Strain Variation Can Significantly Modulate the miRNA Response to Zika Virus Infection.

Zika virus (ZIKV) is a mosquito-transmitted virus that has emerged as a major public health concern due to its association with neurological disorders in humans, including microcephaly in fetuses. ZIKV infection has been shown to alter the miRNA profile in host cells, and these changes can contain elements that are proviral, while others can be antiviral in action. In this study, the expression of 22 miRNAs in human A549 cells infected with two different ZIKV isolates was investigated. All of the investigated miRNAs showed significant changes in expression at at least one time point examined. Markedly, 18 of the miRNAs examined showed statistically significant differences in expression between the two strains examined. Four miRNAs (miR-21, miR-34a, miR-128 and miR-155) were subsequently selected for further investigation. These four miRNAs were shown to modulate antiviral effects against ZIKV, as downregulation of their expression through anti-miRNA oligonucleotides resulted in increased virus production, whereas their overexpression through miRNA mimics reduced virus production. However, statistically significant changes were again seen when comparing the two strains investigated. Lastly, candidate targets of the miRNAs miR-34a and miR-128 were examined at the level of the mRNA and protein. HSP70 was identified as a target of miR-34a, but, again, the effects were strain type-specific. The two ZIKV strains used in this study differ by only nine amino acids, and the results highlight that consideration must be given to strain type variation when examining the roles of miRNAs in ZIKV, and probably other virus infections.

A human neurodevelopmental model for Williams syndrome.

Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes, with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations, leading to behavioural pathologies in humans, remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome, we narrowed this cellular phenotype to a single gene candidate, frizzled 9 (FZD9). At the neuronal stage, layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites, increased numbers of spines and synapses, aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain.

Establishment of Human-Induced Pluripotent Stem Cell-Derived Neurons-A Promising In Vitro Model for a Molecular Study of Rabies Virus and Host Interaction.

Rabies is a deadly viral disease caused by the rabies virus (RABV), transmitted through a bite of an infected host, resulting in irreversible neurological symptoms and a 100% fatality rate in humans. Despite many aspects describing rabies neuropathogenesis, numerous hypotheses remain unanswered and concealed. Observations obtained from infected primary neurons or mouse brain samples are more relevant to human clinical rabies than permissive cell lines; however, limitations regarding the ethical issue and sample accessibility become a hurdle for discovering new insights into virus-host interplays. To better understand RABV pathogenesis in humans, we generated human-induced pluripotent stem cell (hiPSC)-derived neurons to offer the opportunity for an inimitable study of RABV infection at a molecular level in a pathologically relevant cell type. This study describes the characteristics and detailed proteomic changes of hiPSC-derived neurons in response to RABV infection using LC-MS/MS quantitative analysis. Gene ontology (GO) enrichment of differentially expressed proteins (DEPs) reveals temporal changes of proteins related to metabolic process, immune response, neurotransmitter transport/synaptic vesicle cycle, cytoskeleton organization, and cell stress response, demonstrating fundamental underlying mechanisms of neuropathogenesis in a time-course dependence. Lastly, we highlighted plausible functions of heat shock cognate protein 70 (HSC70 or HSPA8) that might play a pivotal role in regulating RABV replication and pathogenesis. Our findings acquired from this hiPSC-derived neuron platform help to define novel cellular mechanisms during RABV infection, which could be applicable to further studies to widen views of RABV-host interaction.

The contribution of GTF2I haploinsufficiency to Williams syndrome.

Williams syndrome (WS) is a neurodevelopmental disorder involving hemideletion of as many as 26-28 genes, resulting in a constellation of unique physical, cognitive and behavior phenotypes. The haploinsufficiency effect of each gene has been studied and correlated with phenotype(s) using several models including WS subjects, animal models, and peripheral cell lines. However, links for most of the genes to WS phenotypes remains unclear. Among those genes, general transcription factor 2I (GTF2I) is of particular interest as its haploinsufficiency is possibly associated with hypersociability in WS. Here, we describe studies of atypical WS cases as well as mouse models focusing on GTF2I that support a role for this protein in the neurocognitive and behavioral profiles of WS individuals. We also review collective studies on diverse molecular functions of GTF2I that may provide mechanistic explanation for phenotypes recently reported in our relevant cellular model, namely WS induced pluripotent stem cell (iPSC)-derived neurons. Finally, in light of the progress in gene-manipulating approaches, we suggest their uses in revealing the neural functions of GTF2I in the context of WS.

The vitamin D receptor agonist EB1089 can exert its antiviral activity independently of the vitamin D receptor.

Vitamin D has been shown to have antiviral activity in a number of different systems. However, few studies have investigated whether the antiviral activity is exerted through the vitamin D receptor (VDR). In this study, we investigated whether the antiviral activity of a vitamin D receptor agonist (EB1089) towards dengue virus (DENV) was modulated by VDR. To undertake this, VDR was successively overexpressed, knocked down and retargeted through mutation of the nuclear localization signal. In no case was an effect seen on the level of the antiviral activity induced by EB1089, strongly indicating that the antiviral activity of EB1089 is not exerted through VDR. To further explore the antiviral activity of EB1089 in a more biologically relevant system, human neural progenitor cells were differentiated from induced pluripotent stem cells, and infected with Zika virus (ZIKV). EB1089 exerted a significant antiviral effect, reducing virus titers by some 2Log10. In support of the results seen with DENV, no expression of VDR at the protein level was observed. Collectively, these results show that the vitamin D receptor agonist EB1089 exerts its antiviral activity independently of VDR.

Hydrogel-based 3D human iPSC-derived neuronal culture for the study of rabies virus infection.

Background: Rabies is a highly fatal infectious disease that poses a significant threat to human health in developing countries. In vitro study-based understanding of pathogenesis and tropism of different strains of rabies virus (RABV) in the central nervous system (CNS) is limited due to the lack of suitable culture models that recapitulate the complex communication pathways among host cells, extracellular matrices, and viruses. Therefore, a three-dimensional (3D) cell culture that mimics cell-matrix interactions, resembling in vivo microenvironment, is necessary to discover relevant underlying mechanisms of RABV infection and host responses. Methods: The 3D collagen-Matrigel hydrogel encapsulating hiPSC-derived neurons for RABV infection was developed and characterized based on cell viability, morphology, and gene expression analysis of neuronal markers. The replication kinetics of two different strains of RABV [wild-type Thai (TH) and Challenge Virus Standard (CVS)-11 strains] in both 2D and 3D neuronal cultures were examined. Differential gene expression analysis (DEG) of the neuropathological pathway of RABV-infected 2D and 3D models was also investigated via NanoString analysis. Results: The 3D hiPSC-derived neurons revealed a more physiologically interconnected neuronal network as well as more robust and prolonged maturation and differentiation than the conventional 2D monolayer model. TH and CVS-11 exhibited distinct growth kinetics in 3D neuronal model. Additionally, gene expression analysis of the neuropathological pathway observed during RABV infection demonstrated a vast number of differentially expressed genes (DEGs) in 3D model. Unlike 2D neuronal model, 3D model displayed more pronounced cellular responses upon infection with CVS-11 when compared to the TH-infected group, highlighting the influence of the cell environment on RABV-host interactions. Gene ontology (GO) enrichment of DEGs in the infected 3D neuronal culture showed alterations of genes associated with the inflammatory response, apoptotic signaling pathway, glutamatergic synapse, and trans-synaptic signaling which did not significantly change in 2D culture. Conclusion: We demonstrated the use of a hydrogel-based 3D hiPSC-derived neuronal model, a highly promising technology, to study RABV infection in a more physiological environment, which will broaden our understanding of RABV-host interactions in the CNS.

SARS-CoV-2 Delta (B.1.617.2) variant replicates and induces syncytia formation in human induced pluripotent stem cell-derived macrophages.

Alveolar macrophages are tissue-resident immune cells that protect epithelial cells in the alveoli from invasion by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the interaction between macrophages and SARS-CoV-2 is inevitable. However, little is known about the role of macrophages in SARS-CoV-2 infection. Here, we generated macrophages from human induced pluripotent stem cells (hiPSCs) to investigate the susceptibility of hiPSC-derived macrophages (iMΦ) to the authentic SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants as well as their gene expression profiles of proinflammatory cytokines during infection. With undetectable angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression, iMΦ were susceptible to productive infection with the Delta variant, whereas infection of iMΦ with the Omicron variant was abortive. Interestingly, Delta induced cell-cell fusion or syncytia formation in iMΦ, which was not observed in Omicron-infected cells. However, iMΦ expressed moderate levels of proinflammatory cytokine genes in response to SARS-CoV-2 infection, in contrast to strong upregulation of these cytokine genes in response to polarization by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overall, our findings indicate that the SARS-CoV-2 Delta variant can replicate and cause syncytia formation in macrophages, suggesting that the Delta variant can enter cells with undetectable ACE2 levels and exhibit greater fusogenicity.

PEDV and PDCoV Pathogenesis: The Interplay Between Host Innate Immune Responses and Porcine Enteric Coronaviruses.

Enteropathogenic porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), members of the coronavirus family, account for the majority of lethal watery diarrhea in neonatal pigs in the past decade. These two viruses pose significant economic and public health burdens, even as both continue to emerge and reemerge worldwide. The ability to evade, circumvent or subvert the host's first line of defense, namely the innate immune system, is the key determinant for pathogen virulence, survival, and the establishment of successful infection. Unfortunately, we have only started to unravel the underlying viral mechanisms used to manipulate host innate immune responses. In this review, we gather current knowledge concerning the interplay between these viruses and components of host innate immunity, focusing on type I interferon induction and signaling in particular, and the mechanisms by which virus-encoded gene products antagonize and subvert host innate immune responses. Finally, we provide some perspectives on the advantages gained from a better understanding of host-pathogen interactions. This includes their implications for the future development of PEDV and PDCoV vaccines and how we can further our knowledge of the molecular mechanisms underlying virus pathogenesis, virulence, and host coevolution.

Modeling Williams syndrome with induced pluripotent stem cells.

The development of induced pluripotent stem cells (iPSCs) like never before has opened novel opportunity to study diseases in relevant cell types. In our recent study, Williams syndrome (WS), a rare genetic neurodevelopmental disorder, that is caused by hemizygous deletion of 25-28 genes on chromosome 7, is of interest because of its unique cognitive and social profiles. Little is known about haploinsufficiency effect of those deleted genes on molecular and cellular phenotypes at the neural level due to the lack of relevant human cellular model. Using the cellular reprogramming approach, we reported that WS iPSC-derived neural progenitor cells (NPCs) has increased apoptosis and therefore increased doubling time, which could be rescued by complementation of frizzled 9, one of the genes typically deleted in WS. Moreover, WS iPSC-derived CTIP2-positive pyramidal neurons exhibit morphologic alterations including longer total dendrites and increasing dendritic spine number. In addition, WS iPSC-derived neurons show an increase in calcium transient frequency and synchronized activity likely due to increased number of dendritic spines and synapses. Our work integrated cross-level data from genetics to behavior of WS individuals and revealed altered cellular phenotypes in WS human NPCs and neurons that could be validated in other model systems such as magnetic resonance imaging (MRI) in live subjects and postmortem brain tissues.

Deciphering the biology of porcine epidemic diarrhea virus in the era of reverse genetics.

