Cellular Replication Research Articles

Genome-wide identification and expression analysis of C-type lectins in discus fish (Symphysodon aequifasciatus) during parental care

Discus fish (Symphysodon aequifasciatus) exhibit a unique parental care behavior: adult discus produces secretion through their skin, on which the larvae live after birth. The immune components in the skin mucus of parental discus would change during different parental care. C-type lectins (CTLs) could identify and eliminate pathogenic microorganisms and play important roles in innate immunity. Studies on CTLs of discus fish especially during parental care, however, are scarce. Here, we identified 186 CTL genes that distributed in 27 linkage groups based on discus genome. Phylogenetic analysis showed that S. aequifasciatus CTL (SaCTL) members were grouped into 14 subfamilies. A total of 80 gene replication events occurred, of which 15 pairs were subjected to segmental duplication and 65 pairs underwent tandem duplication. Ka/Ks ranged from 0.11 (SaCTL25/SaCTL158) to 0.68 (SaCTL36/SaCTL69), all undergoing purifying selection. RNA-seq analysis revealed that SaCTL members, including duplicated genes, in the skin of parental discus show distinct expression patterns in different care stages and between male and female parents. The SaCTL11 was differentially expressed in most care stages and reached the maximum after eggs spawned, but the expression of its paired SaCTL14 was low in each stage. The SaCTL39 increased first and then decreased, reaching a peak in eggs spawned, while paired SaCTL48 first decreased and then increased, reaching a peak in hatched eggs. The SaCTL50 was differentially expressed only in female fish during care, but not in male fish. These results provide new insights into the evolution and potential functional differentiation of CTLs in discus fish during parental care.

Replication elongates short DNA, reduces sequence bias and develops trimer structure.

The origin of molecular evolution required the replication of short oligonucleotides to form longer polymers. Prebiotically plausible oligonucleotide pools tend to contain more of some nucleobases than others. It has been unclear whether this initial bias persists and how it affects replication. To investigate this, we examined the evolution of 12-mer biased short DNA pools using an enzymatic model system. This allowed us to study the long timescales involved in evolution, since it is not yet possible with currently investigated prebiotic replication chemistries. Our analysis using next-generation sequencing from different time points revealed that the initial nucleotide bias of the pool disappeared in the elongated pool after isothermal replication. In contrast, the nucleotide composition at each position in the elongated sequences remained biased and varied with both position and initial bias. Furthermore, we observed the emergence of highly periodic dimer and trimer motifs in the rapidly elongated sequences. This shift in nucleotide composition and the emergence of structure through templated replication could help explain how biased prebiotic pools could undergo molecular evolution and lead to complex functional nucleic acids.

Specificity protein 1 (Sp1) and glucocorticoid receptor (GR) stimulate bovine alphaherpesvirus 1 (BoHV-1) replication and cooperatively transactivate the immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1+ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.

Tau seeding without tauopathy

Neurodegenerative tauopathies such as Alzheimer’s disease (AD) are caused by brain accumulation of tau assemblies. Evidence suggests tau functions as a prion, and cells and animals efficiently propagate unique, transmissible tau pathology. This suggests a dedicated cellular replication machinery, potentially with a normal physiologic function for tau seeds. Consequently, we hypothesized that healthy control brains would have seeding activity. We have recently developed a novel monoclonal antibody (MD3.1) specific for tau seeds. We used this antibody to immunopurify tau from the parietal and cerebellar cortices of 19 healthy subjects without any neuropathology, ranging 19-65 years. We detected seeding in lysates from the parietal cortex, but not in the cerebellum. We also detected no seeding in brain homogenates from wild-type or human tau knockin mice, suggesting that cellular/genetic context dictates development of seed-competent tau. Seeding did not correlate with subject age or brain tau levels. We confirmed our essential findings using an orthogonal assay, RTQuIC, which amplifies tau seeds in vitro. Dot blot analyses revealed no AT8 immunoreactivity above background levels in parietal and cerebellar extracts and ∼1/100 of that present in AD. Based on binding to a panel of antibodies, the conformational characteristics of control seeds differed from AD, suggesting a unique underlying assembly, or structural ensemble. Tau’s ability to adopt self-replicating conformations under non-pathogenic conditions may reflect a normal function that goes awry in disease states.

Insect α-Amylases and Their Application in Pest Management.

Amylase is an indispensable hydrolase in insect growth and development. Its varied enzymatic parameters cause insects to have strong stress resistance. Amylase gene replication is a very common phenomenon in insects, and different copies of amylase genes enable changes in its location and function. In addition, the classification, structure, and interaction between insect amylase inhibitors and amylases have also invoked the attention of researchers. Some plant-derived amylase inhibitors have inhibitory activities against insect amylases and even mammalian amylases. In recent years, an increasing number of studies have clarified the effects of pesticides on the amylase activity of target and non-target pests, which provides a theoretical basis for exploring safe and efficient pesticides, while the exact lethal mechanisms and safety in field applications remain unclear. Here, we summarize the most recent advances in insect amylase studies, including its sequence and characteristics and the regulation of amylase inhibitors (α-AIs). Importantly, the application of amylases as the nanocide trigger, RNAi, or other kinds of pesticide targets will be discussed. A comprehensive foundation will be provided for applying insect amylases to the development of new-generation insect management tools and improving the specificity, stability, and safety of pesticides.

Quencher-free fluorescent assays by controlled DNA partitioning in the aqueous two-phase system with crowding-enhanced kinetics

Fluorescent DNA assays are promising in disease diagnosis, environmental monitoring, and drug screening, encompassing both heterogeneous and homogeneous assay types. Nevertheless, heterogeneous assays suffer from tedious washing steps and slow reaction kinetics, whereas homogenous assays require well-designed fluorophore pairs to modulate signal off/on. Herein, we developed a cost-effective and efficient quencher-free fluorescent DNA assay using an aqueous two-phase system (ATPS). Using a strand-displacement reaction, we showed that similar sensing performance could be achieved at a much lower cost. Furthermore, the unique crowding environment in ATPS accelerated strand-displacement reactions by up to six-fold and reduced DNA amplification time from 120 min to 30 min. Our assay demonstrated robust sensing in serum environments and successful detection of miRNA extracted from cells. This innovative assay format has the potential for biosensor development with both heterogeneous readout and rapid reaction kinetics in various applications.

Exploring the Accessory Genome of Multidrug-Resistant Rhodococcus equi Clone 2287.

Decades of antimicrobial overuse to treat respiratory disease in foals have promoted the emergence and spread of zoonotic multidrug-resistant (MDR) Rhodococcus equi worldwide. Three main R. equi MDR clonal populations-2287, G2106, and G2017-have been identified so far. However, only clones 2287 and G2016 have been isolated from sick animals, with clone 2287 being the main MDR R. equi recovered. The genetic mechanisms that make this MDR clone superior to the others at infecting foals are still unknown. Here, we performed a deep genetic characterization of the accessory genomes of 207 R. equi isolates, and we describe IME2287, a novel genetic element in the accessory genome of clone 2287, potentially involved in the maintenance and spread of this MDR population over time. IME2287 is a putative self-replicative integrative mobilizable element (IME) carrying a DNA replication and partitioning operon and genes encoding its excision and integration from the R. equi genome via a serine recombinase. Additionally, IME2287 encodes a protein containing a Toll/interleukin-1 receptor (TIR) domain that may inhibit TLR-mediated NF-kB signaling in the host and a toxin-antitoxin (TA) system, whose orthologs have been associated with antibiotic resistance/tolerance, virulence, pathogenicity islands, bacterial persistence, and pathogen trafficking. This new set of genes may explain the success of clone 2287 over the other MDR R. equi clones.

Plant-Derived Epi-Nutraceuticals as Potential Broad-Spectrum Anti-Viral Agents.

Although the COVID-19 pandemic appears to be diminishing, the emergence of SARS-CoV-2 variants represents a threat to humans due to their inherent transmissibility, immunological evasion, virulence, and invulnerability to existing therapies. The COVID-19 pandemic affected more than 500 million people and caused over 6 million deaths. Vaccines are essential, but in circumstances in which vaccination is not accessible or in individuals with compromised immune systems, drugs can provide additional protection. Targeting host signaling pathways is recommended due to their genomic stability and resistance barriers. Moreover, targeting host factors allows us to develop compounds that are effective against different viral variants as well as against newly emerging virus strains. In recent years, the globe has experienced climate change, which may contribute to the emergence and spread of infectious diseases through a variety of factors. Warmer temperatures and changing precipitation patterns can increase the geographic range of disease-carrying vectors, increasing the risk of diseases spreading to new areas. Climate change may also affect vector behavior, leading to a longer breeding season and more breeding sites for disease vectors. Climate change may also disrupt ecosystems, bringing humans closer to wildlife that transmits zoonotic diseases. All the above factors may accelerate the emergence of new viral epidemics. Plant-derived products, which have been used in traditional medicine for treating pathological conditions, offer structurally novel therapeutic compounds, including those with anti-viral activity. In addition, plant-derived bioactive substances might serve as the ideal basis for developing sustainable/efficient/cost-effective anti-viral alternatives. Interest in herbal antiviral products has increased. More than 50% of approved drugs originate from herbal sources. Plant-derived compounds offer diverse structures and bioactive molecules that are candidates for new drug development. Combining these therapies with conventional drugs could improve patient outcomes. Epigenetics modifications in the genome can affect gene expression without altering DNA sequences. Host cells can use epigenetic gene regulation as a mechanism to silence incoming viral DNA molecules, while viruses recruit cellular epitranscriptomic (covalent modifications of RNAs) modifiers to increase the translational efficiency and transcript stability of viral transcripts to enhance viral gene expression and replication. Moreover, viruses manipulate host cells' epigenetic machinery to ensure productive viral infections. Environmental factors, such as natural products, may influence epigenetic modifications. In this review, we explore the potential of plant-derived substances as epigenetic modifiers for broad-spectrum anti-viral activity, reviewing their modulation processes and anti-viral effects on DNA and RNA viruses, as well as addressing future research objectives in this rapidly emerging field.

The genomic characteristics and pathogenicity of a mammalian orthoreovirus within a new lineage from wild pika in plateau

Emerging and re-emerging viruses from wild animals have seriously threatened the health of humans and domesticated animals in recent years. Herein, we isolated a new mammalian orthoreovirus (MRV), Pika/MRV/GCCDC7/2019 (PMRV-GCCDC7), in the Qinghai-Tibet Plateau wild pika (Ochotona curzoniae). Though the PMRV-GCCDC7 shows features of a typical reovirus with ten gene segments arranged in 3:3:4 in length, the virus belongs to an independent evolutionary branch compared to other MRVs based on phylogenetic tree analysis. The results of cellular susceptibility, species tropism, and replication kinetics of PMRV-GCCDC7 indicated the virus could infect four human cell lines (A549, Huh7, HCT, and LoVo) and six non-human cell lines, including Vero-E6, LLC-MK2, BHK-21, N2a, MDCK, and RfKT cell, derived from diverse mammals, i.e. monkey, mice, canine and bat, which revealed the potential of PMRV-GCCDC7 to infect a variety of hosts. Infection of BALB/c mice with PMRV-GCCDC7 via intranasal inoculation led to relative weight loss, lung tissue damage and inflammation with the increase of virus titer, but no serious respiratory symptoms and death occurred. The characterization of the new reovirus from a plateau-based wild animal has expanded our knowledge of the host range of MRV and provided insight into its risk of trans-species transmission and zoonotic diseases.

Chronic Mitochondrial Dysfunction Renders Acute Myeloid Leukemia Resistant to Multipronged Inhibition of Mitochondrial Metabolism

