Strategies Shaping the Transcription of Carbohydrate-Active Enzyme Genes in Aspergillus nidulans

Understanding the coordinated regulation of the hundreds of carbohydrate-active enzyme (CAZyme) genes occurring in the genomes of fungi has great practical importance. We recorded genome-wide transcriptional changes of Aspergillus nidulans cultivated on glucose, lactose, or arabinogalactan, as well as under carbon-starved conditions. We determined both carbon-stress-specific changes (weak or no carbon source vs. glucose) and carbon-source-specific changes (one type of culture vs. all other cultures). Many CAZyme genes showed carbon-stress-specific and/or carbon-source-specific upregulation on arabinogalactan (138 and 62 genes, respectively). Besides galactosidase and arabinan-degrading enzyme genes, enrichment of cellulolytic, pectinolytic, mannan, and xylan-degrading enzyme genes was observed. Fewer upregulated genes, 81 and 107 carbon stress specific, and 6 and 16 carbon source specific, were found on lactose and in carbon-starved cultures, respectively. They were enriched only in galactosidase and xylosidase genes on lactose and rhamnogalacturonanase genes in both cultures. Some CAZyme genes (29 genes) showed carbon-source-specific upregulation on glucose, and they were enriched in β-1,4-glucanase genes. The behavioral ecological background of these characteristics was evaluated to comprehensively organize our knowledge on CAZyme production, which can lead to developing new strategies to produce enzymes for plant cell wall saccharification.

The FUT2 Variant c.461G>A (p.Trp154*) Is Associated With Differentially Expressed Genes and Nasopharyngeal Microbiota Shifts in Patients With Otitis Media

Otitis media (OM) is a leading cause of childhood hearing loss. Variants in FUT2, which encodes alpha-(1,2)-fucosyltransferase, were identified to increase susceptibility to OM, potentially through shifts in the middle ear (ME) or nasopharyngeal (NP) microbiotas as mediated by transcriptional changes. Greater knowledge of differences in relative abundance of otopathogens in carriers of pathogenic variants can help determine risk for OM in patients. In order to determine the downstream effects of FUT2 variation, we examined gene expression in relation to carriage of a common pathogenic FUT2 c.461G>A (p.Trp154*) variant using RNA-sequence data from saliva samples from 28 patients with OM. Differential gene expression was also examined in bulk mRNA and single-cell RNA-sequence data from wildtype mouse ME mucosa after inoculation with non-typeable Haemophilus influenzae (NTHi). In addition, microbiotas were profiled from ME and NP samples of 65 OM patients using 16S rRNA gene sequencing. In human carriers of the FUT2 variant, FN1, KMT2D, MUC16 and NBPF20 were downregulated while MTAP was upregulated. Post-infectious expression in the mouse ME recapitulated these transcriptional differences, with the exception of Fn1 upregulation after NTHi-inoculation. In the NP, Candidate Division TM7 was associated with wildtype genotype (FDR-adj-p=0.009). Overall, the FUT2 c.461G>A variant was associated with transcriptional changes in processes related to response to infection and with increased load of potential otopathogens in the ME and decreased commensals in the NP. These findings provide increased understanding of how FUT2 variants influence gene transcription and the mucosal microbiota, and thus contribute to the pathology of OM.

Radiation-Induced Alterations in Proliferation, Migration, and Adhesion in Lens Epithelial Cells and Implications for Cataract Development

The lens of the eye is one of the most radiosensitive tissues. Although the exact mechanism of radiation-induced cataract development remains unknown, altered proliferation, migration, and adhesion have been proposed as factors. Lens epithelial cells were exposed to X-rays (0.1–2 Gy) and radiation effects were examined after 12 h and 7 day. Proliferation was quantified using an MTT assay, migration was measured using a Boyden chamber and wound-healing assay, and adhesion was assessed on three extracellular matrices. Transcriptional changes were also examined using RT-qPCR for a panel of genes related to these processes. In general, a nonlinear radiation response was observed, with the greatest effects occurring at a dose of 0.25 Gy. At this dose, a reduction in proliferation occurred 12 h post irradiation (82.06 ± 2.66%), followed by an increase at 7 day (116.16 ± 3.64%). Cell migration was increased at 0.25 Gy, with rates 121.66 ± 6.49% and 232.78 ± 22.22% greater than controls at 12 h and 7 day respectively. Cell adhesion was consistently reduced above doses of 0.25 Gy. Transcriptional alterations were identified at these same doses in multiple genes related to proliferation, migration, and adhesion. Overall, this research began to elucidate the functional changes that occur in lens cells following radiation exposure, thereby providing a better mechanistic understanding of radiation-induced cataract development.

Transgenerational transcriptional heterogeneity from cytoplasmic chromatin

Transcriptional heterogeneity from plasticity of the epigenetic state of chromatin is thought to contribute to tumor evolution, metastasis, and drug resistance. However, the mechanisms leading to nongenetic cell-to-cell variation in gene expression remain poorly understood. Here we demonstrate that heritable transcriptional changes can result from the formation of micronuclei, aberrations of the nucleus that are common in cancer. Micronuclei have fragile nuclear envelopes (NE) that are prone to spontaneous rupture, which exposes chromosomes to the cytoplasm and disrupts many nuclear activities. Using a combination of long-term live-cell imaging and same-cell, single-cell RNA sequencing (Look-Seq2), we identified significant reduction of gene expression in micronuclei, both before and after NE rupture. Furthermore, chromosomes in micronuclei fail to normally recover histone 3 lysine 27 acetylation, a critical step for the reestablishment of normal transcription after mitosis. These transcription and chromatin defects can persist into the next generation in a subset of cells, even after these chromosomes are incorporated into normal daughter nuclei. Moreover, persistent transcriptional repression is strongly associated with, and may be explained by, surprisingly long-lived DNA damage to these reincorporated chromosomes. Therefore, heritable alterations in transcription can originate from aberrations of nuclear architecture.

The Occurrence of Malignancy in Trypanosoma brucei brucei by Rapid Passage in Mice

Pleomorphic Trypanosoma brucei are best known for their tightly controlled cell growth and developmental program, which ensures their transmissibility and host fitness between the mammalian host and insect vector. However, after long-term adaptation in the laboratory or by natural evolution, monomorphic parasites can be derived. The origin of these monomorphic forms is currently unclear. Here, we produced a series of monomorphic trypanosome stocks by artificially syringe-passage in mice, creating snapshots of the transition from pleomorphism to monomorphism. We then compared these artificial monomorphic trypanosomes, alongside several naturally monomorphic T. evansi and T. equiperdum strains, with the pleomorphic T. brucei. In addition to failing to generate stumpy forms in animal bloodstream, we found that monomorphic trypanosomes from laboratory and nature exhibited distinct differentiation patterns, which are reflected by their distinct differentiation potential and transcriptional changes. Lab-adapted monomorphic trypanosomes could still be induced to differentiate, and showed only minor transcriptional differences to that of the pleomorphic slender forms but some accumulated differences were observed as the passages progress. All naturally monomorphic strains completely fail to differentiate, corresponding to their impaired differentiation regulation. We propose that the natural phenomenon of trypanosomal monomorphism is actually a malignant manifestation of protozoal cells. From a disease epidemiological and evolutionary perspective, our results provide evidence for a new way of thinking about the origin of these naturally monomorphic strains, the malignant evolution of trypanosomes may raise some concerns. Additionally, these monomorphic trypanosomes may reflect the quantitative and qualitative changes in the malignant evolution of T. brucei, suggesting that single-celled protozoa may also provide the most primitive model of cellular malignancy, which could be a primitive and inherent biological phenomenon of eukaryotic organisms from protozoans to mammals.

Neuronal Yin Yang1 in the prefrontal cortex regulates transcriptional and behavioral responses to chronic stress in mice

Although the synaptic alterations associated with the stress-related mood disorder major depression has been well-documented, the underlying transcriptional mechanisms remain poorly understood. Here, we perform complementary bulk nuclei- and single-nucleus transcriptome profiling and map locus-specific chromatin interactions in mouse neocortex to identify the cell type-specific transcriptional changes associated with stress-induced behavioral maladaptation. We find that cortical excitatory neurons, layer 2/3 neurons in particular, are vulnerable to chronic stress and acquire signatures of gene transcription and chromatin structure associated with reduced neuronal activity and expression of Yin Yang 1 (YY1). Selective ablation of YY1 in cortical excitatory neurons enhances stress sensitivity in both male and female mice and alters the expression of stress-associated genes following an abbreviated stress exposure. These findings demonstrate how chronic stress impacts transcription in cortical excitatory neurons and identify YY1 as a regulator of stress-induced maladaptive behavior in mice.

Comparative Analysis on Transcriptomics of Ivermectin Resistant and Sensitive Strains of Haemonchus Contortus

Background: Ivermectin (IVM) is one of the most important and widely used anthelmintics in veterinary medicine. However, its efficacy is increasingly compromised by widespread resistance, and the exact mechanism of IVM resistance remains unclear for most parasitic nematodes including Haemonchus contortus, a blood-sucking parasitic nematode of small ruminants.Methods: In this study, we isolated and assessed an IVM resistant strain from Zhaosu, Xinjiang, China. Subsequently, the comparative analyses on transcriptomics of IVM susceptible and resistant H. contortus adult worms were carried out using RNA sequencing and bioinformatics.Results: In total, 543 and 359 differentially expressed genes (DEGs) were identified in male and female adult worms of the resistant strain compared with the susceptible strain, respectively. The DEGs encode molecules involved in receptor activities, transport, detoxification, lipid metabolism and cuticle collagen formation. In addition, Gene Ontology (GO) analysis revealed that transcriptional changes were dominant in genes associated with ligand-gated channel activity, oxidation-reduction process, lipid metabolic process, and structural constituent of cuticle. The results support previous proposal that the IVM resistant mechanism of H. contortus involved in both neuromuscular and non-neuromuscular pathways. Finally, the quantitative RT-PCR results confirmed that the transcriptional profiles of selected DEGs (male: 8 genes, female: 10 genes) were consistent with those obtained by the RNA-Seq.Conclusions: The findings from this work provided valuable information for further studies on the IVM resistance in H. contortus.

Interactions Between Streptococcus gordonii and Fusobacterium nucleatum Altered Bacterial Transcriptional Profiling and Attenuated the Immune Responses of Macrophages

Interspecies coaggregation promotes transcriptional changes in oral bacteria, affecting bacterial pathogenicity. Streptococcus gordonii (S. gordonii) and Fusobacterium nucleatum (F. nucleatum) are common oral inhabitants. The present study investigated the transcriptional profiling of S. gordonii and F. nucleatum subsp. polymorphum in response to the dual-species coaggregation using RNA-seq. Macrophages were infected with both species to explore the influence of bacterial coaggregation on both species’ abilities to survive within macrophages and induce inflammatory responses. Results indicated that, after the 30-min dual-species coaggregation, 116 genes were significantly up-regulated, and 151 genes were significantly down-regulated in S. gordonii; 97 genes were significantly down-regulated, and 114 genes were significantly up-regulated in F. nucleatum subsp. polymorphum. Multiple S. gordonii genes were involved in the biosynthesis and export of cell-wall proteins and carbohydrate metabolism. F. nucleatum subsp. polymorphum genes were mostly associated with translation and protein export. The coaggregation led to decreased expression levels of genes associated with lipopolysaccharide and peptidoglycan biosynthesis. Coaggregation between S. gordonii and F. nucleatum subsp. polymorphum significantly promoted both species’ intracellular survival within macrophages and attenuated the production of pro-inflammatory cytokines IL-6 and IL-1β. Physical interactions between these two species promoted a symbiotic lifestyle and repressed macrophage’s killing and pro-inflammatory responses.

Regulation of gene expression by the APP family in the adult cerebral cortex

Amyloid precursor protein (APP) is associated with both familial and sporadic forms of Alzheimer’s disease. APP has two homologs, amyloid precursor-like protein 1 and 2 (APLP1 and APLP2), and they have functional redundancy. APP intracellular c-terminal domain (AICD), produced by sequential α- or β- and γ-secretase cleavages, is thought to control gene expression, similarly as the ICD of Notch. To investigate the role of APP family in transcriptional regulation, we examined gene expression changes in the cerebral cortex of APP/APLP1/APLP2 conditional triple knockout (cTKO) mice, in which APP family members are selectively inactivated in excitatory neurons of the postnatal forebrain. Of the 12 previously reported AICD target genes, only Nep and Npas4 mRNA levels were significantly reduced in the cerebral cortex of cTKO mice, compared to littermate controls. We further examined global transcriptional changes by RNA-seq and identified 189 and 274 differentially expressed genes in the neocortex and hippocampus, respectively, of cTKO mice relative to controls. Gene Ontology analysis indicated that these genes are involved in a variety of cellular functions, including extracellular organization, learning and memory, and ion channels. Thus, inactivation of APP family alters transcriptional profiles of the cerebral cortex and affects wide-ranging molecular pathways.

Longitudinal single-cell transcriptomics reveals distinct patterns of recurrence in acute myeloid leukemia

The heterogeneity and evolution of AML blasts can render therapeutic interventions ineffective in a yet poorly understood patient-specific manner. To gain insight into the clonal heterogeneity of diagnosis (Dx) and relapse (Re) pairs, we employed whole-exome sequencing and single-cell RNA-seq to longitudinally profile two t(8;21) (AML1-ETO = RUNX1-RUNX1T1), and four FLT3-ITD AML cases. The single cell RNA data underpinned the tumor heterogeneity amongst patient blasts. The Dx-Re transcriptomes of high risk FLT3-ITD pairs formed a continuum from extensively changed in the absence of significantly mutational changes in AML-associated genes to rather similar Dx-Re pair of an intermediate risk FLT3-ITD. In one high risk FLT3-ITD pair, a pathway switched from an AP-1 regulated network in Dx to mTOR signaling in Re. The distinct AML1-ETO pairs comprise clusters that share genes related to hematopoietic stem cell maintenance and cell migration suggesting that the Re leukemic stem cell-like (LSC-like) cells probably evolved from the Dx LSC-like cells. In summary, our study revealed a continuum from drastic transcriptional changes to extensive similarities between respective Dx-Re pairs that are poorly explained by the well-established model of clonal evolution. Our results suggest alternative and currently unappreciated and unexplored mechanisms leading to therapeutic resistance and AML recurrence.