Biology subjects

Nielsen, K.

Publications and source records attributed to Nielsen, K..

17 recordsLinked to original sources

bioRxiv2026

The female larval fly CNS connectome: first draft

We release an enhanced FIB-SEM volume and associated neuron, mitochondria, synapse and glia automated segmentations for the complete, gap- and artifact-free central nervous system of the first instar female larval Drosophila melanogaster. Segmentation data is largely unproofread besides select neurons in the premotor and proprioceptive system and select subvolumes where synapses have been manually annotated, serving as training and evaluation data. The dataset represents the first complete central nervous system of the larval fly, and now awaits substantial improvement of machine vision approaches that effectively leverage domain knowledge for the automated yet data-guided proofreading, or substantial and costly human proofreading. All the data and methods are publicly available for further improvement and analysis.

neuroscience↗

bioRxiv2026

Ultra-High Multiplexing Enables Near-Full-Length 16S rRNA Gene Amplicon Sequencing of Over 1,200 Gut Microbiome Samples on a Single Nanopore Flow Cell

Next-generation sequencing (NGS) of the prokaryotic 16S rRNA gene revolutionized gut microbiome research two decades ago. However, short read lengths remain an inherent limitation of platforms such as the widely used Illumina platforms (2 x 150-300 bp). Recent advances in Oxford Nanopore Technologies (ONT) flow cell chemistry (R10.4.1) have substantially improved sequencing accuracy. Combined with a custom multiple-primer strategy that comprehensively targets 16S rRNA gene variants to generate near-full-length amplicons, this approach enables read-by-read taxonomic classification, a feature not feasible with short-read sequencing platforms. Although our multiple-primer strategy could enable parallel sequencing of more than 18,000 samples (192 x 96), current flow cell capacity offers sufficient sequencing depth for approximately 1,000-1,500 samples. To validate the scalability and our per-read classification pipeline, we show that more than a thousand human fecal microbiome samples spiked with two bacterial strains (Imtechella halotolerans and Allobacillus halotolerans), not otherwise present in human fecal samples, can be successfully sequenced on a single flow cell, achieving a per-molecule error rate sufficient for direct per-read classification and at an adequate read depth for downstream analysis. This level of scalability significantly reduces per-sample costs, making the approach more accessible to a broader research community. To embrace these advancements, we have developed RubyRed, a pipeline that processes raw sequencing data and assigns taxonomic classifications on a per-read basis. Using spike-in references (I. halotolerans and A. halotolerans), we demonstrate high mean single-read sequencing accuracy (99% and 98.9%, respectively), with the majority of reads exceeding the canonical threshold required for species-level taxonomic classification based on the 16S rRNA gene.

microbiology↗

bioRxiv2026

Daily Bowel Movements are Associated with Stronger Gut-Brain Phase-Amplitude Coupling and Better Cognitive Performance

Gut-brain communication has emerged as a rapidly expanding field of research, with recent electrophysiological studies revealing rhythmic gut-brain coupling between gastric activity and brain oscillations in humans. Gut motility is a key determinant of gastrointestinal function, but it remains unclear whether individual differences in gut motility reflected by weekly bowel movements (e.g., defecation frequency) are associated with differences in gut-brain coupling. Here, we address this question by examining women with self-reported daily bowel movements (N = 38) and women with less frequent bowel movements (N = 38). We recorded simultaneous electroencephalography (EEG) and electrogastrography (EGG) at fasting state, performed cognitive assessments, and analysed faecal short-chain fatty acids (SCFAs) as markers of colonic fermentation. In a subset of participants, EEG-EGG coupling was assessed twice over an interval of at least eight weeks to assess test-retest reliability. In this group, EEG-EGG coupling showed moderate test-retest reliability (Intraclass Correlation Coefficient (ICC) = 0.50). When comparing the two groups of women, the phase-amplitude coupling (PAC) analysis between EEG and EGG signals revealed a significantly stronger gut-brain coupling in women with daily bowel movements compared to women with less frequent bowel movements (p = 0.03). We additionally found that women with daily bowel movements made less errors in the cognitive tasks and had higher levels of faecal SCFAs. A path analysis suggested that bowel movements significantly affect gut-brain phase-amplitude coupling through faecal SCFAs. However, neither faecal SCFAs nor phase-amplitude coupling significantly predicted cognitive performance, suggesting the existence of alternative pathways for the association between bowel movements and cognitive performance. Together, our findings suggest that the strength of gut-brain coupling is associated with bowel movements and cognitive performance, making EEG-EGG coupling a promising marker of human gut-brain interactions.

neuroscience↗

bioRxiv2026

Paradoxical Th1 activation and CTLA-4 regulation is beneficial during latent cryptococcosis

Cryptococcus neoformans is the predominant causative agent of cryptococcal meningitis in immunocompromised individuals. Conversely in immunocompetent individuals, C. neoformans establishes a latent pulmonary infection characterized by a paucity of clinical symptoms. Using a mouse inhalation model of latent C. neoformans infection, we previously showed that CD4 T-cells are necessary for preventing fungal proliferation in the lungs. In the current study, we performed single cell RNA sequencing (scRNAseq) and found that the CD4 T-cell response was both highly heterogenous and dichotomous during pulmonary C. neoformans infection, with concomitant expression of genes related to Th1 polarization (Tbx21, Ifng) and immune regulation (Ctla4). First, we demonstrated that cells with Th1-like phenotypes are necessary and sufficient to control latent infection via adoptive transfer of T-bet positive cells into infection-matched CD4-depleted recipient mice. Second, scRNAseq analysis revealed the subpopulation of effector CD4 T-cells that co-expressed Ctla4 and Gata3 was significantly higher than a subpopulation that co-expressed Ctla4 and Tbx21. Furthermore, our data suggested that CTLA-4 upregulation is beneficial against C. neoformans infection, as CTLA-4 blockade promoted fungal proliferation. Thus, we propose a model wherein Th1 control of latent C. neoformans infection is supported by CTLA-4 suppression of detrimental Th2 activation. IMPORTANCECD4 T-cells are a critical part of the adaptive immune response in many infections. Upon inhalation of the fungal pathogen Cryptococcus neoformans, CD4 T-cells are necessary to control the initial latent infection and prevent disease. In immunocompromised individuals that lack CD4 T-cells, the initial infection in the lungs can spread to the brain and cause lethal meningitis. In this study, we show that the CD4 T-cells present during latent C. neoformans infection are heterogeneous with expression of diverse polarization and immune regulation markers that function in concert to prevent disease. Our studies highlight the previously unappreciated diversity and complex regulation of the CD4 T-cell response that is required to prevent disease in this important fungal pathogen.

genomics↗

bioRxiv2025

Global proteomic analysis of Cryptococcus neoformans clinical strains reveals significant differences between latent and lethal infection

To predict the outcomes of disseminated fungal disease, a deeper understanding of host-pathogen interactions at the site of infection is needed to identify targets for clinical intervention and diagnostic development. Cryptococcus neoformans is the causative agent of cryptococcosis, the largest infectious killer of individuals living with HIV. Cryptococcal infection begins in the lungs, with loss of immunological control leading to disseminated central nervous system disease and death. Using advanced mass spectrometry-based proteomic techniques, in vivo infection models, and patient-derived clinical strains, we explored the proteomic profiles of C. neoformans infections related to differences in strain virulence. Our findings reveal that non-lethal latent infection produces a proteomic response that differs significantly from the response caused by lethal infections, and that the proteomic profiles of typical and hypervirulent infections are markedly similar despite differences in time-to-death. Overall, the mouse pulmonary proteomic response in latent infection is defined by enrichment of proteins and pathways involved in extracellular matrix organization, cell adhesion, and structural changes, while the lethal infection is dominated by host-defense, translation, and metabolic processes. These results provide clinically relevant information on how infections caused by different Cryptococcus strains may produce significantly different outcomes. We also identified abundant fungal proteins that could be future drug targets in latent and lethal cryptococcal infection. IMPORTANCECryptococcus neoformans is a fungal pathogen that causes substantial morbidity and mortality in immunocompromised individuals. The initial infection begins in the lungs after exposure to inhaled spores after which local immune cells respond by either killing or containing the fungal cells. Immunosuppression weakens the immune system and allows fungal cells in the lungs to escape through the circulatory system and invade the central nervous system and cause fatal disease. However, differences between fungal strains influence the severity of disease manifestation. Our group has previously described genetic differences that contribute to strain-specific disease manifestations. In this study, we expanded our analysis to investigate the proteomic differences between strains of C. neoformans to identify candidate proteins and pathways that contribute to disease manifestation. We found that latent infection differs significantly from lethal disease from both the host and pathogen proteomic perspectives and have identified several fungal protein targets for future study.

