Biology subjects

Ceballos, R. M.

Publications and source records attributed to Ceballos, R. M..

8 recordsLinked to original sources

bioRxiv2026

A multi-electrode array and immunofluorescence workflow to characterize impacts of HHV-6 infection in induced pluripotent stems cells differentiated to mixed neuronal endpoints

Roseoloviruses, notably human herpesviruses 6A and 6B (HHV-6A and HHV-6B), are neurotropic viruses implicated as agents in neurological disorders, including: epilepsy, multiple sclerosis, and chronic fatigue syndrome. However, the effects of roseolovirus infection on neuronal signaling and network activity are not characterized. This is, in part, due to the complexities of monitoring electrical activity in individual neurons and across neuronal connections during viral infection. This protocol describes a human induced pluripotent stem cell (iPSC)-derived neuronal culture system that employs multi-electrode array (MEA) recordings to study neurophysiological changes during viral infection. Two culture platforms are described: (a) NGN2-induced forebrain neuronal cultures; and, (b) progenitor cell-derived neuron-astrocyte mixed cultures. Cell composition in cultures is validated by immunofluorescence staining using neuronal, glial, and viral markers. Functional activity is assessed using extracellular recordings detected via the MEA2100 system. Mean firing rate, inter-spike interval, single-electrode bursting, and network burst activity are characterized between roseolovirus-infected versus uninfected (control) states. Pharmacological treatment with bicuculline, gabazine, and nicotine in conjunction with immunofluorescence is used to confirm functional responsiveness of defined neuronal neurotransmitter chemotypes. Here, we show that HHV-6A infection changes neuronal firing compared to uninfected controls. The methods/workflows described provide a strategy to study how virus infection alters neuronal excitability and may be adapted to compare the effects of different viruses on nerve cell function, infection time courses, different multiplicities of infection (MOI), and therapeutic interventions.

microbiology↗

bioRxiv2026

PhageTAILor leverages machine learning for phage tail-like elements detection and classification in plant-associated bacteria

Phage tail-like elements (PTEs) -- tailocins, bacterial type VI secretion systems (T6SS), and extracellular contractile injection systems (eCIS) -- are contractile nanomachines that bacteria use to kill their neighbors and compete within their micro-ecosystems. PTEs help shape microbial community composition. Most PTE detection tools only detect a single PTE class. Moreover, most tailocin detection methods are largely restricted to Pseudomonas, leaving a key part of tailocin diversity uncharacterized. In this work, we present PhageTAILor (https://github.com/hjcho-bio/PhageTAILor), an integrative and fully automated pipeline that detects and classifies prophages and 3 PTE classes from bacterial genomes. PhageTAILor combines a 6-detector homology-based candidate search (geNomad, tail-gene, PHROGs-tail, SecReT6, eCIStem, and a divergence-tolerant tail-HMM detector) with a LightGBM classifier comprising 1 multiclass and 3 binary heads, trained on 6,501 bacterial genomes carrying 13,082 prophages and PTEs. A phylogeny-free feature matrix used in our model keeps predictions reproducible between model construction and user inference. PhageTAILor performs strongly at the genome level and generalizes beyond its Pseudomonas-rich training set. On a 76-strain cross-clade benchmark, PhageTAILor detected tailocins at F1 = 0.955. Furthermore, it identified 12 of 13 experimentally validated tailocins spanning five genera versus 2 of 13 for a Pseudomonas-restricted tool TattleTail. PhageTAILor also demonstrated sensitivity equivalent to viral detection tool geNomad while avoiding its higher false-positive rate. Applied to 7,925 plant- and soil-associated bacterial isolates, PhageTAILor showed that prophages in the phyllosphere and tailocins in plant-associated bacteria, whereas eCIS are enriched in soil. PhageTAILor is distributed as an open-source, modular pipeline with a command-line interface.

microbiology↗

bioRxiv2022

Virtually the same? Evaluating the effectiveness of remote undergraduate research experiences

In-person undergraduate research experiences (UREs) promote students integration into careers in life science research. In 2020, the COVID-19 pandemic prompted institutions hosting summer URE programs to offer them remotely, raising questions about whether undergraduates who participate in remote research can experience scientific integration. To address this, we investigated indicators of scientific integration for students who participated in remote life science URE programs in summer 2020. We found that these students experienced gains in their scientific self-efficacy and scientific identity similar to results reported for in-person UREs. We also found that these students perceived high benefits and low costs of doing research at the outset of their programs, and their perceptions did not change despite the remote circumstances. Yet, their perceptions differed by program, indicating that programs differentially affected students perceptions of the costs of doing research. Finally, we observed that students with prior research experience made greater gains in self-efficacy and identity, as well as in their perceptions of the alignment of their values with those of the scientific community, in comparison to students with no prior research experience. This finding suggests that additional programming may be needed for undergraduates with no prior experience to benefit from remote research.

scientific communication and education↗

bioRxiv2021

Stability Comparisons between Natural versus Engineered Archaeal Heat Shock Proteins

Crenarchaeal group II chaperonins (a.k.a., "heat shock" proteins, HSPs) are abundantly expressed in species of the family Sulfolobaceae. HSP and HSP{beta} expression is upregulated during thermal shock. HSPs are subunits of larger octadecameric complexes that function to protect intracellular proteins during thermal stress. Engineered HSPs have been constructed with the idea of protecting enzymes in industrial reactions. HSP{beta}-coh, a fusion protein comprised of HSP{beta} and type 1 cohesin from Clostridium thermocellum was used for proof-of-concept. Dockerin-endowed cellulolytic enzymes bind to the complex via cohesin-dockerin interactions. Enzymatic activity (i.e., hydrolysis of lignocellulose) is retained when the platform is used at high temperatures (e.g., 85-88{degrees}C). Moreover, enhancement persists on acid-pretreated substrates prompting the question: Are HSPs acid tolerant? In this study, HSP structural integrity is examined at different temperatures and pH. Far-UV circular dichroism and intrinsic fluorescence indicate HSP and HSP{beta} retain structural integrity at neutral pH over a range of temperatures (25-90{degrees}C) while HSP{beta}-coh is less tolerant to thermal stress. Structural integrity is compromised for all subunits at ultra-low pH (i.e., pH 2) with HSP showing the most susceptibility. Secondary structures of all HSPs are resilient under mildly acidic conditions (pH 4). ANS binding assays indicate a shift in tertiary structure for all subunits at ultra-low pH. Limited trypsin digestion reveals that the backbone of HSP{beta}-coh is the most flexible and HSP{beta} is the most resistant. Results suggest that HSP and HSP{beta} are more resilient than HSP{beta}-coh under thermal challenge and that there are limits to the acid tolerance of all HSPs.

biochemistry↗

bioRxiv2021

"How do we do this at a distance?!" A descriptive study of remote undergraduate research programs during COVID-19

The COVID-19 pandemic shut down undergraduate research programs across the U.S. Twenty-three sites offered remote undergraduate research programs in the life sciences during summer 2020. Given the unprecedented offering of remote research experiences, we carried out a study to describe and evaluate these programs. Using structured templates, we documented how programs were designed and implemented, including who participated. Through focus groups and surveys, we identified programmatic strengths and shortcomings as well as recommendations for improvements from the perspectives of participating students. Strengths included the quality of mentorship, opportunities for learning and professional development, and development of a sense of community. Weaknesses included limited cohort building, challenges with insufficient structure, and issues with technology. Although all programs had one or more activities related to diversity, equity, inclusion, and justice, these topics were largely absent from student reports even though programs coincided with a peak in national consciousness about racial inequities and structural racism. Our results provide evidence for designing remote REUs that are experienced favorably by students. Our results also indicate that remote REUs are sufficiently positive to further investigate their affordances and constraints, including the potential to scale up offerings, with minimal concern about disenfranchising students.

scientific communication and education↗

bioRxiv2021

Differential susceptibility of neuronal neurotransmitter phenotypes to HHV6 infection

Within the family Herpesviridae, sub-family {beta}-herpesvirinae, and genus Roseolovirus, there are only three human herpesviruses that have been discovered and described: HHV-6A, HHV-6B, and HHV-7. Initially, HHV-6A and HHV-6B were considered to be simply two variants of the same virus (i.e., HHV6). Despite high overall genetic sequence identity (~90%), HHV-6A and HHV-6B are now recognized as two distinct viruses of the genus. Limited sequence identity (e.g., <70%) in key coding regions as well as significant differences in physiological and biochemical profiles (e.g., preferential use of different receptors for viral entry into susceptible hosts) underscore the conclusion that HHV-6A and HHV-6B are distinct virus species. Likewise, each virus appears to differentially contribute as putative etiologic agents to a variety of neurological disorders, including: multiple sclerosis, epilepsy, and chronic fatigue syndrome. Despite being implicated as causative agents in nervous system dysfunction, mechanisms of action and relative contributions of each virus to neural disorders remain elusive. Unresolved questions regarding: cell receptor use and binding affinity (i.e., CD49 versus CD134); cell tropism; the role of HHV-7 superinfection; and, relative virulence between HHV-6A versus HHV-6B - prevent a complete characterization. Although it has been demonstrated that both HHV-6A and HHV-6B can infect glia and, more recently, cerebellar Purkinje cells, cell tropism of HHV-6A versus HHV-6B for different nerve cell types remains vague. In this study, we demonstrate that both HHV-6A and HHV-6B can infect different nerve cell types (i.e., glia versus neurons) and different neuronal neurotransmitter phenotypes derived from the differentiation of human neural stem cells. We further show that both HHV-6A and HHV-6B induce cytopathic effects (CPEs) in susceptible nerve cells. However, the time-course and severity of CPEs appear to differ between HHV-6A versus HHV-6B infections and are dependent upon multiplicity of infection (MOI). As demonstrated by immunofluorescence, although both the HHV-6A and HHV-6B viruses productively infected VGluT1-containing cells (i.e., glutamatergic neurons) and dopamine-containing cells (i.e., dopaminergic neurons), neither HHV-6A nor HHV-6B challenge resulted in the productive infection of GAD67-containing cells (i.e., GABAergic cells). The reason underlying the apparent resistance of GABAergic cells to HHV-6A and HHV-6B infection remains unclear. Morphometric and image analyses of neurite extension and retraction dynamics as well as the time-course of cell aggregation phenomena (e.g., syncytia formation) during infection also indicate that HHV-6A induces more severe CPEs than HHV-6B at the same time-point and MOI. These data suggest that HHV-6A is more virulent than HHV-6B on susceptible human neural stem cells (HNSCs) differentiated into neuronal phenotypes, while neither virus is able to infect GABAergic cells. If these in vitro data hold in vivo, the inhibitory interneuron dysfunction hypothesis for HHV6-driven seizures may be ruled out as a potential mechanism for HHV6-induced epileptogenesis.

microbiology↗

bioRxiv2020

Quantifying relative virulence: When μmax fails and AUC alone just isn't enough

AO_SCPLOWBSTRACTC_SCPLOWOne of the more challenging aspects in quantitative virology is quantifying relative virulence between two (or more) viruses that have different replication dynamics in a given susceptible host. Host growth curve analysis is often used to detail virus-host interactions and to determine the impact of viral infection on a host. Quantifying relative virulence using canonical parameters such as maximum specific growth rate (max) can fail to provide accurate information regarding experimental infection, especially for non-lytic viruses. Although area-under-the-curve (AUC) can be more robust by through calculation of a percent inhibition (PIAUC), this metric can be sensitive to limit selection. In this study, using empirical and extrapolated data from Sulfolobus Spindle-shaped Virus (SSV) infections, we introduce a novel, simple metric that is proven to be more robust and less sensitive than traditional measures for determining relative virulence. This metric (ISC) more accurately aligns biological phenomena with quantified metrics from growth curve analysis to determine trends in relative virulence. It also addresses a major gap in virology by allowing comparisons between non-lytic single-virus/single-host (SVSH) infections and between non-lytic versus lytic virus infection on a given host. How ISC may be applied to polymicrobial infection - both coinfection of a host culture and superinfection of a single cell with more than one virus (or other pathogen type) is a topic of ongoing investigation.

microbiology↗

bioRxiv2020

Host-dependent differences in replication strategy of the Sulfolobus Spindle-shaped Virus strain SSV9 (a.k.a., SSVK1): Lytic replication in hosts of the family Sulfolobaceae

The Sulfolobus Spindle-shaped Virus (SSV) system has become a model for studying thermophilic virus biology, including archaeal host-virus interactions and biogeography. Several factors make the SSV system amenable to studying archaeal genetic mechanisms (e.g., CRISPRs) as well as virus-host interactions in high temperature acidic environments. First, it has been shown that endemic populations of Sulfolobus, the reported SSV host, exhibit biogeographic structure. Second, the acidic (pH<4.5) high temperature (65-88{degrees}C) SSV habitats have low biodiversity, thus, diminishing opportunities for host switching. Third, SSVs and their hosts are readily cultured in liquid media and on gellan gum plates. Fourth, given the wide geographic separation between the various SSV-Sulfolobus habitats, the system is amenable for studying allopatric versus sympatric virus-host interactions. Previously, we reported that SSVs exhibit differential infectivity on allopatric and sympatric hosts. We also noticed a wide host range for virus strain SSV9 (a.k.a., SSVK1). For decades, SSVs have been described as "non-lytic" dsDNA viruses that infect species of the genus Sulfolobus and release virions via "blebbing" or "budding" as a preferred strategy over host lysis. Here, we show that SSVs infect more than one genus of the family Sulfolobaceae and, in allopatric hosts, SSV9 does not appear to release virions by blebbing. Instead, SSV9 appears to lyse all susceptible allopatric hosts tested, while exhibiting canonical non-lytic viral release via "blebbing" (historically reported for all other SSVs), on a single sympatric host. Lytic versus non-lytic virion release does not appear to be driven by multiplicity of infection (MOI). Greater relative stability of SSV9 compared to other SSVs (i.e., SSV1) in high temperature, low pH environments may contribute to higher transmission rates. However, neither higher transmission rate nor relative virulence in SSV9 infection drives replication profile (i.e., lytic versus non-lytic) in susceptible hosts. Although it is known that CRISPR-Cas systems offer protection against viral infection in prokaryotes, CRISPRS are not reported to be a determinant virus replication strategy. Thus, the genetic/molecular mechanisms underlying SSV9-induced lysis are unknown. These results suggest that there are unknown genetic elements, resulting from allopatric evolution, that drive virion release strategy in specific host strain-SSV strain pairings.

microbiology↗