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Background/Objectives: Sepsis is characterized by a dysregulated immune response to infection and is associated with high lethality. Extracellular DNA (ecDNA) has drawn significant interest as a damage-associated molecular pattern because of its potential involvement in the pathophysiology of sepsis. Methods: In this study, we examined the ecDNA concentration in 27 adult patients admitted to the intensive care unit. Fluorometry and quantitative PCR were used for the assessment of ecDNA. In addition, deoxyribonuclease activity was measured as a potential modulator of ecDNA. Results: Our findings reveal nearly 5-fold higher concentrations of ecDNA in non-survivors, suggesting its potential as a prognostic indicator for sepsis outcomes on day 7. Interestingly, the subcellular origin of ecDNA was similar between patients diagnosed with systemic inflammatory response syndrome, sepsis, and septic shock. Deoxyribonuclease activity, implicated in the cleavage of ecDNA, was comparable across all patient groups. Conclusions: To establish the prognostic value of ecDNA as a biomarker, further investigations within a larger patient cohort are needed. Nevertheless, our results suggest that high ecDNA in sepsis patients represents a negative prognostic biomarker. [ABSTRACT FROM AUTHOR]

Sepsis continues to be a significant public health challenge despite advances in understanding its pathophysiology and management strategies. Therefore, this study evaluated the value of cell-free nuclear DNA (cf-nDNA) and cell-free mitochondrial DNA (cf-mtDNA) for assessing the severity and prognosis of sepsis. Ninety-four patients were divided into three groups: infection (n = 32), sepsis (n = 30), and septic shock (n = 32). Plasma samples were collected at the time of diagnosis, and cfDNA concentrations were determined by qPCR assay. The results showed that plasma cfDNA levels increased with the severity of the disease. To distinguish between patients with infection and those with sepsis, the biomarker L1PA290 achieved the highest AUC of 0.817 (95% CI: 0.725–0.909), demonstrating a sensitivity of 77.0% and a specificity of 79.3%. When cf-nDNA was combined with the SOFA score, there was a significant improvement in the AUC (0.916 (0.853–0.979)), sensitivity (88.1%), and specificity (80.0%). Moreover, patients admitted to the ICU after being diagnosed with sepsis had significantly higher cf-nDNA concentrations. In patients admitted to the ICU, combining cf-nDNA with the SOFA score yielded an AUC of 0.753 (0.622–0.857), with a sensitivity of 95.2% and a specificity of 50.0%. cfDNA can differentiate between patients with infection and those with sepsis. It can also identify patients who are likely to be admitted to the ICU by predicting those with indications for intensive care, suggesting its potential as a biomarker for sepsis. [ABSTRACT FROM AUTHOR]

Background/Objectives: Sepsis is characterized by a dysregulated immune response to infection and is associated with high lethality. Extracellular DNA (ecDNA) has drawn significant interest as a damage-associated molecular pattern because of its potential involvement in the pathophysiology of sepsis. Methods: In this study, we examined the ecDNA concentration in 27 adult patients admitted to the intensive care unit. Fluorometry and quantitative PCR were used for the assessment of ecDNA. In addition, deoxyribonuclease activity was measured as a potential modulator of ecDNA. Results: Our findings reveal nearly 5-fold higher concentrations of ecDNA in non-survivors, suggesting its potential as a prognostic indicator for sepsis outcomes on day 7. Interestingly, the subcellular origin of ecDNA was similar between patients diagnosed with systemic inflammatory response syndrome, sepsis, and septic shock. Deoxyribonuclease activity, implicated in the cleavage of ecDNA, was comparable across all patient groups. Conclusions: To establish the prognostic value of ecDNA as a biomarker, further investigations within a larger patient cohort are needed. Nevertheless, our results suggest that high ecDNA in sepsis patients represents a negative prognostic biomarker.

