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The construction of biosensors for specific, sensitive, and rapid detection of tumor biomarkers significantly contributes to biomedical research and early cancer diagnosis. However, conventional assays often involve large sample consumption and poor sensitivity, limiting their further application in real samples. In recent years, single-molecule biosensing has emerged as a robust tool for detecting and characterizing biomarkers due to its unique advantages including simplicity, low sample consumption, ultra-high sensitivity, and rapid assay time. This review summarizes the recent advances in the construction of single-molecule biosensors for the measurement of various tumor biomarkers, including DNAs, DNA modifications, RNAs, and enzymes. We give a comprehensive review about the working principles and practical applications of these single-molecule biosensors. Additionally, we discuss the challenges and limitations of current single-molecule biosensors, and highlight the future directions. [ABSTRACT FROM AUTHOR]

Simple Summary: Leptospirosis is an infectious zoonotic disease caused by pathogenic Leptospira and affects both humans and animals throughout the world. Domestic animals, such as dogs, can act as potential reservoirs of leptospires for human or animal infections and environmental contamination. Due to the different varieties of the predominant local circulating pathogenic serovars in several regions, serodiagnosis tests, such as conventional microscopic agglutination tests (MATs) or others that do not include local serovars and their protein antigens, have limitations, and may fail to detect the disease and carry out antibody surveillance in certain regions. This study aimed to develop a more accurate antibody detection tool for canine leptospirosis in our region by using an indirect enzyme-linked immunosorbent assay (ELISA) using relevant local isolates of leptospiral serovars from asymptomatic dogs. All the modified IgG-ELISAs with the local isolates gave positive results for all infected dogs, especially outer membrane protein (OMP)-based IgG-ELISAs that showed negative results for all dogs from non-endemic areas, demonstrating improved accuracy and reduced limitations over those of the standard MAT and providing an enhanced method for leptospirosis detection in the study area. This improvement is crucial when investigating the epidemiology of the disease and preventing its spread. The article highlights the requirement and significance of utilising local circulating isolates in serological approaches to accurately diagnose and monitor leptospirosis. Leptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance. [ABSTRACT FROM AUTHOR]

The pathologic consequences of Coronavirus Disease-2019 (COVID-19) include elevated inflammation and dysregulated vascular functions associated with thrombosis. In general, disruption of vascular homeostasis and ensuing prothrombotic events are driven by activated platelets, monocytes, and macrophages, which form aggregates (thrombi) attached to the endothelium lining of vessel walls. However, molecular pathways underpinning the pathological interactions between myeloid cells and endothelium during COVID-19 remain undefined. Here, we tested the hypothesis that modulations in the expression of cellular receptors angiotensin-converting enzyme 2 (ACE2), CD147, and glucose-regulated protein 78 (GRP78), which are involved in homeostasis and endothelial performance, are the hallmark responses induced by SARS-CoV-2 infection. Cultured macrophages and lungs of hamster model systems were used to test this hypothesis. The results indicate that while macrophages and endothelial cells are less likely to support SARS-CoV-2 proliferation, these cells may readily respond to inflammatory stimuli generated by the infected lung epithelium. SARS-CoV-2 induced modulations of tested cellular receptors correlated with corresponding changes in the mRNA expression of coagulation cascade regulators and endothelial integrity components in infected hamster lungs. Among these markers, tissue factor (TF) had the best correlation for prothrombotic events during SARS-CoV-2 infection. Furthermore, the single-molecule fluorescence in situ hybridization (smFISH) method alone was sufficient to determine the peak and resolution phases of SARS-CoV-2 infection and enabled screening for cellular markers co-expressed with the virus. These findings suggest possible molecular pathways for exploration of novel drugs capable of blocking the prothrombotic shift events that exacerbate COVID-19 pathophysiology and control the disease. [ABSTRACT FROM AUTHOR]