Acute Myeloid Leukemia (AML) is a genetically diverse and aggressive myeloid neoplasm that awaits novel therapeutics to improve patient survival. Targeting AML metabolic aberrations as a genotype-agnostic strategy has shown promise, evidenced by the clinical success of venetoclax and the rapid pre-clinical development of other mitochondrial (mt) metabolism targeted therapies. However, the mechanism of how AML develops resistance to drugs targeting key mt metabolic pathways is not fully understood. We previously identified sirtuin 5 (SIRT5), a deacylase that regulates several pathways of mt metabolism in AML including oxidative phosphorylation and glutamine metabolism. In AML, it functions as a master metabolic regulator that is necessary for leukemic stem cell survival. Genetic or pharmacological inhibition of SIRT5 leads to rapid AML cell apoptosis in vitro and disease regression in multiple AML mouse models. However, a subset of AML cell lines and primary samples can survive SIRT5 inhibition, indicating an unusual intrinsic resistance to mt dysfunction. To provide a mechanistic explanation, we first compared the molecular features between SIRT5-dependent and independent cases. Among the 25 primary AML samples we tested, NPM1c mutation, but not FLT3-ITD, TP53, WT1, NRAS, or IDH1 mutations, is significantly enriched in SIRT5-independent samples. Accordingly, among the panel of 22 AML cell lines we tested, the only NPM1 mutant cell line (OCI-AML3) sits at the most SIRT5-independent extreme of the spectrum. NPM1 mutant AML cells have increased cytoplasmic mitochondrial DNA (mtDNA) and fragmented mt morphology, indicating chronic mt damage (Wu H et al. Cancer Discovery. 2021). Because SIRT5 exerts its metabolic maintenance functions within the mt, we hypothesize that AML cells that can adapt to chronic, sublethal mt dysfunction will be capable of proliferation without SIRT5. To test this, we took three orthogonal approaches: (1) introducing NPM1c mutation, (2) using low concentrations of ethidium bromide (EtBr) to selectively block mtDNA replication, or (3) genetically inducing chronic mt dysfunction by silencing the essential mt transcription factor TFAM in 3 AML cell lines that are heavily dependent on SIRT5 - OCI-AML2, CMK, and SKM-1. SIRT5-dependent cells expressing ectopic NPM1c exhibit mt dysfunction as evidenced by decreased oxygen consumption rate (OCR) measured using the Seahorse metabolic flux assay. Also, the lethal effect of SIRT5 depletion is rescued in these cells. Pharmacologically blocking NPM1c nuclear export with selinexor in the typically SIRT5-independent OCI-AML3 cells sensitizes them to the lethal effect of SIRT5 depletion. After culturing in 100 ng/mL EtBr for 3, 5, or 11 days, cells had significantly decreased mt abundance as measured by mtDNA content relative to nuclear DNA and had significantly decreased OCR and extracellular acidification rate (ECAR). SIRT5-dependent cells cultured in EtBr survived shRNA-induced SIRT5 depletion, in contrast to the rapid lethality of cells not cultured in EtBr. Additionally, cells initially cultured in EtBr and allowed to recover in the absence of EtBr assume their original phenotype of SIRT5 dependence. However, inhibiting specific subunits of the mt using potent complex I and III inhibitors (rotenone and antimycin A) shows no effect on SIRT5 dependence. Together these data suggest that mt damage, rather than loss of function in one pathway, is needed to convert SIRT5-dependent cells to SIRT5 independence. TFAM, a nuclearly coded transcription factor for mtDNA, is necessary for the replication and transcription of mt genes. We used cas13d-mediated knockdown of TFAM to deplete mtDNA and induce mt dysfunction. Experiments that investigate how this method of inducing mt dysfunction affects OCR, ECAR, and SIRT5-dependent cell line proliferation are currently underway and will be presented at the ASH conference. In summary, we present a mechanism of how NPM1 mutation-associated or experimentally induced chronic sublethal mt dysfunction renders AML resistant to therapies targeting vital mt metabolic pathways, such as SIRT5 inhibitors, demonstrating AML's metabolic flexibility. Experiments are scheduled to validate these findings in primary samples and additional metabolomic and transcriptional studies will be done to deduce the mechanism for SIRT5 dependence in AML.

RNA-sequencing of peripheral whole blood of individuals at ultra-high-risk for psychosis - A longitudinal perspective.

The peripheral blood is an attractive source of prognostic biomarkers for psychosis conversion. There is limited research on the transcriptomic changes associated with psychosis conversion in the peripheral whole blood. We performed RNA-sequencing of peripheral whole blood from 65 ultra-high-risk (UHR) participants and 70 healthy control participants recruited in the Longitudinal Youth-at-Risk Study (LYRIKS) cohort. 13 UHR participants converted in the study duration. Samples were collected at 3 timepoints, at 12-months interval across a 2-year period. We examined whether the genes differential with psychosis conversion contain schizophrenia risk loci. We then examined the functional ontologies and GWAS associations of the differential genes. We also identified the overlap between differentially expressed genes across different comparisons. Genes containing schizophrenia risk loci were not differentially expressed in the peripheral whole blood in psychosis conversion. The differentially expressed genes in psychosis conversion are enriched for ontologies associated with cellular replication. The differentially expressed genes in psychosis conversion are associated with non-neurological GWAS phenotypes reported to be perturbed in schizophrenia and psychosis but not schizophrenia and psychosis phenotypes themselves. We found minimal overlap between the genes differential with psychosis conversion and the genes that are differential between pre-conversion and non-conversion samples. The associations between psychosis conversion and peripheral blood-based biomarkers are likely to be indirect. Further studies to elucidate the mechanism behind potential indirect associations are needed.

Plant virology: an RNA treasure trove.

Key principles pertaining to RNA biology not infrequently have their origins in plant virology. Examples have arisen from studies on viral RNA-intrinsic properties and the infection process from gene expression, replication, movement, and defense evasion to biotechnological applications. Since RNA is at the core of the central dogma in molecular biology, how plant virology assisted in the reinforcement or adaptations of this concept, while at other instances shook up elements of the doctrine, is discussed. Moreover, despite the negative effects of viral diseases in agriculture worldwide, plant viruses can be considered a scientific treasure trove. Today they remain tools of discovery for biotechnology, studying evolution, cell biology, and host-microbe interactions.

Epigenetics of Autism Spectrum Disorders: A Multi-level Analysis Combining Epi-signature, Age Acceleration, Epigenetic Drift and Rare Epivariations Using Public Datasets.

Epigenetics of Autism Spectrum Disorders (ASD) is still an understudied field. The majority of the studies on the topic used an approach based on mere classification of cases and controls. The present study aimed at providing a multi-level approach in which different types of epigenetic analysis (epigenetic drift, age acceleration) are combined. We used publicly available datasets from blood (n = 3) and brain tissues (n = 3), separately. Firstly, we evaluated for each dataset and meta-analyzed the differential methylation profile between cases and controls. Secondly, we analyzed age acceleration, epigenetic drift and rare epigenetic variations. We observed a significant epi-signature of ASD in blood but not in brain specimens. We did not observe significant age acceleration in ASD, while epigenetic drift was significantly higher compared to controls. We reported the presence of significant rare epigenetic variations in 41 genes, 35 of which were never associated with ASD. Almost all genes were involved in pathways linked to ASD etiopathogenesis (i.e., neuronal development, mitochondrial metabolism, lipid biosynthesis and antigen presentation). Our data support the hypothesis of the use of blood epi-signature as a potential tool for diagnosis and prognosis of ASD. The presence of an enhanced epigenetic drift, especially in brain, which is linked to cellular replication, may suggest that alteration in epigenetics may occur at a very early developmental stage (i.e., fetal) when neuronal replication is still high.

PacBio sequencing of human fecal samples uncovers the DNA methylation landscape of 22 673 gut phages.

Gut phages have an important impact on human health. Methylation plays key roles in DNA recognition, gene expression regulation and replication for phages. However, the DNA methylation landscape of gut phages is largely unknown. Here, with PacBio sequencing (2120×, 4785 Gb), we detected gut phage methylation landscape based on 22673 gut phage genomes, and presented diverse methylation motifs and methylation differences in genomic elements. Moreover, the methylation rate of phages was associated with taxonomy and host, and N6-methyladenine methylation rate was higher in temperate phages than in virulent phages, suggesting an important role for methylation in phage-host interaction. In particular, 3543 (15.63%) phage genomes contained restriction-modification system, which could aid in evading clearance by the host. This study revealed the DNA methylation landscape of gut phage and its potential roles, which will advance the understanding of gut phage survival and human health.

Porcine IGFBP3 promotes porcine circovirus type 2 replication via PERK/eIF2α mediated DNA damage

The infection of porcine circovirus type 2 (PCV2) triggers activation of the protein kinase RNA–like endoplasmic reticulum kinase (PERK) pathway and leads to DNA damage. Insulin-like growth factor-binding protein 3 (IGFBP3) may interact with the endoplasmic reticulum (ER). It remains unclear whether IGFBP3 regulates DNA damage via ER stress to mediate PCV2 replication. In this study, we observed an upregulation of porcine IGFBP3 expression during PCV2 infection, and overexpression of IGFBP3 enhanced the expression of PCV2 Cap protein, PCV2 DNA copy number, and viral titers in PK-15 B6 cells and 3D4/21 cells. Additionally, overexpression of IGFBP3 induced an increase in the DNA damage marker γH2AX by activating the PERK/eIF2α pathway without concomitant activation of ATF4, IRE1α, and ATF6α/GRP78 pathways in PK-15 B6 cells and 3D4/21 cells. Knockdown of IGFBP3 had a reverse effect on PCV2 replication in PK-15 B6 cells and 3D4/21 cells. Furthermore, treatment with etoposide enhanced PCV2 replication while KU57788 decreased it. GSK2606414 and salubrinal limited both DNA damage and viral replication. Therefore, our findings suggest that porcine IGFBP3 promotes PCV2 replication through the PERK/eIF2α pathway-mediated induction of DNA damage in PK-15 B6 cells and 3D4/21 cells. Our study provides a basis for exploring novel antiviral strategies via the extensive understanding of the relationships between host cellular proteins and viral replication.

Comprehensive metagenomic and enzyme activity analysis reveals the inhibitory effects and potential toxic mechanism of tetracycline on denitrification in groundwater

The widespread use of tetracycline (TC) inevitably leads to its increasing emission into groundwater. However, the potential risks of TC to denitrification in groundwater remain unclear. In this study, the effects of TC on denitrification in groundwater were systematically investigated at both the protein and gene levels from the electron behavior aspect for the first time. The results showed that increasing TC from 0 to 10 µg·L−1 decreased the nitrate removal rate from 0.41 to 0.26 mg·L−1·h−1 while enhancing the residual nitrite concentration from 0.52 mg·L−1 to 50.60 mg·L−1 at the end of the experiment. From a macroscopic view, 10 µg·L−1 TC significantly inhibited microbial growth and altered microbial community structure and function in groundwater, which induced the degeneration of denitrification. From the electron behavior aspect (the electron production, electron transport and electron consumption processes), 10 µg·L−1 TC decreased the concentration of electron donors (nicotinamide adenine dinucleotide, NADH), electron transport system activity, and denitrifying enzyme activities at the protein level. At the gene level, 10 µg·L−1 TC restricted the replication of genes related to carbon metabolism, the electron transport system and denitrification. Moreover, discrepant inhibitory effects of TC on individual denitrification steps, which led to the accumulation of nitrite, were observed in this study. These results provide the information that is necessary for evaluating the potential environmental risk of antibiotics on groundwater denitrification and bring more attention to their effects on geochemical nitrogen cycles.

Invasive non-typhoidal Salmonella (iNTS) aminoglycoside-resistant ST313 isolates feature unique pathogenic mechanisms to reach the bloodstream

Invasive non-typhoidal Salmonella (iNTS) from the clonal type ST313 (S. Typhimurium ST313) is the primary cause of invasive salmonellosis in Africa. Recently, in Brazil, iNTS ST313 strains have been isolated from different sources, but there is a lack of understanding of the mechanisms behind how these gut bacteria can break the gut barrier and reach the patient's bloodstream. Here, we compare 13 strains of S. Typhimurium ST313, previously unreported isolates, from human blood cultures, investigating aspects of virulence and mechanisms of resistance. Initially, RNAseq analyses between ST13-blood isolate and SL1344 (ST19) prototype revealed 15 upregulated genes directly related to cellular invasion and replication, such as sopD2, sifB, and pipB. Limited information is available about S. Typhimurium ST313 pathogenesis and epidemiology, especially related to the global distribution of strains. Herein, the correlation of strains isolated from different sources in Brazil was employed to compare clinical and non-clinical isolates, a total of 22 genomes were studied by single nucleotide polymorphism (SNPs). The epidemiological analysis of 22 genomes of S. Typhimurium ST313 strains grouped them into three distinct clusters (A, B, and C) by SNP analysis, where cluster A comprised five, group B six, and group C 11. The 13 clinical blood isolates were all resistant to streptomycin, 92.3% of strains were resistant to ampicillin and 15.39% were resistant to kanamycin. The resistance genes acrA, acrB, mdtK, emrB, emrR, mdsA, and mdsB related to the production of efflux pumps were detected in all (100%) strains studied, similar to pathogenic traits investigated. In conclusion, we evidenced that S. Typhimurium ST313 strains isolated in Brazil have unique epidemiology. The elevated frequencies of virulence genes such as sseJ, sopD2, and pipB are a major concern in these Brazilian isolates, showing a higher pathogenic potential.

MiR-HCC2 suppresses hepatitis B virus replication by inhibiting the activity of the enhancer I/X promoter.

miR-HCC2 has been reported to markedly promote the growth, metastasis, and stemness of hepatocellular carcinoma (HCC) cells in vitro and in vivo. Deep sequencing showed that miR-HCC2 was significantly upregulated in hepatitis B virus (HBV)-positive (HBV+) HCC tissue samples compared with HBV-negative (HBV-) HCC tissue samples. miR-HCC2 expression was further evaluated in HCC tissues and cells, and the expression of miR-HCC2 was found to be significantly higher in HBV+ HCC tissues and cells than in HBV- HCC tissues and cells, suggesting that high miR-HCC2 expression could be induced by HBV infection. To explore the relationship between miR-HCC2 and HBV, we investigated the effect of miR-HCC2 on HBV antigen expression, transcription, and replication. We found that miR-HCC2 was involved in the negative feedback regulation of HBV replication. Further mechanistic studies revealed that miR-HCC2 suppressed HBV replication by inhibiting the activity of the enhancer I/Xpromoter. Our study demonstrates the effect of the inhibition of miR-HCC2 on HBV gene expression and replication, which can help to illustrate the complex regulatory network involving host miRNAs and HBV.

Nuclear interferon-stimulated gene product maintains heterochromatin on the herpes simplex viral genome to limit lytic infection.

Interferons (IFN) are expressed in and secreted from cells in response to virus infection, and they induce the expression of a variety of genes called interferon-stimulated genes (ISGs) in infected and surrounding cells to block viral infection and limit spread. The mechanisms of action of a number of cytoplasmic ISGs have been well defined, but little is known about the mechanism of action of nuclear ISGs. Constitutive levels of nuclear interferon-inducible protein 16 (IFI16) serve to induce innate signaling and epigenetic silencing of herpes simplex virus (HSV), but only when the HSV infected cell protein 0 (ICP0) E3 ligase, which promotes IFI16 degradation, is inactivated. In this study, we found that following IFN induction, the pool of IFI16 within the infected cell remains high and can restrict wild-type viral gene expression and replication due to both the induced levels of IFI16 and the IFI16-mediated repression of ICP0 levels. Restriction of viral gene expression is achieved by IFI16 promoting the maintenance of heterochromatin on the viral genome, which silences it epigenetically. These results indicate that a nuclear ISG can restrict gene expression and replication of a nuclear DNA virus by maintaining or preventing the removal of repressive heterochromatin associated with the viral genome.

Interaction and assembly of the DNA replication core proteins of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes six highly conserved core replication proteins essential for the viral lytic DNA synthesis, including ORF6 (single-stranded DNA-binding protein), ORF9 (DNA polymerase), ORF40/41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor). Since the protein-protein interactions among KSHV core replication proteins are largely unknown, this study aimed to decipher their interrelationships. Herein, we propose a protein-protein interaction network of these six core replication proteins according to the results obtained from confocal fluorescence microscopy, coimmunoprecipitation, and mammalian two-hybrid (GAL4/VP16) reporter assays. In this interaction network, ORF40/41 plays a central role in the connection of different replication subcomplexes. In addition to the well-conserved helicase-primase subcomplex (consisting of ORF44, ORF56, and ORF40/41) and the replisome subcomplex (consisting of ORF9 and ORF59), our data also suggest that several discrete, stable subcomplexes exist in the cell nucleus. Among these replication subcomplexes in the nucleus, the tetrameric subcomplex composed of ORF44, ORF56, ORF40/41, and ORF6 exhibited the ability to trigger a DNA damage response. By using an established GAL4/VP16-based reporter system and confocal fluorescence microscopy, we additionally found that the heat shock protein 90 (Hsp90) inhibitor, radicicol, significantly inhibited the formation of both the helicase-primase subcomplex and the replisome subcomplex in a dose-dependent manner. Collectively, these data not only provide further insights into the interaction and assembly of KSHV-encoded core replication proteins but also suggest a critical role of Hsp90 in assisting the construction of the viral core replication machinery.IMPORTANCEEukaryotic DNA replication is a highly regulated process that requires multiple replication enzymes assembled onto DNA replication origins. Due to the complexity of the cell's DNA replication machinery, most of what we know about cellular DNA replication has come from the study of viral systems. Herein, we focus our study on the assembly of the Kaposi's sarcoma-associated herpesvirus core replication complex and propose a pairwise protein-protein interaction network of six highly conserved viral core replication proteins. A detailed understanding of the interaction and assembly of the viral core replication proteins may provide opportunities to develop new strategies against viral propagation.