Emergence of the porcine epidemic diarrhea virus (PEDV) as a global threat to the swine industry underlies the urgent need for deeper understanding of this virus. To date, we have yet to identify functions for all the major gene products, much less grasp their implications for the viral life cycle and pathogenic mechanisms. A major reason is the lack of genetic tools for studying PEDV. In this review, we discuss the reverse genetics approaches that have been successfully used to engineer infectious clones of PEDV as well as other potential and complementary methods that have yet to be applied to PEDV. The importance of proper cell culture for successful PEDV propagation and maintenance of disease phenotype are addressed in our survey of permissive cell lines. We also highlight areas of particular relevance to PEDV pathogenesis and disease that have benefited from reverse genetics studies and pressing questions that await resolution by such studies. In particular, we examine the spike protein as a determinant of viral tropism, entry and virulence, ORF3 and its association with cell culture adaptation, and the nucleocapsid protein and its potential role in modulating PEDV pathogenicity. Finally, we conclude with an exploration of how reverse genetics can help mitigate the global impact of PEDV by addressing the challenges of vaccine development.

Systematic optimization of human pluripotent stem cells media using Design of Experiments.

Human pluripotent stem cells (hPSC) are used to study the early stages of human development in vitro and, increasingly due to somatic cell reprogramming, cellular and molecular mechanisms of disease. Cell culture medium is a critical factor for hPSC to maintain pluripotency and self-renewal. Numerous defined culture media have been empirically developed but never systematically optimized for culturing hPSC. We applied design of experiments (DOE), a powerful statistical tool, to improve the medium formulation for hPSC. Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin-1 beta 1 (NRG1ß1). The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming. It also enhances efficient hPSC plating as single cells. Altogether, iDEAL potentially allows scalable and controllable hPSC culture routine in translational research. Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.

Micro RNA detection in long-term fixed tissue of cortical glutamatergic pyramidal neurons after targeted laser-capture neuroanatomical microdissection.

BACKGROUND: Formalin fixation (FF) is the standard and most common method for preserving postmortem brain tissue. FF stabilizes cellular morphology and tissue architecture, and can be used to study the distinct morphologic and genetic signatures of different cell types. Although the procedure involved in FF degrades messenger RNA over time, an alternative approach is to use small RNAs (sRNAs) for genetic analysis associated with cell morphology. Although genetic analysis is carried out on fresh or frozen tissue, there is limited availability or impossibility on targeting specific cell populations, respectively. NEW METHOD: The goal of this study is to detect miRNA and other classes of sRNA stored in formalin or in paraffin embedded for over decades. Two brain samples, one formed by a mixed population of cortical and subcortical cells, and one formed by pyramidal shaped cells collected by laser-capture microdissection, were subjected to sRNA sequencing. RESULTS: Performing bioinformatics analysis over the sequenced sRNA from brain tissue, we detected several classes of sRNA, such as miRNAs that play key roles in brain neurodevelopmental and maintenance pathways, and hsa-mir-155 expression in neurons. Comparison with existing method: Our method is the first to combine the approaches for: laser-capture of pyramidal neurons from long-term formalin-fixed brain; extract sRNA from laser-captured pyramidal neurons; apply a suite of bioinformatics tools to detect miRNA and other classes of sRNAs on sequenced samples having high levels of RNA degradation. CONCLUSION: This is the first study to show that sRNA can be rescued from laser-captured FF pyramidal neurons.

Modeling neurodevelopmental disorders using human neurons.

The cellular and molecular mechanisms of neurodevelopmental conditions such as autism spectrum disorders have been studied intensively for decades. The unavailability of live patient neurons for research, however, has represented a major obstacle in the elucidation of the disease etiologies. Recently, the development of induced pluripotent stem cell (iPSC) technology allows for the generation of human neurons from somatic cells of patients. We review ongoing studies using iPSCs as an approach to model neurodevelopmental disorders, the promise and caveats of this technique and its potential for drug screening. The reproducible findings of relevant phenotypes in Rett syndrome iPSC-derived neurons suggest that iPSC technology offers a novel and unique opportunity for the understanding of and the development of therapeutics for other autism spectrum disorders.