Aims: We hypothesized that critically ill patients with SARS-CoV-2 infection and insulin resistance would present a reduced Heat Shock Response (HSR), which is a pathway involved in proteostasis and anti-inflammation, subsequently leading to worse outcomes and higher inflammation. In this work we aimed: (i) to measure the concentration of extracellular HSP72 (eHSP72) in patients with severe COVID-19 and in comparison with noninfected patients; (ii) to compare the HSR between critically ill patients with COVID-19 (with and without diabetes); and (iii) to compare the HSR in these patients with noninfected individuals. Methods: Sixty critically ill adults with acute respiratory failure with SARS-CoV-2, with or without diabetes, were selected. Noninfected subjects were included for comparison (healthy, n = 19 and patients with diabetes, n = 22). Blood samples were collected to measure metabolism (glucose and HbA1c); oxidative stress (lypoperoxidation and carbonyls); cytokine profile (IL-10 and TNF); eHSP72; and the HSR (in vitro). Results: Patients with severe COVID-19 presented higher plasma eHSP72 compared with healthy individuals and noninfected patients with diabetes. Despite the high level of plasma cytokines, no differences were found between critically ill patients with COVID-19 with or without diabetes. Critically ill patients, when compared to noninfected, presented a blunted HSR. Oxidative stress markers followed the same pattern. No differences in the HSR (extracellular/intracellular level) were found between critically ill patients, with or without diabetes. Conclusions: We demonstrated that patients with severe COVID-19 have elevated plasma eHSP72 and that their HSR is blunted, regardless of the presence of diabetes. These results might explain the uncontrolled inflammation and also provide insights on the increased risk in developing type 2 diabetes after SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

The outbreak of coronavirus disease 19 (COVID-19), caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in an unprecedented amount of infection cases and deaths, leading to the global health crisis. Despite many research efforts, our understanding of COVID-19 remains elusive. Recent studies have suggested that cell surface glucose-regulated protein 78 (GRP78) acts as a host co-receptor for SARS-CoV-2 infection and is related to COVID-19 risks, such as older age, obesity, and diabetes. Given its significance in a wide range of biological processes, such as protein homeostasis and cellular signaling, GRP78 might also play an important role in various stages of the viral life cycle and pathology of SARS-CoV-2. In this perspective, we explore the emerging and potential roles of GRP78 in SARS-CoV-2 infection. Additionally, we discuss the association with COVID-19 risks and symptoms. We hope this review article will be helpful to understand COVID-19 pathology and promote attention and study of GRP78 from many clinical and basic research fields. [ABSTRACT FROM AUTHOR]

Pathogens of bacterial and viral origin hijack pathways operating in eukaryotic cells in many ways in order to gain access into the host, to establish themselves and to eventually produce their progeny. The detailed molecular characterization of the subversion mechanisms devised by pathogens to infect host cells is crucial to generate targets for therapeutic intervention. Here we review recent data indicating that coronaviruses probably co-opt membranous carriers derived from the endoplasmic reticulum, which contain proteins that regulate disposal of misfolded polypeptides, for their replication. In addition, we also present models describing potential mechanisms that coronaviruses could employ for this hijacking [ABSTRACT FROM AUTHOR]

Many viruses, including coronaviruses, induce host translational shutoff, while maintaining synthesis of their own gene products. In this study we performed genome-wide microarray analyses of the expression patterns of mouse hepatitis coronavirus (MHV)-infected cells. At the time of MHV-induced host translational shutoff, downregulation of numerous mRNAs, many of which encode protein translation-related factors, was observed. This downregulation, which is reminiscent of a cellular stress response, was dependent on viral replication and caused by mRNA decay. Concomitantly, phosphorylation of the eukaryotic translation initiation factor 2α was increased in MHV-infected cells. In addition, stress granules and processing bodies appeared, which are sites for mRNA stalling and degradation respectively. We propose that MHV replication induces host translational shutoff by triggering an integrated stress response. However, MHV replication per se does not appear to benefit from the inhibition of host protein synthesis, at least in vitro, since viral replication was not negatively affected but rather enhanced in cells with impaired translational shutoff. [ABSTRACT FROM AUTHOR]

In the past year and a half, SARS-CoV-2 has caused 240 million confirmed cases and 5 million deaths worldwide. Autophagy is a conserved process that either promotes or inhibits viral infections. Although coronaviruses are known to utilize the transport of autophagy-dependent vesicles for the viral life cycle, the underlying autophagy-inducing mechanisms remain largely unexplored. Using several autophagy-deficient cell lines and autophagy inhibitors, we demonstrated that SARS-CoV-2 ORF3a was able to induce incomplete autophagy in a FIP200/Beclin-1-dependent manner. Moreover, ORF3a was involved in the induction of the UPR (unfolded protein response), while the IRE1 and ATF6 pathways, but not the PERK pathway, were responsible for mediating the ORF3a-induced autophagy. These results identify the role of the UPR pathway in the ORF3a-induced classical autophagy process, which may provide us with a better understanding of SARS-CoV-2 and suggest new therapeutic modalities in the treatment of COVID-19. [ABSTRACT FROM AUTHOR]