microbiology↗

bioRxiv2024

Pulmonary granuloma formation during latent Cryptococcus neoformans infection in C3HeB/FeJ mice involves progression through three immunological phases

Cryptococcus neoformans is a fungal pathogen that can cause lethal disease in immunocompromised patients. Immunocompetent host immune responses, such as formation of pulmonary granulomas, control the infection and prevent disseminated disease. Little is known about the immunological conditions establishing the latent infection granuloma in the lungs. To investigate this, we performed an analysis of pulmonary immune cell populations, cytokine changes, and granuloma formation during infection with a latent disease-causing clinical isolate in C3HeB/FeJ mice over 360 days. We found that latently infected mice progress through three phases of granuloma formation where different immune profiles dominate: an early phase characterized by eosinophilia, high IL-4/IL-13, and C. neoformans proliferation in the lungs; an intermediate phase characterized by multinucleated giant cell formation, high IL-1/IFN{gamma}, granuloma expansion, and increased blood antigen levels; and a late phase characterized by a significant expansion of T cells, granuloma condensation, and decreases in lung fungal burden and blood antigen levels. These findings highlight a complex series of immune changes that occur during the establishment of granulomas that control C. neoformans in the lungs and lay the foundation for studies to identify critical beneficial immune responses to Cryptococcus infections. IMPORTANCECryptococcus neoformans is a fungal pathogen that disseminates from the lungs to the brain to cause fatal disease. Latent C. neoformans infection in the lungs is controlled by organized collections of immune cells called granulomas. The formation and structure of Cryptococcus granulomas are poorly understood due to inconsistent human pathology results and disagreement between necrotic granuloma-forming rat models and non-necrotic granuloma-forming mouse models. To overcome this, we investigated granuloma formation during latent C. neoformans infection in the C3HeB/FeJ mouse strain which forms necrotic lung granulomas in response to other pathogens. We found that latent C. neoformans granuloma formation progresses through phases that we described as early, intermediate, and late with different immune response profiles and granulomatous characteristics. Ultimately, we show that C3HeB/FeJ mice latently infected with C. neoformans form non-necrotic granulomas and could provide a novel mouse model to investigate host immune response profiles.

microbiology↗

bioRxiv2024

M1-linked Ubiquitination by LUBAC Regulates AMPK Activity and the Response to Energetic Stress

Methionine-1 (M1)-linked ubiquitin chains, assembled by the ubiquitin ligase LUBAC and cleaved by the deubiquitinase OTULIN, are critical regulators of inflammation and immune homeostasis. Genetic loss of either LUBAC or OTULIN causes autoinflammatory syndromes, which are associated with defects in glycogen and lipid metabolism. However, how LUBAC and OTULIN regulate metabolic signalling remains unknown. Here, we demonstrate that LUBAC promotes, while OTULIN restricts, activation of the key metabolic regulator AMP-activated protein kinase (AMPK) in cells, mice, and human samples. LUBAC and OTULIN interact with AMPK, control its M1-ubiquitination, and regulate its activation in response to glucose starvation and allosteric activation. During starvation, LUBAC deficiency impairs autophagy induction and hinders the shift from oxidative phosphorylation to glycolysis. Strikingly, LUBAC-deficient Drosophila have a strongly reduced survival rate after starvation. Our work identifies LUBAC and OTULIN as physiological regulators of AMPK, providing the first mechanism by which M1-linked ubiquitin chains regulate metabolic signalling.

