A Viral Immunoevasin Controls Innate Immunity by Targeting the Prototypical Natural Killer Cell Receptor Family.

Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via paired NK cell receptors (NKRs). Despite identification of numerous NKR-ligand interactions, physiological ligands for the prototypical NK1.1 orphan receptor remain elusive. Here, we identify a viral ligand for the inhibitory and activating NKR-P1 (NK1.1) receptors. This murine cytomegalovirus (MCMV)-encoded protein, m12, restrains NK cell effector function by directly engaging the inhibitory NKR-P1B receptor. However, m12 also interacts with the activating NKR-P1A/C receptors to counterbalance m12 decoy function. Structural analyses reveal that m12 sequesters a large NKR-P1 surface area via a "polar claw" mechanism. Polymorphisms in, and ablation of, the viral m12 protein and host NKR-P1B/C alleles impact NK cell responses in vivo. Thus, we identify the long-sought foreign ligand for this key immunoregulatory NKR family and reveal how it controls the evolutionary balance of immune recognition during host-pathogen interplay.

mRNA translation in astrocytes controls hippocampal long-term synaptic plasticity and memory.

Activation of neuronal protein synthesis upon learning is critical for the formation of long-term memory. Here, we report that learning in the contextual fear conditioning paradigm engenders a decrease in eIF2α (eukaryotic translation initiation factor 2) phosphorylation in astrocytes in the hippocampal CA1 region, which promotes protein synthesis. Genetic reduction of eIF2α phosphorylation in hippocampal astrocytes enhanced contextual and spatial memory and lowered the threshold for the induction of long-lasting plasticity by modulating synaptic transmission. Thus, learning-induced dephosphorylation of eIF2α in astrocytes bolsters hippocampal synaptic plasticity and consolidation of long-term memories.

Impact of COVID-19 infections and vaccination on menstrual cycle symptoms in the south of Jordan: a cross-sectional study.

BACKGROUND: Several recent studies have highlighted the need for more evaluation of the impact of COVID-19 infections and vaccines on the reproductive system and menstruation. This study aimed to assess the impact of COVID-19 infection and vaccines on menstrual symptoms. METHODS: A cross-sectional survey utilizing face-to-face interviews from January 1 to 31 March 2022 was conducted in the city of Al-Karak in southern Jordan. The questionnaire included sociodemographic characteristics, medical and reproductive history, the contraceptive method used if any, menstrual cycle (MC) status, previous medical and drug history, and the impact of infection and vaccination on the MC. RESULTS: The study questionnaire was completed by 400 participants with a mean age of 32.1±12.6 years. Regarding the history of COVID-19 infections, 33.8% of the participants reported a history of confirmed COVID-19 infections, 77.8% of them did not report any menstrual changes following the infection, while the remaining 22.2% reported changes in menstruation. The most commonly reported post-COVID-19 manifestations were irregular (27.6%) and light menstrual cycle (MC) (24.15) or dysmenorrhea (24.1%). Heavy menstruation was reported by 17.2% of participants post-COVID-19 infection. Two-thirds of the study participants (66.6%) reported no changes in the MC following the administration of the COVID-19 vaccine. The most reported symptoms for those who experienced changes in the MC following the vaccination were irregular cycle (13.1%), heavy menstruation (7%), and light menstruation (7%). Other reported symptoms were dysmenorrhea (4.6%), intermenstrual bleeding (1.2%), and amenorrhea (0.5%). CONCLUSION: This study revealed minor changes in the MC following COVID-19 infections and administration of the COVID-19 vaccine. These findings are consistent with published reports. It is recommended that future clinical trials for new vaccines for women of childbearing age include outcomes related to sex hormones and MC. Women should be encouraged to take the vaccines and report symptoms to healthcare professionals for further assessment.

Genome analysis of novel Apilactobacillus sp. isolate from butterfly (Pieris canidia) gut reveals occurrence of unique glucanogenic traits and probiotic potential.

This study was conducted with a perception that fructose-rich niches may inhabit novel species of lactic acid bacteria that are gaining importance as probiotics and for the production of exopolysaccharides that have applications in food and pharmaceuticals. Recently, some Lactobacillus species have been reclassified as fructophilic lactic acid bacteria due to their preference for fructose over glucose as a carbon source. These bacteria are likely to be found in fructose rich niches such as flower nectar and insects that feed on it. We explored the butterfly gut and acquired a new isolate, designated as F1, of fructophilic lactic acid bacteria, which produces a glucan-type exopolysaccharide. Whole genome sequencing and in silico analysis revealed that F1 has significantly lower average nucleotide identity and DNA-DNA hybridization values as compared to its closest Apilactobacillus neighbors in phylogenetic analysis. Therefore, we declare the isolate F1 as a novel Apilactobacillus species with the proposed name of Apilactobacillus iqraium F1. Genome mining further revealed that F1 harbors genes for exopolysaccharide synthesis and health-promoting attributes. To this end, F1 is the only Apilactobacillus species harboring three diverse α-glucan-synthesis genes that cluster with different types of dextransucrases in the dendrogram. Moreover, many nutritional marker genes, as well as genes for epithelial cell adhesion and antimicrobial synthesis, were also detected suggesting the probiotic attributes of F1. Overall analysis suggests A. iqraium sp. F1 be a potential candidate for various health beneficial and pharmaceutical applications.

The Total Synthesis of Glycolipids from Streptococcus pneumoniae and a Re-evaluation of Their Immunological Activity.

Invariant natural killer (iNK) T cells, Type I iNKTs, are responsible for the production of pro-inflammatory cytokines which induce a systemic immune response. They are distinctive in possessing an semi-invariant T-cell receptor that recognizes glycolipid antigens presented by CD1d, a protein closely related to the class I major histocompatibility complex, conserved across multiple mammalian species in a class of proteins well-renowned for their high degree of polymorphism. This receptor's first potent identified antigen is the α-galactosylceramide, KRN7000, a synthetic glycosphingolipid closely related to those isolated from bacteria that were found on a Japanese marine sponge. A corresponding terrestrial antigen remained unidentified until two specific diacylglycerol-containing glycolipids, reported to activate iNKT cells, were isolated from Streptococcus pneumoniae. We report the total synthesis and immunological re-evaluation of these two glycolipids. The compounds are unable to meaningfully activate iNKT cells. Computational modelling shows that these ligands, while being capable of interacting with the CD1d receptor, create a different surface for the binary complex that makes formation of the ternary complex with the iNKT T-cell receptor difficult. Together these results suggest that the reported activity might have been due to an impurity in the original isolated sample and highlights the importance of taking care when reporting biological activity from isolated natural products.

Characterization of rotavirus strains isolated from children with acute gastroenteritis in Rawalpindi, Pakistan.

Diarrhea is the leading cause of childhood morbidity and mortality particularly in developing countries and rotavirus has been identified as the major pathogen associated with diarrheal infections. This study was conducted to detect genotypic distribution of predominant rotavirus strains circulating in children suffering from acute gastroenteritis in Rawalpindi, Pakistan. Stool specimens were collected from children ≤5 years of age, visiting Military Hospital, Rawalpindi, with signs and symptoms of acute gastroenteritis. Two hundred and eighty-four specimens were collected during the period from April 2017 to March 2018. Enzyme immunoassay was performed for detection of rotavirus and reverse transcription-PCR (RT-PCR) was carried out for amplification of VP7 and VP4 gene segments followed by multiplex PCR using genotype-specific primers. Out of 284 children, 71 were found rotavirus positive and among them, 54% were females and 46% males. Our findings showed 92% of infection among children ≤2 years of age, while, the peak age of rotavirus incidence was found to be 6-12 months. Although, rotavirus infection was observed throughout the year but frequency increased in winter. Subtype G1P[8] was more prevalent followed by G2P[4], G3P[8], and G4P[6] subtypes. The results of this study provide insight into the disease burden as well as information on rotavirus diversity which will be useful to develop future strategies to control and prevent diarrheal infections among children.

A Central Role for Ly49 Receptors in NK Cell Memory.

In the past decade, the study of NK cells was transformed by the discovery of three ways these "innate" immune cells display adaptive immune behavior, including the ability to form long-lasting, Ag-specific memories of a wide variety of immunogens. In this review, we examine these types of NK cell memory, highlighting their unique features and underlying similarities. We explore those similarities in depth, focusing on the role that Ly49 receptors play in various types of NK cell memory. From this Ly49 dependency, we will build a model by which we understand the three types of NK cell memory as aspects of what is ultimately the same adaptive immune process, rather than separate facets of NK cell biology. We hope that a defined model for NK cell memory will empower collaboration between researchers of these three fields to further our understanding of this surprising and clinically promising immune response.

Production of a high molecular weight levan by Bacillus paralicheniformis, an industrially and agriculturally important isolate from the buffalo grass rhizosphere.

A new exopolysaccharide (EPS) producing Gram-positive bacterium was isolated from the rhizosphere of Bouteloua dactyloides (buffalo grass) and its EPS product was structurally characterized. The isolate, designated as LB1-1A, was identified as Bacillus paralicheniformis based on 16S rRNA gene sequence and phylogenetic tree analysis. The EPS produced by LB1-1A was identified as a levan, having ß(2 → 6) linked backbone with ß(2 → 1) linkages at the branch points (4.66%). The isolate LB1-1A yielded large amount (~ 42 g/l) of levan having high weight average molecular weight (Mw) of 5.517 × 107 Da. The relatively low degree of branching and high molecular weight of this levan makes B. paralicheniformis LB1-1A a promising candidate for industrial applications.

Evaluating the Suitability of 3D Bioprinted Samples for Experimental Radiotherapy: A Pilot Study.

Radiotherapy is an important component in the treatment of lung cancer, one of the most common cancers worldwide, frequently resulting in death within only a few years of diagnosis. In order to evaluate new therapeutic approaches and compare their efficiency with regard to tumour control at a pre-clinical stage, it is important to develop standardized samples which can serve as inter-institutional outcome controls, independent of differences in local technical parameters or specific techniques. Recent developments in 3D bioprinting techniques could provide a sophisticated solution to this challenge. We have conducted a pilot project to evaluate the suitability of standardized samples generated from 3D printed human lung cancer cells in radiotherapy studies. The samples were irradiated at high dose rates using both broad beam and microbeam techniques. We found the 3D printed constructs to be sufficiently mechanically stable for use in microbeam studies with peak doses up to 400 Gy to test for cytotoxicity, DNA damage, and cancer cell death in vitro. The results of this study show how 3D structures generated from human lung cancer cells in an additive printing process can be used to study the effects of radiotherapy in a standardized manner.

Defective Influenza A Virus RNA Products Mediate MAVS-Dependent Upregulation of Human Leukocyte Antigen Class I Proteins.

Influenza A virus (IAV) increases the presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms for these processes have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947(H1N1) IAV significantly increased the presentation of HLA-B, -C, and -E on lung epithelial cells. Virus entry was not sufficient to induce HLA upregulation because UV-inactivated virus had no effect. Aberrant internally deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs) expressed from an IAV minireplicon were sufficient for inducing HLA upregulation. These defective RNAs bind to retinoic acid-inducible gene I (RIG-I) and initiate mitochondrial antiviral signaling (MAVS) protein-dependent antiviral interferon (IFN) responses. Indeed, MAVS was required for HLA upregulation in response to IAV infection or ectopic mvRNA/DI RNA expression. The effect was partially due to paracrine signaling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-ß and IFN-λ1 and conditioned media from these cells elicited a modest increase in HLA surface levels in naive epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by the Janus kinase (JAK) inhibitor ruxolitinib. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein; we determined that NS1 limits cell-intrinsic and paracrine mechanisms of HLA upregulation. Taken together, our findings indicate that aberrant IAV RNAs stimulate HLA presentation, which may aid viral evasion of innate immunity.IMPORTANCE Human leukocyte antigens (HLAs) are cell surface proteins that regulate innate and adaptive immune responses to viral infection by engaging with receptors on immune cells. Many viruses have evolved ways to evade host immune responses by modulating HLA expression and/or processing. Here, we provide evidence that aberrant RNA products of influenza virus genome replication can trigger retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS)-dependent remodeling of the cell surface, increasing surface presentation of HLA proteins known to inhibit the activation of an immune cell known as a natural killer (NK) cell. While this HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.

Critical role for the Ly49 family of class I MHC receptors in adaptive natural killer cell responses.

Adaptive natural killer (NK) cell memory represents a new frontier in immunology. Work over the last decade has discovered and confirmed the existence of NK cells with antigen-specific memories, which had previously been considered a unique property of T and B cells. These findings have shown that antigen-specific NK cells gain their specificity without the use of RAG proteins, representing a novel mechanism for generating antigen specificity, but the details of this mechanism have remained a mystery. We have discovered that members of the Ly49 family of surface receptors are critically involved in both the sensitization and the challenge phases of an NK cell memory response, as is antigen presentation from their binding partner, the class I MHC. Moreover, we demonstrate that the Ly49-interacting component of a presented antigen dictates the specificity of the NK cell memory response, implicating Ly49 receptors themselves in antigen-specific recognition. Finally, we demonstrate that adaptive NK cell memories can protect against an otherwise lethal melanoma without T cell or B cell support. These findings offer insight into the mechanism behind NK cell antigen specificity and demonstrate the clinical potential of this adaptive immune cell.

Correlation between the clinically diagnosed inflammatory process and periapical index scores in severely painful endodontically involved teeth.

AIM: To assess and correlate three distinct states of severely painful endodontically derived inflammation with their depiction on periapical radiographs using periapical index (PAI) scores. METHODOLOGY: During a period of 15 months, 368 consecutively enrolled patients with suspected endodontic emergency conditions were examined at the University of Zurich, Center of Dental Medicine. Cases with a severely painful (numeric rating scale, NRS-11 > 6) endodontically involved tooth and a clear pulpal and apical diagnosis (n = 162) were selected (one tooth per patient). Teeth were divided into three groups according to the clinically diagnosed main location of the inflammatory process: level 1: pulp (positive response to cold test), level 2: periodontium (no response to cold without swelling) and level 3: periapical tissues (no response to cold with swelling). Periapical radiographs were obtained using a digital unit and analysed by two calibrated observers. For level 2, which had the highest PAI variance (n = 76), the PAI scores were further scrutinized regarding their dependence on tooth location and the duration of pain. Data were analysed using chi-squared and non-parametric tests, alpha = 0.05. RESULTS: Overall, the PAI scores correlated well with the clinically diagnosed main location of periapical inflammation (Spearman's rho = 0.5131, P < 0.001), with level 1 having the lowest scores by far (P < 0.001) and level 2 having significantly lower scores compared to level 3 (P < 0.05). However, a PAI score of 5 was found in merely 3 teeth within the entire cohort, and 49% of the teeth in the level 2 group had no radiolucency (PAI < 3). Within level 2, the PAI scores were not dependent on tooth location but were substantially (P < 0.001) higher for teeth which had hurt for more than one week, and for root filled teeth. CONCLUSIONS: For the analysed, severely painful endodontically involved teeth, the clinically diagnosed main location of inflammation was reflected by the periapical index. PAI scores were not significantly influenced by anatomical noise, yet in some cases under-estimated the clinical situation.

Hidden Role of Gut Microbiome Dysbiosis in Schizophrenia: Antipsychotics or Psychobiotics as Therapeutics?

Schizophrenia is a chronic, heterogeneous neurodevelopmental disorder that has complex symptoms and uncertain etiology. Mounting evidence indicates the involvement of genetics and epigenetic disturbances, alteration in gut microbiome, immune system abnormalities, and environmental influence in the disease, but a single root cause and mechanism involved has yet to be conclusively determined. Consequently, the identification of diagnostic markers and the development of psychotic drugs for the treatment of schizophrenia faces a high failure rate. This article surveys the etiology of schizophrenia with a particular focus on gut microbiota regulation and the microbial signaling system that correlates with the brain through the vagus nerve, enteric nervous system, immune system, and production of postbiotics. Gut microbially produced molecules may lay the groundwork for further investigations into the role of gut microbiota dysbiosis and the pathophysiology of schizophrenia. Current treatment of schizophrenia is limited to psychotherapy and antipsychotic drugs that have significant side effects. Therefore, alternative therapeutic options merit exploration. The use of psychobiotics alone or in combination with antipsychotics may promote the development of novel therapeutic strategies. In view of the individual gut microbiome structure and personalized response to antipsychotic drugs, a tailored and targeted manipulation of gut microbial diversity naturally by novel prebiotics (non-digestible fiber) may be a successful alternative therapeutic for the treatment of schizophrenia patients.

Bioprinted Cancer Model of Neuroblastoma in a Renal Microenvironment as an Efficiently Applicable Drug Testing Platform.

Development of new anticancer drugs with currently available animal models is hampered by the fact that human cancer cells are embedded in an animal-derived environment. Neuroblastoma is the most common extracranial solid malignancy of childhood. Major obstacles include managing chemotherapy-resistant relapses and resistance to induction therapy, leading to early death in very-high-risk patients. Here, we present a three-dimensional (3D) model for neuroblastoma composed of IMR-32 cells with amplified genes of the myelocytomatosis viral related oncogene MYCN and the anaplastic lymphoma kinase (ALK) in a renal environment of exclusively human origin, made of human embryonic kidney 293 cells and primary human kidney fibroblasts. The model was produced with two pneumatic extrusion printheads using a commercially available bioprinter. Two drugs were exemplarily tested in this model: While the histone deacetylase inhibitor panobinostat selectively killed the cancer cells by apoptosis induction but did not affect renal cells in the therapeutically effective concentration range, the peptidyl nucleoside antibiotic blasticidin induced cell death in both cell types. Importantly, differences in sensitivity between two-dimensional (2D) and 3D cultures were cell-type specific, making the therapeutic window broader in the bioprinted model and demonstrating the value of studying anticancer drugs in human 3D models. Altogether, this cancer model allows testing cytotoxicity and tumor selectivity of new anticancer drugs, and the open scaffold design enables the free exchange of tumor and microenvironment by any cell type.

Mouse Cytomegalovirus m153 Protein Stabilizes Expression of the Inhibitory NKR-P1B Ligand Clr-b.

Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germ line-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand Clr-b (encoded by Clec2d) on infected cells. Here we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using m153-deficient MCMV mutants (Δm144-m158 and Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during Δm153 mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of Clec2d transcription, imaging experiments revealed that the m153 and Clr-b proteins only minimally colocalize within the same subcellular compartments, and tagged versions of the proteins were refractory to coimmunoprecipitation under mild-detergent conditions. Surprisingly, the Δm153 mutant possesses enhanced virulence in vivo, independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlight a unique mechanism by which MCMV modulates NK ligand expression.IMPORTANCE Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells ("missing-self"). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.

Growth Inhibition of Common Enteric Pathogens in the Intestine of Broilers by Microbially Produced Dextran and Levan Exopolysaccharides.

Antibiotics are generally applied for treatment or as subtherapeutic agents to overcome diseases caused by pathogenic bacteria including Escherichia coli, Salmonella and Enterococcus species in poultry. However, due to their possible adverse effects on animal health and to maintain food safety, probiotics, prebiotics, and synbiotics have been proposed as alternatives to antibiotic growth promoters (AGPs) in poultry production. In this study, the effects of prebiotics on the augmentation of broiler's indigenous gut microbiology were studied. Day old 180 broilers chicks were divided into four treatment groups: G, L, C1, and C2. The groups G and L were fed with basal diet containing 3% dextran and 3% levan, respectively. Control groups were fed with basal diets without antibiotic (C1) and with antibiotics (C2). The experimental groups showed decreased mortality as compared to control groups. After 35 days, the chickens were euthanized and intestinal fluid was analyzed for enteric pathogens on chromogenic agar plates and by 16S rRNA gene sequencing. Inhibition of the growth of E. coli and Enterococcus was observed in groups G and L, respectively, whereas Salmonella was only present in group C1. Also, high populations of lactic acid bacteria were detected in the intestine of prebiotic fed birds as compared to controls. These results depict that dextran and levan have the potential to replace the use of antibiotics in poultry feed for inhibiting the growth of common enteric pathogens. To the best of our knowledge, this is the first study where effects of dextran and levan on intestinal microbiota of broilers have been reported.

Current Challenges of Digital Health Interventions in Pakistan: Mixed Methods Analysis.

BACKGROUND: Digital health is well-positioned in low and middle-income countries (LMICs) to revolutionize health care due, in part, to increasing mobile phone access and internet connectivity. This paper evaluates the underlying factors that can potentially facilitate or hinder the progress of digital health in Pakistan. OBJECTIVE: The objective of this study is to identify the current digital health projects and studies being carried out in Pakistan, as well as the key stakeholders involved in these initiatives. We aim to follow a mixed-methods strategy and to evaluate these projects and studies through a strengths, weaknesses, opportunities, and threats (SWOT) analysis to identify the internal and external factors that can potentially facilitate or hinder the progress of digital health in Pakistan. METHODS: This study aims to evaluate digital health projects carried out in the last 5 years in Pakistan with mixed methods. The qualitative and quantitative data obtained from field surveys were categorized according to the World Health Organization's (WHO) recommended building blocks for health systems research, and the data were analyzed using a SWOT analysis strategy. RESULTS: Of the digital health projects carried out in the last 5 years in Pakistan, 51 are studied. Of these projects, 46% (23/51) used technology for conducting research, 30% (15/51) used technology for implementation, and 12% (6/51) used technology for app development. The health domains targeted were general health (23/51, 46%), immunization (13/51, 26%), and diagnostics (5/51, 10%). Smartphones and devices were used in 55% (28/51) of the interventions, and 59% (30/51) of projects included plans for scaling up. Artificial intelligence (AI) or machine learning (ML) was used in 31% (16/51) of projects, and 74% (38/51) of interventions were being evaluated. The barriers faced by developers during the implementation phase included the populations' inability to use the technology or mobile phones in 21% (11/51) of projects, costs in 16% (8/51) of projects, and privacy concerns in 12% (6/51) of projects. CONCLUSIONS: We conclude that while digital health has a promising future in Pakistan, it is still in its infancy at the time of this study. However, due to the coronavirus disease 2019 (COVID-19) pandemic, there is an increase in demand for digital health and implementation of health outcomes following global social distancing protocols, especially in LMICs. Hence, there is a need for active involvement by public and private organizations to regulate, mobilize, and expand the digital health sector for the improvement of health care systems in countries.

Deciphering the Enigmatic Biological Functions of RNA Guanine-Quadruplex Motifs in Human Cells.

Guanine-rich sequences in nucleic acids can form noncanonical structures known as guanine quadruplexes (G-quadruplexes), which constitute a not yet fully elucidated layer of regulatory function for central cellular processes. RNA G-quadruplexes have been shown to be involved in the modulation of translation, the regulation of (alternative) splicing, and the subcellular transport of mRNAs, among other processes. However, in living cells, an equilibrium between the formation of G-quadruplex structures and their unwinding by RNA helicases is likely. The extent to which G-rich sequences adopt G-quadruplex structures in living eukaryotic cells is currently a matter of debate. Multiple lines of evidence confirm the intracellular formation of G-quadruplex structures, such as their detection by immunochemical approaches, fluorogenic probes, and in vivo nuclear magnetic resonance. However, intracellular chemical probing suggests most if not all are in an unfolded state. It is therefore tempting to speculate that some G-quadruplex structures are only temporarily formed when they are required to contribute to the fine-tuning of the processes mentioned above. Future research should focus on the analysis of G-quadruplex formation under physiological conditions, which will allow the re-evaluation of the biological function of G-quadruplex motifs in regulatory processes in their natural environment and at physiological expression levels. This will help in the elucidation of their significance in the regulation of central processes in molecular biology and the exploitation of their potential as therapeutic targets.

Alternatively spliced variants of the 5'-UTR of the ARPC2 mRNA regulate translation by an internal ribosome entry site (IRES) harboring a guanine-quadruplex motif.

The 5'-UTR of the actin-related protein 2/3 complex subunit 2 (ARPC2) mRNA exists in two variants. Using a bicistronic reporter construct, the present study demonstrates that the longer variant of the 5'-UTR harbours an internal ribosome entry site (IRES) which is lacking in the shorter one. Multiple control assays confirmed that only this variant promotes cap-independent translation. Furthermore, it includes a guanine-rich region that is capable of forming a guanine-quadruplex (G-quadruplex) structure which was found to contribute to the IRES activity. To investigate the cellular function of the IRES element, we determined the expression level of ARPC2 at various cell densities. At high cell density, the relative ARPC2 protein level increases, supporting the presumed function of IRES elements in driving the expression of certain genes under stressful conditions that compromise cap-dependent translation. Based on chemical probing experiments and computer-based predictions, we propose a structural model of the IRES element, which includes the G-quadruplex motif exposed from the central stem-loop element. Taken together, our study describes the functional relevance of two alternative 5'-UTR splice variants of the ARPC2 mRNA, one of which contains an IRES element with a G-quadruplex as a central motif, promoting translation under stressful cellular conditions.