• Cell-free mitochondrial DNA (cf-mtDNA) is an emerging biomarker of psychobiological stress. • MitoQuicLy was developed to perform high-throughput quantification of cf-mtDNA in clinically relevant biofluids. • Within a person, cf-mtDNA levels vary up to ∼100-fold between biofluid types. • Correlations between cf-mtDNA and clinical biomarkers is biofluid dependent. • These results raise critical questions around the biological nature of cf-mtDNA in different biofluids. Circulating cell-free mitochondrial DNA (cf-mtDNA) is an emerging biomarker of psychobiological stress and disease which predicts mortality and is associated with various disease states. To evaluate the contribution of cf-mtDNA to health and disease states, standardized high-throughput procedures are needed to quantify cf-mtDNA in relevant biofluids. Here, we describe MitoQuicLy: Mito chondrial DNA Qu antification i n c ell-free samples by Ly sis. We demonstrate high agreement between MitoQuicLy and the commonly used column-based method, although MitoQuicLy is faster, cheaper, and requires a smaller input sample volume. Using 10 µL of input volume with MitoQuicLy, we quantify cf-mtDNA levels from three commonly used plasma tube types, two serum tube types, and saliva. We detect, as expected, significant inter-individual differences in cf-mtDNA across different biofluids. However, cf-mtDNA levels between concurrently collected plasma, serum, and saliva from the same individual differ on average by up to two orders of magnitude and are poorly correlated with one another, pointing to different cf-mtDNA biology or regulation between commonly used biofluids in clinical and research settings. Moreover, in a small sample of healthy women and men (n = 34), we show that blood and saliva cf-mtDNAs correlate with clinical biomarkers differently depending on the sample used. The biological divergences revealed between biofluids, together with the lysis-based, cost-effective, and scalable MitoQuicLy protocol for biofluid cf-mtDNA quantification, provide a foundation to examine the biological origin and significance of cf-mtDNA to human health. [ABSTRACT FROM AUTHOR]

Sepsis continues to be a significant public health challenge despite advances in understanding its pathophysiology and management strategies. Therefore, this study evaluated the value of cell-free nuclear DNA (cf-nDNA) and cell-free mitochondrial DNA (cf-mtDNA) for assessing the severity and prognosis of sepsis. Ninety-four patients were divided into three groups: infection (n = 32), sepsis (n = 30), and septic shock (n = 32). Plasma samples were collected at the time of diagnosis, and cfDNA concentrations were determined by qPCR assay. The results showed that plasma cfDNA levels increased with the severity of the disease. To distinguish between patients with infection and those with sepsis, the biomarker L1PA290 achieved the highest AUC of 0.817 (95% CI: 0.725–0.909), demonstrating a sensitivity of 77.0% and a specificity of 79.3%. When cf-nDNA was combined with the SOFA score, there was a significant improvement in the AUC (0.916 (0.853–0.979)), sensitivity (88.1%), and specificity (80.0%). Moreover, patients admitted to the ICU after being diagnosed with sepsis had significantly higher cf-nDNA concentrations. In patients admitted to the ICU, combining cf-nDNA with the SOFA score yielded an AUC of 0.753 (0.622–0.857), with a sensitivity of 95.2% and a specificity of 50.0%. cfDNA can differentiate between patients with infection and those with sepsis. It can also identify patients who are likely to be admitted to the ICU by predicting those with indications for intensive care, suggesting its potential as a biomarker for sepsis.

Currently, non-communicable diseases (NCDs) have emerged as potential risks for humans due to adopting a sedentary lifestyle and inaccurate diagnoses. The early detection of NCDs using point-of-care technologies significantly decreases the burden and will be poised to transform clinical intervention and healthcare provision. An imbalance in the levels of circulating cell-free microRNAs (ccf-miRNA) has manifested in NCDs, which are passively released into the bloodstream or actively produced from cells, improving the efficacy of disease screening and providing enormous sensing potential. The effective sensing of ccf-miRNA continues to be a significant technical challenge, even though sophisticated equipment is needed to analyze readouts and expression patterns. Nanomaterials have come to light as a potential solution as they provide significant advantages over other widely used diagnostic techniques to measure miRNAs. Particularly, CNDs-based fluorescence nano-biosensors are of great interest. Owing to the excellent fluorescence characteristics of CNDs, developing such sensors for ccf-microRNAs has been much more accessible. Here, we have critically examined recent advancements in fluorescence-based CNDs biosensors, including tools and techniques used for manufacturing these biosensors. Green synthesis methods for scaling up high-quality, fluorescent CNDs from a natural source are discussed. The various surface modifications that help attach biomolecules to CNDs utilizing covalent conjugation techniques for multiple applications, including self-assembly, sensing, and imaging, are analyzed. The current review will be of particular interest to researchers interested in fluorescence-based biosensors, materials chemistry, nanomedicine, and related fields, as we focus on CNDs-based nano-biosensors for ccf-miRNAs detection applications in the medical field. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is one of the most common and deadly tumors worldwide. Management of HCC depends on reliable biomarkers for screening, diagnosis, and monitoring of the disease, as well as predicting response towards therapy and safety. To date, imaging has been the established standard technique in the diagnosis and follow-up of HCC. However, imaging techniques have their limitations, especially in the early detection of HCC. Therefore, there is an urgent need for reliable, non/minimal invasive biomarkers. To date, alpha-fetoprotein (AFP) is the only serum biomarker used in clinical practice for the management of HCC. However, AFP is of relatively rather low quality in terms of specificity and sensitivity. Liquid biopsies as a source for biomarkers have become the focus of clinical research. Our review highlights alternative biomarkers derived from liquid biopsies, including circulating tumor cells, proteins, circulating nucleic acids, and exosomes, and their potential for clinical application. Using defined combinations of different biomarkers will open new perspectives for diagnosing, treating, and monitoring HCC. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) represents the third most prevalent cancer worldwide and a leading cause of mortality among the population of western countries. However, CRC is frequently a preventable malignancy due to various screening tests being available. While failing to obtain real-time data, current screening methods (either endoscopic or stool-based tests) also require disagreeable preparation protocols and tissue sampling through invasive procedures, rendering adherence to CRC screening programs suboptimal. In this context, the necessity for novel, less invasive biomarkers able to identify and assess cancer at an early stage is evident. Liquid biopsy comes as a promising minimally invasive diagnostic tool, able to provide comprehensive information on tumor heterogeneity and dynamics during carcinogenesis. This review focuses on the potential use of circulating tumor cells (CTCs), circulating nucleic acids (CNAs) and extracellular vesicles as emerging liquid biopsy markers with clinical application in the setting of CRC screening. The review also examines the opportunity to implement liquid biopsy analysis during everyday practice and provides highlights on clinical trials researching blood tests designed for early cancer diagnosis. Additionally, the review explores potential applications of liquid biopsies in the era of immunotherapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostate cancer (PCa) is a widespread malignancy, representing the second leading cause of cancer-related death in men. In the last years, liquid biopsy has emerged as an attractive and promising strategy complementary to invasive tissue biopsy to guide PCa diagnosis, follow-up and treatment response. Liquid biopsy is employed to assess several body fluids biomarkers, including circulating tumor cells (CTCs), extracellular vesicles (EVs), circulating tumor DNA (ctDNA) and RNA (ctRNA). This review dissects recent advancements and future perspectives of liquid biopsy, highlighting its strength and weaknesses in PCa management. Although appreciable attempts in screening and diagnostic approaches have been achieved, prostate cancer (PCa) remains a widespread malignancy, representing the second leading cause of cancer-related death in men. Drugs currently used in PCa therapy initially show a potent anti-tumor effect, but frequently induce resistance and PCa progresses toward metastatic castration-resistant forms (mCRPC), virtually incurable. Liquid biopsy has emerged as an attractive and promising strategy complementary to invasive tissue biopsy to guide PCa diagnosis and treatment. Liquid biopsy shows the ability to represent the tumor microenvironment, allow comprehensive information and follow-up the progression of the tumor, enabling the development of different treatment strategies as well as permitting the monitoring of therapy response. Liquid biopsy, indeed, is endowed with a significant potential to modify PCa management. Several blood biomarkers could be analyzed for diagnostic, prognostic and predictive purposes, including circulating tumor cells (CTCs), extracellular vesicles (EVs), circulating tumor DNA (ctDNA) and RNA (ctRNA). In addition, several other body fluids may be adopted (i.e., urine, sperm, etc.) beyond blood. This review dissects recent advancements and future perspectives of liquid biopsies, highlighting their strength and weaknesses in PCa management. [ABSTRACT FROM AUTHOR]

Despite many advances in assisted reproductive technologies (ART), only 10-15% of the embryos replaced give birth to a child. Some of the failures are directly attributable to the embryo itself, but the main limiting factor is the implantation failure, often associated with desynchronizations of dialogue between embryonic and maternal tissue. Over the past decade, Omics techniques (high-throughput techniques) have been widely used in the fertility field. Notably, the study of circulating nucleic acids (microRNAs, free DNA) has been the subject of important scientific and medical advances. Indeed, they currently occupy a primordial place as biomarkers of interest in human pathology, especially in cancer. Easily accessible in biological fluids or in the embryonic culture medium, they represent tools of choice in ART for the development of new noninvasive diagnostic and prognostic tests. The use of these biomarkers both in the assessment of the ovary reserve, embryonic quality and in the prevention of implantation failures, corresponds to an innovative and extremely promising approach in the field. On the other hand, Omics techniques have enabled major advances in the identification of endometrial receptivity biomarkers, which have therefore allowed the establishment of an endometrial receptivity test, prompting practitioners to revisit their practices. [ABSTRACT FROM AUTHOR]

Currently, non-communicable diseases (NCDs) have emerged as potential risks for humans due to adopting a sedentary lifestyle and inaccurate diagnoses. The early detection of NCDs using point-of-care technologies significantly decreases the burden and will be poised to transform clinical intervention and healthcare provision. An imbalance in the levels of circulating cell-free microRNAs (ccf-miRNA) has manifested in NCDs, which are passively released into the bloodstream or actively produced from cells, improving the efficacy of disease screening and providing enormous sensing potential. The effective sensing of ccf-miRNA continues to be a significant technical challenge, even though sophisticated equipment is needed to analyze readouts and expression patterns. Nanomaterials have come to light as a potential solution as they provide significant advantages over other widely used diagnostic techniques to measure miRNAs. Particularly, CNDs-based fluorescence nano-biosensors are of great interest. Owing to the excellent fluorescence characteristics of CNDs, developing such sensors for ccf-microRNAs has been much more accessible. Here, we have critically examined recent advancements in fluorescence-based CNDs biosensors, including tools and techniques used for manufacturing these biosensors. Green synthesis methods for scaling up high-quality, fluorescent CNDs from a natural source are discussed. The various surface modifications that help attach biomolecules to CNDs utilizing covalent conjugation techniques for multiple applications, including self-assembly, sensing, and imaging, are analyzed. The current review will be of particular interest to researchers interested in fluorescence-based biosensors, materials chemistry, nanomedicine, and related fields, as we focus on CNDs-based nano-biosensors for ccf-miRNAs detection applications in the medical field.

Introduction: Circulating nucleic acids can be obtained by minimally invasive procedures based on liquid biopsy, which has emerged as a promising area of investigation for screening and monitoring cancer treatment. Currently, tests based on circulating nucleic acid analysis, specifically cellfree DNA (cfDNA), are commercially available for diagnostic and prognostic investigation of a number of neoplasms. Objective: To describe the research on circulating nucleic acid markers for cancer prospecting in Brazil, since this area has advanced rapidly in recent years. Methods: In this systematic review, we surveyed Brazilian publications in cancer research focused on cfDNA and cfRNA present in different fluids. Both MEDLINE-PUBMED and EMBASE databases were inspected using terms such as "circulating nucleic acids", "cancer", and "Brazil". Results: The search returned 326 articles, in which 28 Brazilian translational studies were eligible. Different methodologies were reported for different types of cancer, in which cfDNA from plasma was the most investigated biological material. Molecular investigations included quantification, somatic mutation, RNA expression, genotyping, microsatellites, blood protein interaction, and methylation. Discrepancies in the regional distribution of the studies were also observed. Conclusion: Studies on circulating nucleic acid markers have advanced significantly in the oncology field, but many others are needed to better address the clinical practice in Brazil. [ABSTRACT FROM AUTHOR]

Gliomas are diffusely growing tumours arising from progenitors within the central nervous system. They encompass a range of different molecular types and subtypes, many of which have a well‐defined profile of driver mutations, copy number changes and DNA methylation patterns. A majority of gliomas will require surgical intervention to relieve raised intracranial pressure and reduce tumour burden. A proportion of tumours, however, are located in neurologically sensitive areas and a biopsy poses a significant risk of a deficit. A majority of gliomas recur after surgery, and monitoring tumour burden of the recurrence is currently achieved by imaging. However, most imaging modalities have limitations in assessing tumour burden and infiltration into adjacent brain, and sometimes imaging is unable to discriminate between tumour recurrence and pseudo‐progression. Liquid biopsies, obtained from body fluids such as cerebrospinal fluid or blood, contain circulating nucleic acids or extracellular vesicles containing tumour‐derived components. The studies for this systematic review were selected according to PRISMA criteria, and suggest that the detection of circulating tumour‐derived nucleic acids holds great promises as biomarker to aid diagnosis and prognostication by monitoring tumour progression, and thus can be considered a pathway towards personalized medicine. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the most common malignancies and is a major cause of cancer-related deaths worldwide. Thus, there is a clinical need to improve early detection of CRC and personalize therapy for patients with this disease. In the era of precision oncology, liquid biopsy has emerged as a major approach to characterize the circulating tumor elements present in body fluids, including cell-free DNA and RNA, circulating tumor cells, and extracellular vesicles. This non-invasive tool has allowed the identification of relevant molecular alterations in CRC patients, including some indicating the disruption of epigenetic mechanisms. Epigenetic alterations found in solid and liquid biopsies have shown great utility as biomarkers for early detection, prognosis, monitoring, and evaluation of therapeutic response in CRC patients. Here, we summarize current knowledge of the most relevant epigenetic mechanisms associated with cancer development and progression, and the implications of their deregulation in cancer cells and liquid biopsy of CRC patients. In particular, we describe the methodologies used to analyze these epigenetic alterations in circulating tumor material, and we focus on the clinical utility of epigenetic marks in liquid biopsy as tumor biomarkers for CRC patients. We also discuss the great challenges and emerging opportunities of this field for the diagnosis and personalized management of CRC patients.

Neurological diseases are responsible for approximately 6.8 million deaths every year. They affect up to 1 billion people worldwide and cause significant disability and reduced quality of life. In most neurological disorders, the diagnosis can be challenging; it frequently requires long-term investigation. Thus, the discovery of better diagnostic methods to help in the accurate and fast diagnosis of neurological disorders is crucial. Circulating nucleic acids (CNAs) are defined as any type of DNA or RNA that is present in body biofluids. They can be found within extracellular vesicles or as cell-free DNA and RNA. Currently, CNAs are being explored as potential biomarkers for diseases because they can be obtained using non-invasive methods and may reflect unique characteristics of the biological processes involved in several diseases. CNAs can be especially useful as biomarkers for conditions that involve organs or structures that are difficult to assess, such as the central nervous system. This review presents a critical assessment of the most current literature about the use of plasma and serum CNAs as biomarkers for several aspects of neurological disorders: defining a diagnosis, establishing a prognosis, and monitoring the disease progression and response to therapy. We explored the biological origin, types, and general mechanisms involved in the generation of CNAs in physiological and pathological processes, with specific attention to neurological disorders. In addition, we present some of the future applications of CNAs as non-invasive biomarkers for these diseases.

Imaging-based monitoring of disease burden in glioma patients is frequently confounded by treatment effects. