Biology subjects

Vergara, R.

Publications and source records attributed to Vergara, R..

12 recordsLinked to original sources

bioRxiv2025

LuxSit Pro and sPro: Next-Generation Designed Luciferases for Bioluminescent Reporting and Complementation-Based Detection

Bioluminescent systems are widely used as molecular reporters in life sciences due to their low background, broad dynamic range, and the key advantage of not requiring external excitation. However, natural luciferases often lack the brightness, stability, and long-lasting light emission needed to support a wide range of high-sensitivity applications. Here, we describe our next-generation luciferase solution: LuxSit Pro and its substrate, Luxterazine (LTZ). LuxSit Pro is a de novo luciferase optimized for steady brightness and structural stability through an iterative combination of computational and experimental methods. Composed of only 117 amino acids, LuxSit Pro is currently the smallest highest-performing known luciferase, exhibiting exceptional brightness and biochemical stability with robust recombinant expression in both mammalian and bacterial systems. Notably, LuxSit Pro was deliberately designed devoid of lysine and cysteine residues which enormously expands its potential applications. This property renders the reporter largely resistant to post-translational modifications such as ubiquitination and also enables site-directed chemical modifications, as lysines or cysteines can be reintroduced at specific sites to serve as chemical handles for augmenting or adapting the enzymes function. The novel synthetic substrate, Luxterazine (LTZ), is a compound that enables LuxSit Pro to deliver bright, steady, blue light emission ([~]490 nm) and offers improved solubility compared to existing Coelenterazine analogs. Finally, we also developed LuxSit sPro, a highly sensitive two-component complementation system for the quantitative detection of molecular interactions. The broad potential applications of LuxSit Pro and sPro, in combination with Luxterazine, are poised to drive the development of next-generation reporters and biosensor systems for the research and in vitro diagnostics fields.

molecular biology↗

bioRxiv2024

Distinct immune profiles in children living with HIV based on timing and duration of suppressive antiretroviral treatment

Timely initiation of antiretroviral therapy (ART) remains a major challenge in the effort to treat children living with HIV ("CLH") and little is known regarding the dynamics of immune normalization following ART in CLH with varying times to and durations of ART. Here, we leveraged two cohorts of virally-suppressed CLH from Nairobi, Kenya to examine differences in the peripheral immune systems between two cohorts of age-matched children (to control for immune changes with age): one group which initiated ART during early HIV infection and had been on ART for 5-6 years at evaluation (early, long-term treated; "ELT" cohort), and one group which initiated ART later and had been on ART for approximately 9 months at evaluation (delayed, short-term treated; "DST" cohort). We profiled PBMC and purified NK cells from these two cohorts by mass cytometry time-of-flight (CyTOF). Although both groups of CLH had undetectable viral RNA load at evaluation, there were marked differences in both immune composition and immune phenotype between the ELT cohort and the DST cohort. DST donors had reduced CD4 T cell percentages, decreased naive to effector memory T cell ratios, and markedly higher expression of stress-induced markers. Conversely, ELT donors had higher naive to effector memory T cell ratios, low expression of stress-induced markers, and increased expression of markers associated with an effective antiviral response and resolution of inflammation. Collectively, our results demonstrate key differences in the immune systems of virally-suppressed CLH with different ages at ART initiation and durations of treatment and provide further rationale for emphasizing early onset of ART. AUTHOR SUMMARYMany children living with HIV lack access to both antiviral treatments and testing for HIV infection and are therefore unable to initiate treatment in a timely manner. When children do begin treatment, their immune systems take time to recover from the uncontrolled HIV infection. In this study, we examine how the immune systems of children living with HIV normalize after treatment onset by looking at two groups of children whose HIV is well-controlled by treatment and who therefore dont have virus replicating in their blood. One group started treatment within the first year of life and has been on treatment for 5-6 years, while the other began treatment after the first year and has been treated for around 9 months. Although both of these groups are virally-suppressed, we found significant differences in their immune profiles, with the children who had delayed and short-term treatment showing signs of inflammation and immune dysfunction. Collectively, our study helps us understand how variation in the timing and duration of ART treatment impacts the immune system in children with viral suppression and therefore provides clinicians with additional knowledge that can inform the care of children living with HIV, improving their health and quality of life.

immunology↗

bioRxiv2023

Unveiling the role of local metabolic constraints on the structure and activity of spiking neural networks

Understanding the intricate interplay between neural dynamics and metabolic constraints is crucial for unraveling the mysteries of the brain. Despite the significance of this relationship, specific details concerning the impact of metabolism on neuronal dynamics and neural network architecture remain elusive, creating a notable gap in the existing literature. This study employs an energy-dependent neuron and plasticity model to analyze the role of local metabolic constraints in shaping both the dynamics and structure of Spiking Neural Networks (SNN). Specifically, an energy-dependent version of the leaky integrate-and-fire model is utilized, along with a three-factor learning rule that incorporates postsynaptic available energy as the third factor. These models allow for fine-tuning sensitivity in the presence of energy imbalances. Analytical expressions predicting the networks activity and structure are derived, and a fixed-point analysis reveals the emergence of attractor states characterized by neuronal and synaptic sensitivity to energy imbalances. Analytical findings are validated through numerical simulations using an excitatory-inhibitory network. Furthermore, these simulations enable the study of SNN activity and structure under conditions simulating metabolic impairment. In conclusion, by employing energy-dependent models with adjustable sensitivity to energy imbalances, our study advances the understanding of how metabolic constraints shape SNN dynamics and structure. Moreover, in light of compelling evidence linking neuronal metabolic impairment to neurodegenerative diseases, the incorporation of local metabolic constraints into the investigation of neuronal network structure and activity opens an intriguing avenue for inspiring the development of therapeutic interventions.

neuroscience↗

bioRxiv2023

Cross-species transmission and PB2 mammalian adaptations of highly pathogenic avian influenza A/H5N1 viruses in Chile

H5N1 highly pathogenic avian influenza viruses (HPAIV) emerged in wild birds in Chile in December 2022 and spilled over into poultry, marine mammals, and one human. Between December 9, 2022 - March 14, 2023, a coordinated government/academic response detected HPAIV by real-time RT-PCR in 8.5% (412/4735) of samples from 23 avian and 3 mammal orders. Whole-genome sequences obtained from 77 birds and 8 marine mammals revealed that all Chilean H5N1 viruses belong to lineage 2.3.4.4b and cluster monophyletically with viruses from Peru, indicating a single introduction from North America into Peru/Chile. Mammalian adaptations were identified in the PB2 segment: D701N in two sea lions, one human, and one shorebird, and Q591K in the human and one sea lion. Minor variant analysis revealed that D701N was present in 52.9 - 70.9% of sequence reads, indicating the presence of both genotypes within hosts. Further surveillance of spillover events is warranted to assess the emergence and potential onward transmission of mammalian adapted H5N1 HPAIV in South America.

microbiology↗

bioRxiv2023

Thermodynamic and kinetic analysis of the LAO binding protein and its isolated domains reveal non-additivity in stability, folding, and function

Substrate-binding proteins (SBP) are used by organisms from the three domains of life for transport and signaling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here we describe the ligand binding, conformational stability, and folding kinetics of the Lysine Arginine Ornithine binding protein (LAO) from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behavior based on their connectivity, the discontinuous domain shows a stable native-like structure that binds L-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability, and folding pathway of the lobes on their covalent association, is most likely the result of the coevolution of both domains as a single unit.

biochemistry↗

bioRxiv2023

Cochlear dysfunction as an early biomarker for cognitive decline in normal hearing and mild hearing loss

Age-related hearing loss (presbycusis) at moderate levels (>40 dB HL) has been recognized as an important risk factor for cognitive decline. However, whether individuals with mild hearing loss (audiogram thresholds between 25 and 40 dB HL) or even those with normal audiograms (<25 dB HL) have a higher risk of dementia, is still debated. Importantly, these early stages of presbycusis are the most common among the elderly, indicating the need to screen and identify individuals with early presbycusis that have an increased risk of cognitive decline. Unfortunately, in this group of patients, audiogram thresholds are not sufficiently sensitive to detect all the hearing impairments that are related to cognitive decline. Consequently, at the individual level, audiogram thresholds are not good estimators of the dementia risk in the group with mild hearing loss or normal hearing thresholds. Here, we propose to use distortion product otoacoustic emissions (DPOAE), as an objective and sensitive tool to estimate the risk of clinically relevant cognitive decline in elders with normal hearing o mild hearing loss. We assessed neuropsychological, brain magnetic resonance imaging, and auditory analyses on 94 subjects aged >64 years old. In addition, cognitive and functional performance was evaluated with the Clinical Dementia Rating Sum of Boxes (CDR SoB), assessed through structured interviews conducted by neurologists, who were blind to the DPOAE results. We found that cochlear dysfunction, measured by DPOAE -and not by conventional audiometry-, was associated with CDR SoB classification and brain atrophy in the group with mild hearing loss (25 to 40 dB), and normal hearing (<25 dB). Our findings suggest that DPOAE may be a non-invasive tool for detecting neurodegeneration and cognitive decline in the elderly, potentially allowing for early intervention.

neuroscience↗

bioRxiv2020

De novo design of ACE2 protein decoys to neutralize SARS-CoV-2

There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins ([~]18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.

biochemistry↗

bioRxiv2020

Mass cytometry analysis of the NK cell receptor-ligand repertoire reveals unique differences between dengue-infected children and adults

Dengue virus (DENV) is a significant cause of morbidity in many regions of the world, with children at the greatest risk of developing severe dengue. Natural killer (NK) cells, characterized by their ability to rapidly recognize and kill virally infected cells, are activated during acute DENV infection. However, their role in viral clearance versus pathogenesis has not been fully elucidated. Our goal was to profile the NK cell receptor-ligand repertoire to provide further insight into the function of NK cells during pediatric and adult DENV infection. We used mass cytometry (CyTOF) to phenotype isolated NK cells and peripheral blood mononuclear cells (PBMCs) from a cohort of DENV-infected children and adults. Using unsupervised clustering, we found that pediatric DENV infection leads to a decrease in total NK cell frequency with a reduction in the percentage of CD56dimCD38bright NK cells and an increase in the percentage of CD56dimperforinbright NK cells. No such changes were observed in adults. Next, we identified markers predictive of DENV infection using a differential state test. In adults, NK cell expression of activation markers, including CD69, perforin, and Fas-L, and myeloid cell expression of activating NK cell ligands, namely Fas, were predictive of infection. In contrast, NK cell expression of the maturation marker CD57 and increased myeloid cell expression of inhibitory ligands, such as HLA class I molecules, were predictive of pediatric DENV infection. These findings suggest that acute pediatric DENV infection may result in diminished NK cell activation, which could contribute to enhanced pathogenesis and disease severity.

immunology↗

bioRxiv2020

Charge-Altering Releasable Transporters enable specific phenotypic manipulation of resting primary natural killer cells

Natural killer (NK) cells are capable of rapid and robust cytotoxicity, making them excellent tools for immunotherapy. However, their recalcitrance to standard transfection techniques has limited both mechanistic studies and clinical applications. Current approaches for NK cell manipulation rely on viral transduction or methods requiring NK cell activation, which can alter NK cell function. Here, we report that non-viral Charge-Altering Releasable Transporters (CARTs) efficiently transfect primary human NK cells with mRNA without relying on NK cell activation. Compared to electroporation, CARTs transfect NK cells two orders of magnitude more efficiently, better preserve cell viability, and cause minimal reconfiguration of NK cell phenotype and function. Finally, we use CARTs to generate highly cytotoxic primary human chimeric antigen receptor NK cells, indicating potential therapeutic utility of this technique. To our knowledge, CARTs represent the first efficacious transfection technique for resting primary NK cells that preserves NK cell phenotype, and can drive new biological discoveries and clinical applications of this understudied lymphocyte subset.

immunology↗

bioRxiv2019

TIGIT is upregulated by HIV-1 infection and marks a highly functional adaptive and mature subset of natural killer cells

ObjectiveOur objective was to investigate the mechanisms that govern natural killer (NK) cell responses to HIV, with a focus on specific receptor-ligand interactions involved in HIV recognition by NK cells.\n\nDesign and MethodsWe first performed a mass cytometry-based screen of NK cell receptor expression patterns in healthy controls and HIV+ individuals. We then focused mechanistic studies on the expression and function of T cell immunoreceptor with Ig and ITIM domains (TIGIT).\n\nResultsThe mass cytometry screen revealed that TIGIT is upregulated on NK cells of untreated HIV+ women, but not in antiretroviral-treated women. TIGIT is an inhibitory receptor that is thought to mark exhausted NK cells; however, blocking TIGIT did not improve anti-HIV NK cell responses. In fact, the TIGIT ligands CD112 and CD155 were not upregulated on CD4+ T cells in vitro or in vivo, providing an explanation for the lack of benefit from TIGIT blockade. TIGIT expression marked a unique subset of NK cells that express significantly higher levels of NK cell activating receptors (DNAM-1, NTB-A, 2B4, CD2) and exhibit a mature/adaptive phenotype (CD57hi, NKG2Chi, LILRB1hi, FcR{gamma}lo, Syklo). Furthermore, TIGIT+ NK cells had increased responses to mock-infected and HIV-infected autologous CD4+ T cells, and to PMA/ionomycin, cytokine stimulation and the K562 cancer cell line.\n\nConclusionsTIGIT expression is increased on NK cells from untreated HIV+ individuals. Although TIGIT does not participate directly in NK cell recognition of HIV, it marks a population of mature/adaptive NK cells with increased functional responses.

immunology↗

bioRxiv2019

HLA upregulation during dengue virus infection suppresses the natural killer cell response

Dengue virus (DENV) is the most prevalent mosquito-borne virus in the world and a major cause of morbidity in the tropics and subtropics. Upregulation of HLA class I molecules has long been considered a feature of DENV infection, yet this has not been evaluated in the setting of natural infection. Natural killer (NK) cells, an innate immune cell subset critical for mounting an early response to viral infection, are inhibited by self HLA class I, suggesting that upregulation of HLA class I during DENV infection could dampen the NK cell response. Here we addressed whether upregulation of HLA class I molecules occurs during in vivo DENV infection and, if so, whether this suppresses the NK cell response. We found that HLA class I expression was indeed upregulated during acute DENV infection across multiple cell lineages in vivo. To better understand the role of HLA class I upregulation, we infected primary human monocytes, a major target of DENV infection, in vitro. Upregulation of total HLA class I is dependent on active viral replication and is mediated in part by cytokines and other soluble factors induced by infection, while upregulation of HLA-E occurs in the presence of replication-incompetent virus. Importantly, blocking DENV-infected monocytes with a pan-HLA class I Fab nearly doubles the frequency of degranulating NK cells, while blocking HLA-E does not significantly improve the NK cell response. These findings demonstrate that upregulation of HLA class I during DENV infection suppresses the NK cell response, potentially contributing to disease pathogenesis.

immunology↗

bioRxiv2017

Strain-Specific Human Natural Killer Cell Recognition of Influenza A Virus

AbstractInnate Natural killer (NK) cells employ an array of surface receptors to detect altered self induced by infection or malignancy. Despite their decisive role in early antiviral immunity, the cellular mechanisms governing if or how they discriminate between viral infections remain unresolved. Here, we demonstrate that while human NK cells are capable of reducing infection levels of distinct influenza A strains, the A/California/07/2009 (pH1N1) strain induces a significantly more robust IFN-{gamma} response than A/Victoria/361/2011 (H3N2) and all other strains tested. This surprising degree of strain specificity results in part from the inability of the pH1N1 strain to downregulate the activating ligands CD112 (Nectin-2) and CD54 (ICAM-1) as efficiently as the H3N2 strain, leading to enhanced NK cell detection and IFN-{gamma} secretion. A network analysis of differentially expressed transcripts identifies the interferon /{beta} receptor (IFNAR) pathway as an additional, critical determinant of this strain-specific response. Strain-specific downregulation of NK cell activating ligands and modulation of type I IFN production represents a previously unrecognized influenza immunoevasion tactic and could present new opportunities to modulate the quality and quantity of the innate antiviral response for therapeutic benefit.\n\nOne Sentence SummaryHuman natural killer cells distinguish between Influenza A strains using a combinatorial cytokine priming and receptor-ligand signaling mechanism.

immunology↗