The innate immune system relies on families of pattern recognition receptors (PRRs) that detect distinct conserved molecular motifs from microbes to initiate antimicrobial responses. Activation of PRRs triggers a series of signaling cascades, leading to the release of pro-inflammatory cytokines, chemokines and antimicrobials, thereby contributing to the early host defense against microbes and regulating adaptive immunity. Additionally, PRRs can detect perturbation of cellular homeostasis caused by pathogens and fine-tune the immune responses. Among PRRs, nucleotide binding oligomerization domain (NOD)-like receptors (NLRs) have attracted particular interest in the context of cellular stress-induced inflammation during infection. Recently, mechanistic insights into the monitoring of cellular homeostasis perturbation by NLRs have been provided. We summarize the current knowledge about the disruption of cellular homeostasis by pathogens and focus on NLRs as innate immune sensors for its detection. We highlight the mechanisms employed by various pathogens to elicit cytoskeleton disruption, organelle stress as well as protein translation block, point out exemplary NLRs that guard cellular homeostasis during infection and introduce the concept of stress-associated molecular patterns (SAMPs). We postulate that integration of information about microbial patterns, danger signals, and SAMPs enables the innate immune system with adequate plasticity and precision in elaborating responses to microbes of variable virulence. [ABSTRACT FROM AUTHOR]

The ongoing COVID-19 pandemic is a clear and present threat to global public health. Research into how the causative SARS-CoV-2 virus together with its individual constituent genes and proteins interact with target host cells can facilitate the development of improved strategies to manage the acute and long-term complications of COVID-19. In this study, to better understand the biological roles of critical SARS-CoV-2 proteins, we determined and compared the host transcriptomic responses of the HL-CZ human pro-monocytic cell line upon transfection with key viral genes encoding the spike S1 subunit, S2 subunit, nucleocapsid protein (NP), NSP15 (endoribonuclease), and NSP16 (2′-O-ribose-methyltransferase). RNA sequencing followed by gene set enrichment analysis and other bioinformatics tools revealed that host genes associated with topologically incorrect protein, virus receptor activity, heat shock protein binding, endoplasmic reticulum stress, antigen processing and presentation were up-regulated in the presence of viral spike S1 expression. With spike S2 expression, pro-monocytic genes associated with the interferon-gamma-mediated signaling pathway, regulation of phosphatidylinositol 3-kinase activity, adipocytokine signaling pathway, and insulin signaling pathway were down-regulated, whereas those associated with cytokine-mediated signaling were up-regulated. The expression of NSP15 induced the up-regulation of genes associated with neutrophil degranulation, neutrophil-mediated immunity, oxidative phosphorylation, prion disease, and pathways of neurodegeneration. The expression of NSP16 resulted in the down-regulation of genes associated with S-adenosylmethionine-dependent methyltransferase activity. The expression of NP down-regulated genes associated with positive regulation of neurogenesis, nervous system development, and heart development. Taken together, the complex transcriptomic alterations arising from these viral-host gene interactions offer useful insights into host genes and their pathways that potentially contribute to SARS-CoV-2 pathogenesis. [ABSTRACT FROM AUTHOR]

Mitochondria emerged from bacterial ancestors during endosymbiosis and are crucial for cellular processes such as energy production and homeostasis, stress responses, cell survival, and more. They are the site of aerobic respiration and adenosine triphosphate (ATP) production in eukaryotes. However, oxidative phosphorylation (OXPHOS) is also the source of reactive oxygen species (ROS), which are both important and dangerous for the cell. Human mitochondria contain mitochondrial DNA (mtDNA), and its integrity may be endangered by the action of ROS. Fortunately, human mitochondria have repair mechanisms that allow protecting mtDNA and repairing lesions that may contribute to the occurrence of mutations. Mutagenesis of the mitochondrial genome may manifest in the form of pathological states such as mitochondrial, neurodegenerative, and/or cardiovascular diseases, premature aging, and cancer. The review describes the mitochondrial structure, genome, and the main mitochondrial repair mechanism (base excision repair (BER)) of oxidative lesions in the context of common features between human mitochondria and bacteria. The authors present a holistic view of the similarities of mitochondria and bacteria to show that bacteria may be an interesting experimental model for studying mitochondrial diseases, especially those where the mechanism of DNA repair is impaired. [ABSTRACT FROM AUTHOR]