Viral noncoding RNAs have acquired increasing prominence as important regulators of infection and mediators of pathogenesis. Circular RNAs (circRNAs) generated by backsplicing events have been ...identified in several oncogenic human DNA viruses. Here, we show that Merkel cell polyomavirus (MCV), the etiologic cause of ∼80% of Merkel cell carcinomas (MCCs), also expresses circular RNAs. By RNase R-resistant RNA sequencing, four putative circRNA backsplice junctions (BSJs) were identified from the MCV early region (ER). The most abundantly expressed MCV circRNA, designated circMCV-T, is generated through backsplicing of all of ER exon II to form a 762-nucleotide (nt) circular RNA molecule. Curiously, circMCV-T, as well as two other less abundantly expressed putative MCV circRNAs, overlaps in a complementary fashion with the MCV microRNA (miRNA) locus that encodes MCV-miR-M1. circMCV-T is consistently detected in concert with linear T antigen transcripts throughout infection, suggesting a crucial role for this RNA molecule in the regulatory functions of the early region, known to be vital for viral replication. Knocking out the hairpin structure of MCV-miR-M1 in genomic early region expression constructs and using a new high-efficiency, recombinase-mediated, recircularized MCV molecular clone demonstrates that circMCV-T levels decrease in the presence of MCV-miR-M1, underscoring the interplay between MCV circRNA and miRNA. Furthermore, circMCV-T partially reverses the known inhibitory effect of MCV-miR-M1 on early gene expression. RNase R-resistant RNA sequencing of lytic rat polyomavirus 2 (RatPyV2) identified an analogously located circRNA, stipulating a crucial, conserved regulatory function of this class of RNA molecules in the family of polyomaviruses.
Covalently closed circular RNAs were recently described in the human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV). Here, we show that MCV, another DNA tumor virus, generates circRNAs from its early regulatory region in concert with T antigen linear transcripts. MCV circMCV-T interacts with another MCV noncoding RNA, miR-M1, to functionally modulate early region transcript expression important for viral replication and long-term episomal persistence. This work describes a dynamic regulatory network integrating circRNA/miRNA/mRNA biomolecules and underscores the intricate functional modulation between several classes of polyomavirus-encoded RNAs in the control of viral replication.
Cyclin-dependent kinase 1 (CDK1)/cyclin B1 phosphorylates many of the same substrates as mTORC1 (a key regulator of glucose metabolism), including the eukaryotic initiation factor 4E-binding protein ...1 (4E-BP1). Only mitotic CDK1 phosphorylates 4E-BP1 at residue S82 in mice (S83 in humans), in addition to the common 4E-BP1 phospho-acceptor sites phosphorylated by both CDK1 and mTORC1. We examined glucose metabolism in mice having a single aspartate phosphomimetic amino acid knock in substitution at the 4E-BP1 serine 82 (4E-BP1S82D) mimicking constitutive CDK1 phosphorylation.
Knock-in homozygous 4E-BP1S82D and 4E-BP1S82A C57Bl/6N mice were assessed for glucose tolerance testing (GTT) and metabolic cage analysis on regular and on high-fat chow diets. Gastrocnemius tissues from 4E-BP1S82D and WT mice were subject to Reverse Phase Protein Array analysis. Since the bone marrow is one of the few tissues typically having cycling cells that transit mitosis, reciprocal bone-marrow transplants were performed between male 4E-BP1S82D and WT mice, followed by metabolic assessment, to determine the role of actively cycling cells on glucose homeostasis.
Homozygous knock-in 4E-BP1S82D mice showed glucose intolerance that was markedly accentuated with a diabetogenic high-fat diet (p = 0.004). In contrast, homozygous mice with the unphosphorylatable alanine substitution (4E-BP1S82A) had normal glucose tolerance. Protein profiling of lean muscle tissues, largely arrested in G0, did not show protein expression or signaling changes that could account for these results. Reciprocal bone-marrow transplantation between 4E-BP1S82D and wild-type littermates revealed a trend for wild-type mice with 4E-BP1S82D marrow engraftment on high-fat diets to become hyperglycemic after glucose challenge.
4E-BP1S82D is a single amino acid substitution that induces glucose intolerance in mice. These findings indicate that glucose metabolism may be regulated by CDK1 4E-BP1 phosphorylation independent from mTOR and point towards an unexpected role for cycling cells that transit mitosis in diabetic glucose control.
Ion detection in a DNA nanopore FET device Livernois, William; Cao, Purunc Simon; Saha, Soumyadeep ...
Nanotechnology,
08/2024, Letnik:
35, Številka:
32
Journal Article
Recenzirano
An ion detection device that combines a DNA-origami nanopore and a field-effect transistor (FET) was designed and modeled to determine sensitivity of the nanodevice to the local cellular environment. ...Such devices could be integrated into a live cell, creating an abiotic-biotic interface integrated with semiconductor electronics. A continuum model is used to describe the behavior of ions in an electrolyte solution. The drift-diffusion equations are employed to model the ion distribution, taking into account the electric fields and concentration gradients. This was matched to the results from electric double layer theory to verify applicability of the model to a bio-sensing environment. The FET device combined with the nanopore is shown to have high sensitivity to ion concentration and nanopore geometry, with the electrical double layer behavior governing the device characteristics. A logarithmic relationship was found between ion concentration and a single FET current, generating up to 200 nA of current difference with a small applied bias.
Cutaneous T-cell lymphomas are a heterogeneous group of neoplasms originating in the skin, with mycosis fungoides (MF) and Sézary syndrome (SS) representing the most common variants. The cellular ...origin of cutaneous lymphomas has remained controversial owing to their immense phenotypic heterogeneity that obfuscates lineage reconstruction on the basis of classical surface biomarkers. To overcome this heterogeneity and reconstruct the differentiation trajectory of malignant cells in MF and SS, TCR sequencing was performed in parallel with targeted transcriptomics at the single-cell resolution among cutaneous samples in MF and SS. Unsupervised lineage reconstruction showed that Sézary cells exist as a population of CD4+ T cells distinct from those in patch, plaque, and tumor MF. Further investigation of malignant cell heterogeneity in SS showed that Sézary cells phenotypically comprised at least 3 subsets on the basis of differential proliferation potentials and expression of exhaustion markers. A T helper 1–polarized cell type, intermediate cell type, and exhausted T helper 2–polarized cell type were identified, with T helper 1– and T helper 2–polarized cells displaying divergent proliferation potentials. Collectively, these findings provide evidence to clarify the relationship between MF and SS and reveal cell subsets in SS that suggest a possible mechanism for therapeutic resistance.
SARS‐CoV‐2 NSP12, the viral RNA‐dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID‐19. To facilitate research on SARS‐CoV‐2 NSP12 protein, ...we developed a rat monoclonal antibody (CM12.1) against the NSP12 N‐terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID‐19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club‐like, vascular endothelial cells, and a range of immune cells, despite wide‐spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady‐state expression or extensive posttranslation modifications that limit antibody reactivity during SARS‐CoV‐2 replication.
Variable selection on high throughput biological data, such as gene expression or single nucleotide polymorphisms (SNPs), becomes inevitable to select relevant information and, therefore, to better ...characterize diseases or assess genetic structure. There are different ways to perform variable selection in large data sets. Statistical tests are commonly used to identify differentially expressed features for explanatory purposes, whereas Machine Learning wrapper approaches can be used for predictive purposes. In the case of multiple highly correlated variables, another option is to use multivariate exploratory approaches to give more insight into cell biology, biological pathways or complex traits.
A simple extension of a sparse PLS exploratory approach is proposed to perform variable selection in a multiclass classification framework.
sPLS-DA has a classification performance similar to other wrapper or sparse discriminant analysis approaches on public microarray and SNP data sets. More importantly, sPLS-DA is clearly competitive in terms of computational efficiency and superior in terms of interpretability of the results via valuable graphical outputs. sPLS-DA is available in the R package mixOmics, which is dedicated to the analysis of large biological data sets.
Organ-on-A-chip (OoAC) devices are miniaturized, functional, in vitro constructs that aim to recapitulate the in vivo physiology of an organ using different cell types and extracellular matrix, while ...maintaining the chemical and mechanical properties of the surrounding microenvironments. From an end-point perspective, the success of a microfluidic OoAC relies mainly on the type of biomaterial and the fabrication strategy employed. Certain biomaterials, such as PDMS (polydimethylsiloxane), are preferred over others due to their ease of fabrication and proven success in modelling complex organ systems. However, the inherent nature of human microtissues to respond differently to surrounding stimulations has led to the combination of biomaterials ranging from simple PDMS chips to 3D-printed polymers coated with natural and synthetic materials, including hydrogels. In addition, recent advances in 3D printing and bioprinting techniques have led to the powerful combination of utilizing these materials to develop microfluidic OoAC devices. In this narrative review, we evaluate the different materials used to fabricate microfluidic OoAC devices while outlining their pros and cons in different organ systems. A note on combining the advances made in additive manufacturing (AM) techniques for the microfabrication of these complex systems is also discussed.
The Nadanhada Terrane, located along the eastern margin of Eurasia, contains a typical accretionary complex related to paleo‐Pacific plate subduction‐accretion. The Yuejinshan Complex is the first ...stage accretion complex that consists of meta‐clastic rocks and metamafic‐ultramafic rocks, whereas the Raohe Complex forms the main parts of the terrane and consists of limestone, bedded chert, and mafic‐ultramafic rocks embedded as olistolith blocks in a weakly sheared matrix of clastic meta‐sedimentary rocks. Geochemical data indicate that the Yuejinshan metabasalts have normal mid‐ocean ridge basalt (N‐MORB) affinity, whereas the Raohe basaltic pillow lavas have an affinity to ocean island basalts (OIB). Sensitive high‐resolution ion microprobe (SHRIMP) U‐Pb zircon analyses of gabbro in the Raohe Complex yield a weighted mean 206Pb/238U zircon age of 216 ± 5 Ma, whereas two samples of granite intruded into the complex yield weighted mean 206Pb/238U zircon ages of 128 ± 2 and 129 ± 2 Ma. Laser ablation inductively coupled plasma mass spectrometry (LA‐ICPMS) U‐Pb zircon analyses of basaltic pillow lava in the Raohe Complex define a weighted mean age of 167 ± 1 Ma. Two sandstone samples in the Raohe Complex record younger concordant zircon weighted mean ages of 167 ± 17 and 137 ± 3 Ma. These new data support the view that accretion of the Raohe Complex was between 170 and 137 Ma, and that final emplacement of the Raohe Complex took place at 137–130 Ma. The accretion of the Yuejinshan Complex probably occurred between the 210 and 180 Ma, suggesting that paleo‐Pacific plate subduction was initiated in the Late Triassic to Early Jurassic.
Key Points
Nadanhada Terrane is an accretionary complexBasaltic rocks have OIB and N‐MORB affinitiesOnset of Pacific accretion at 210–180 Ma, final emplacement at 137–130 Ma