cell biology↗

bioRxiv2023

Transcriptional Control of Neocortical Size and Microcephaly

The mammalian neocortex differs vastly in size and complexity between mammalian species, yet the mechanisms that lead to an increase in brain size during evolution are not known. We show here that two transcription factors coordinate gene expression programs in progenitor cells of the neocortex to regulate their proliferative capacity and neuronal output in order to determine brain size. Comparative studies in mice, ferrets and macaques demonstrate an evolutionary conserved function for these transcription factors to regulate progenitor behaviors across the mammalian clade. Strikingly, the two transcriptional regulators control the expression of large numbers of genes linked to microcephaly suggesting that transcriptional deregulation as an important determinant of the molecular pathogenesis of microcephaly, which is consistent with the finding that genetic manipulation of the two transcription factors leads to severe microcephaly.

neuroscience↗

bioRxiv2023

Systematic comparison of risky choices in humans and monkeys

The past decades have seen tremendous progress in fundamental studies on economic choice in humans. However, elucidation of the underlying neuronal processes requires invasive neurophysiological studies that are met with difficulties in humans. Monkeys as evolutionary closest relatives offer a solution. The animals display sophisticated and well-controllable behavior that allows to implement key constructs of proven economic choice theories. However, the similarity of economic choice between the two species has never been systematically investigated. We investigated compliance with the independence axiom (IA) of expected utility theory as one of the most demanding choice tests and compared IA violations between humans and monkeys. Using generalized linear modeling and cumulative prospect theory (CPT), we found that humans and monkeys made comparable risky choices, although their subjective values (utilities) differed. These results suggest similar fundamental choice mechanism across these primate species and encourage to study their underlying neurophysiological mechanisms.

neuroscience↗

bioRxiv2022

Cell-specific regulation of gene expression using splicing-dependent frameshifting.

Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, sequence conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED vectors can leverage the strong expression of constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. We generated SLED vectors to selectively target all neurons, photoreceptors, or excitatory neurons, and demonstrated that specificity was retained in vivo when delivered using AAVs. We further demonstrated the utility of SLED by creating what would otherwise be unobtainable research tools, specifically a GluA2 flip/flop reporter and a dual excitatory/inhibitory neuronal calcium indicator. Finally, we show the translational potential of SLED by rescuing photoreceptor degeneration in Prph2rds/rds mice and by developing an oncolytic vector that can selectively induce apoptosis in SF3B1 mutant cancer cells. The flexibility of SLED technology enables new avenues for basic and translational research.

molecular biology↗

bioRxiv2021

The cyclin Cln1 controls polyploid titan cell formation following a stress-induced G2 arrest in Cryptococcus

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the non-essential cyclin Cln1 resulted in over-production of titan cells in vivo, and transient morphology defects upon release from stationary phase in vivo. Using a copper-repressible promoter PCTR4-CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen. ImportanceDysregulation of the cell cycle underlies many human genetic diseases and cancers. Yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. We analyzed the C. neoformans in vivo and in vitro cell cycles to identify changes required to generate the polyploid titan cells. C. neoformans paused cell cycle progression in response to various environmental stresses after DNA replication and before morphological changes associated with cell division, referred to as a G2 arrest. Release from this G2 arrest was coordinated by the cyclin Cln1. Reduced CLN1 expression after the G2 arrest was associated with polyploid titan cell production. These results demonstrate a mechanism to generate genomic diversity in eukaryotic cells through manipulation of the cell cycle that has broad disease implications.

