequence-provide a means to stabilize an engineered gene by directly linking its evolutionary fate with that of an overlapped gene. However, creating overlapping gene pairs is challenging as it requires redesign of both protein products to accommodate overlap constraints. Here, we present a new "overlapping, alternate-frame insertion" (OAFI) method for creating overlapping genes by insertion of an "inner" gene, encoded in an alternate frame, into a flexible region of an "outer" gene. Using OAFI, we create new overlapping gene pairs of bacterial toxins within an antibiotic resistance gene. We show that both the inner and outer genes retain functionality despite redesign, with translation of the inner gene is influenced by its overlap position in the outer gene. Additionally, we show that selection for the outer gene alters the permitted inactivating mutations in the inner gene and that overlapping toxins can restrict horizontal gene transfer of the antibiotic resistance gene. Overall, OAFI offers a versatile tool for synthetic biology, expanding the applications of overlapping genes in gene stabilization and biocontainment.

synthetic biology↗

bioRxiv2024

Posture-dependent modulation of marmoset cortical motor maps detected via rapid multichannel epidural stimulation

In this study, rapid topographical changes were detected in the forelimb motor maps in the primary motor cortex (M1) of awake marmoset monkeys using our previously developed accurate short-time stimulation mapping procedure (Takemi et al. 2017; Kosugi et al. 2018). The results revealed that although the hotspot (the location in M1 that elicited a forelimb muscle twitch with the lowest stimulus intensity) remained constant across postures, the stimulus intensity required to elicit the forelimb muscle twitch in the perihotspot region and the size of motor representations were posture-dependent. Hindlimb posture was particularly effective in inducing these modulations. The angle of the body axis relative to the gravitational vertical line did not alter the motor maps. These results provide a proof of concept that a rapid stimulation mapping system with chronically implanted cortical electrodes can capture the dynamic regulation of forelimb motor maps in natural conditions. The flexible nature of the motor maps necessitates the reconsideration of the results of motor control and neuroplasticity studies. Neural mechanisms regulating forelimb muscle representations in M1 by the hindlimb sensorimotor state warrant further exploration.

neuroscience↗

bioRxiv2023

Increased activity of IRE1 improves the clinical presentation of EAE

Activation of the ER stress sensor IRE1 contributes to neuronal development and is known to induce neuronal remodeling in vitro and in vivo. On the other hand, excessive IRE1 activity is often detrimental and may contribute to neurodegeneration. To determine the consequences of increased activation of IRE1, we used a mouse model expressing a C148S variant of IRE1 with increased and sustained activation. Surprisingly, the mutation did not affect the differentiation of highly secretory antibody-producing cells, but exhibited a strong protective effect in a mouse model of experimental autoimmune encephalomyelitis (EAE). Significant improvement in motor function was found in IRE1C148S mice with EAE relative to WT mice. Coincident with this improvement, there was reduced microgliosis in the spinal cord of IRE1C148S mice, with reduced expression of pro-inflammatory cytokine genes. This was accompanied by reduced axonal degeneration and enhanced CNPase levels, suggestiing improved myelin integrity. Interestingly, while the IRE1C148S mutation is expressed in all cells, the reduction in proinflammatory cytokines and in the activation of microglial activation marker IBA1, along with preservation of phagocytic gene expression, all point to microglia as the cell type contributing to the clinical improvement in IRE1C148S animals. Our data suggest that sustained increase in IRE1 activity can be protective in vivo, and that this protection is cell type and context dependent. Considering the overwhelming but conflicting evidence for the role of the ER stress in neurological diseases, a better understanding of the function of ER stress sensors in physiological contexts is clearly needed.

neuroscience↗

bioRxiv2023

Spatiotemporal dynamics across visual cortical laminae support a predictive coding framework for interpreting mismatch responses

AbstractContext modulates neocortical processing of sensory data. Unexpected visual stimuli elicit large responses in primary visual cortex (V1) -- a phenomenon known as deviance detection (DD) at the neural level, or "mismatch negativity" (MMN) when measured with EEG. It remains unclear how visual DD/MMN signals emerge across cortical layers, in temporal relation to the onset of deviant stimuli, and with respect to brain oscillations. Here we employed a visual "oddball" sequence - a classic paradigm for studying aberrant DD/MMN in neuropsychiatric populations - and recorded local field potentials in V1 of awake mice with 16-channel multielectrode arrays. Multiunit activity and current source density profiles showed that while basic adaptation to redundant stimuli was present early (50ms) in layer 4 responses, DD emerged later (150-230ms) in supragranular layers (L2/3). This DD signal coincided with increased delta/theta (2-7Hz) and high-gamma (70-80Hz) oscillations in L2/3 and decreased beta oscillations (26-36hz) in L1. These results clarify the neocortical dynamics elicited during an oddball paradigm at a microcircuit level. They are consistent with a predictive coding framework, which posits that predictive suppression is present in cortical feed-back circuits, which synapse in L1, while "prediction errors" engage cortical feed-forward processing streams, which emanate from L2/3.

neuroscience↗

bioRxiv2022

Dually biofortified cisgenic tomatoes with increased flavonoids and branched-chain amino acids content

Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched-chain amino acids (BCAAs) in vegan diets can reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant-derived foods contain only small amounts of BCAAs and those plants with high levels of flavonoids are not eaten broadly. Here we describe the generation of metabolically-engineered cisgenic tomatoes enriched in both flavonoids and BCAAs. In this approach, coding and regulatory DNA elements, all derived from the tomato genome, were combined to obtain a herbicide-resistant version of an acetolactate synthase (mSlALS) gene expressed broadly, and a MYB12-like transcription factor (SlMYB12) expressed in a fruit-specific manner. The mSlALS played a dual role, as a selectable marker as well as being key enzyme in BCAA enrichment. The resulting cisgenic tomatoes were highly enriched in Leucine (21-fold compared to wild type levels), Valine (9-fold), Isoleucine (3-fold), and concomitantly biofortified in several antioxidant flavonoids including kaempferol (64-fold) and quercetin (45-fold). Comprehensive metabolomic and transcriptomic analysis of the biofortified cisgenic tomatoes revealed marked differences to wild type and could serve to evaluate the safety of these biofortified fruits for human consumption.

bioengineering↗

bioRxiv2022

Computationally restoring the potency of a clinical antibody against SARS-CoV-2 Omicron subvariants

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3, but also revealed how quickly viral escape can curtail effective options4, 5. With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including EvusheldTM and its constituent, cilgavimab4, 6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protection in vivo against the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities.

microbiology↗

bioRxiv2021

Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients. Author summarySARS-CoV-2 pandemic has resulted in millions of infections and deaths worldwide, yet the role of host innate immune responses in COVID-19 pathogenesis remains only partially characterized. Innate immunity represents the first line of host defense against viruses. Upon viral recognition, the secretion of type I and III interferons (IFN) establishes the cellular state of viral resistance, and contributes to induce the specific adaptive immune responses. Moving from in vitro evidences on the protective role played by plasmacytoid dendritic cells (pDC)-released type I IFN in the early phase of SARS-CoV-2 infection, here we characterized ex vivo the pDC phenotype and the balance between anti-viral and pro-inflammatory cytokines of COVID-19 patients stratified according to disease severity. Our study confirms in COVID-19 the crucial and protective role of pDC/type I IFN axis, whose deeper understanding may contribute to the development of novel pharmacological strategies and/or host-directed therapies aimed at boosting pDC response since the early phases of SARS-CoV-2 infection.

immunology↗

bioRxiv2020

The connector between the kinase and RNase domains of IRE1α transmits the conformational change that underlies ER stress-induced activation.

The unfolded protein response (UPR) plays an evolutionarily conserved role in homeostasis, and its dysregulation often leads to human disease, including diabetes and cancer. IRE1 is a major transducer that conveys endoplasmic reticulum (ER) stress to biochemical signals, yet major gaps persist in our understanding of how the detection of stress is converted to one of several molecular outcomes. It is known that upon sensing unfolded proteins via its ER luminal domain, IRE1 dimerizes and oligomerizes (often visualized as clustering), and then trans-autophosphorylates. The IRE1 kinase activity is required for activation of its RNase effector domain and for clustering of IRE1. It is not yet clear if IRE1 clustering is a platform for the RNase activity, or if the two represent distinct biological functions. Here, we uncover a previously unrecognized role for helix K between IRE1 kinase and RNase domains in conveying critical conformational changes. Using mutants within this inter-domain helix, we show for the first time that: 1) distinct substitutions (specifically, of Leu827) selectively affect oligomerization, RNase activity, and, unexpectedly, the kinase activity of IRE1; 2) RNase activation can be uncoupled from IRE1 oligomerization, and phosphorylation of S729 marks the former but not the latter; 3) The nature of residue 827 determines the conformation that the IRE1 protein adopts, leading to different patterns of biochemical activities. In summary, this work reveals a previously unappreciated role for the inter-domain helix as a pivotal conduit for attaining the stress-responsive conformation of IRE1.

biochemistry↗