microbiology↗

bioRxiv2021

FIND-IT: Ultrafast mining of genome diversity

Novel crop improvement methodologies, including the exploitation of natural genetic variation, are urgently required to feed our rapidly growing human population in the context of global climate change. Here we describe a Fast Identification of Nucleotide variants by DigITal PCR (FIND-IT) method for the rapid identification of pre-targeted genetic variants or rare alleles in large genomic populations. Libraries of 500,000 individuals can be screened and desired variants isolated within two weeks. FIND-IT is widely applicable for mining valuable diversity in any genomic population, including elite breeding and wild germplasm collections. The method provides single nucleotide resolution that has been validated by identifying and isolating knockout lines, non-synonymous codon changes and variants of miRNA and transcription factor binding sites in the agronomically important crop barley. In contrast to existing methods, FIND-IT does not require transformation, cloning or enzymatic steps, and is exempt from GMO regulations. Thus, FIND-IT can be applied immediately to elite crop cultivars and can be tailored to minimize or eliminate time-consuming backcrossing requirements.

genetics↗

bioRxiv2021

Proteomic portraits reveal evolutionarily conserved and divergent responses to spinal cord injury

Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of CSF and serum samples from 111 acute SCI patients and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at six months with an AUC of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response, and establish the CSF abundance of glial fibrillary acidic protein (GFAP) as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.

neuroscience↗

bioRxiv2019

Identification of pathogen genomic differences that impact human immune response and disease during Cryptococcus neoformans infection

Patient outcomes during infection are due to a complex interplay between the quality of medical care, host immunity factors, and the infecting pathogens characteristics. To probe the influence of pathogen genotype on human immune response and disease, we examined Cryptococcus neoformans isolates collected during the Cryptococcal Optimal ART Timing (COAT) trial in Uganda. We measured human participants immunologic phenotypes, meningitis disease parameters, and survival. We compared this clinical data to whole genome sequences from 38 C. neoformans isolates of the most frequently observed sequence type (ST) ST93 in our Ugandan participant population, and an additional 18 strains from 9 other sequence types representing the known genetic diversity within the Ugandan Cryptococcus clinical isolates. We focused our analyses on 652 polymorphisms that: were variable among the ST93 genomes, were not in centromeres or extreme telomeres, and were predicted to have a fitness effect. Logistic regression and principal component analyses identified 40 candidate Cryptococcus genes and 3 hypothetical RNAs associated with human immunologic response or clinical parameters. We infected mice with 17 available KN99 gene deletion strains for these candidate genes and found that 35% (6/17) directly influenced murine survival. Four of the six gene deletions that impacted murine survival were novel. Such bedside-to-bench translational research provides important candidate genes for future studies on virulence-associated traits in human Cryptococcus infections.\n\nAuthor SummaryEven with the best available care, mortality rates in cryptococcal meningitis range from 20-60%. Disease is often due to infection by the fungus Cryptococcus neoformans and involves a complex interaction between the human host and the fungal pathogen. Although previous studies have suggested genetic differences in the pathogen impact human disease, it has proven quite difficult to identify the specific C. neoformans genes that impact the outcome of the human infection. Here, we take advantage of a Ugandan patient cohort infected with closely related C. neoformans strains to examine to role of pathogen genetic variants on several human disease characteristics. Using a pathogen whole genome sequencing approach, we showed that 40 C. neoformans genes are associated with human disease. Surprisingly, many of these genes are specific to Cryptococcus and have unknown functions. We also show deletion of these genes alters disease in a mouse model of infection, confirming their role in disease. These findings are particularly important because they are the first to identify C. neoformans genes associated with human cryptococcal meningitis and lay the foundation for future studies that may lead to new treatment strategies aimed at reducing patient mortality.

genetics↗

bioRxiv2019

Closely related Cryptococcus neoformans strains possess differential virulence both in humans and the mouse inhalation model

Cryptococcal meningitis (CM) causes high rates of HIV-related mortality, yet Cryptococcus factors influencing patient outcome are not well understood. Pathogen-specific traits, such as the strain genotype and degree of antigen shedding, are associated with clinical outcome but the underlying biology remains elusive. In this study, we examined factors determining disease outcome in HIV-infected cryptococcal meningitis patients infected with C. neoformans strains with the same multi-locus sequence type. Both patient mortality and survival were observed during infections with the same sequence type. Disease outcome did not correlate with underlying patient immune deficiencies. Patient mortality was associated with higher antigen levels, fungal burden in the CSF, and low CSF fungal clearance. Virulence of a subset of clinical strains with the same sequence type were analyzed using the mouse inhalation model of cryptococcosis. We showed a strong correlation between human and mouse mortality rates, demonstrating the mouse inhalation model recapitulates human infection. Similar to human infection, the ability to multiply in vivo, demonstrated by high fungal burden in the lung and brain tissues, was associated with mouse mortality. Mortality rate was not associated with single C. neoformans virulence factors in vitro or in vivo; rather, a trend in mortality rate correlated with a suite of traits. These observations show that genotype similarities between C. neoformans strains do not necessarily translate into similar virulence either in the mouse model or in human patients. In addition, our results show that in vitro assays do not fully reproduce in vivo conditions that influence C. neoformans virulence.

microbiology↗

bioRxiv2018

Diverse Pathophysiological Processes Converge on Network Disruption in Mania

BackgroundNeuroimaging of psychiatric disease is challenged by the difficulty of establishing the causal role of neuroimaging abnormalities. Lesions that cause mania present a unique opportunity to understand how brain network disruption may cause mania in both lesions and in bipolar disorder.\n\nMethodsA literature search revealed 23 case reports with imaged lesions that caused mania in patients without history of bipolar disorder. We traced these lesions and examined resting-state functional Magnetic Resonance Imaging (rsfMRI) connectivity to these lesions and control lesions to find networks that would be disrupted specifically by mania-causing lesions. The results were then used as regions-of-interest to examine rsfMRI connectivity in patients with bipolar disorder (n=16) who underwent imaging longitudinally across states of both mania and euthymia alongside a cohort of healthy participants scanned longitudinally. We then sought to replicate these results in independent cohorts of manic (n=26) and euthymic (n=21) participants with bipolar disorder.\n\nResultsMania-inducing lesions overlap significantly in network connectivity. Mania-causing lesions selectively disrupt networks that include orbitofrontal cortex, dorsolateral prefrontal cortex, and temporal lobes. In bipolar disorder, the manic state was reflected in strong, significant, and specific disruption in network communication between these regions and regions implicated in bipolar pathophysiology: the amygdala and ventro-lateral prefrontal cortex.\n\nLimitationsThe was heterogeneity in the clinical characterization of mania causing lesions.\n\nConclusionsLesions causing mania demonstrate shared and specific network disruptions. These disruptions are also observed in bipolar mania and suggest a convergence of multiple disorders on shared circuit dysfunction to cause mania.

neuroscience↗

bioRxiv2017

Identification of environmental and genetic factors important for Cryptococcus neoformans titan cell formation using new in vitro inducing conditions

The pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 {micro}m cells and large titan cells (> 10 {micro}m and up to 100 {micro}m). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 {micro}m), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways.\n\nAuthor SummaryCryptococcus neoformans is a yeast that is capable of morphological change upon interaction with the host. Particularly, in the lungs of infected mice, a subpopulation of yeast enlarges, producing cells up to 100 {micro}m in cell body diameter - referred to as titan cells. Along with their large size, the titan cells have other unique characteristics such as thickened cell wall, dense capsule, polyploidization, large vacuole with peripheral nucleus and cellular organelles. The generation of a large number of such cells outside the lungs of mice has been described but was not reproducible nor standardized. Here we report standardized, reproducible, robust conditions for generation of titan cells and explored the environmental and genetic factors underlying the genesis of these cells. We showed that titan cells were generated upon stresses such as change in the incubation medium, nutrient deprivation, hypoxia and low pH. Using collections of well characterized reference strains and clinical isolates, we validated with our model that the cAMP/PKA/Rim101 pathway is a major genetic determinant of titan cell formation. This study opens the way for a more comprehensive picture of the ontology of morphological changes in Cryptococcus neoformans and its impact on pathobiology of this deadly pathogen.

microbiology↗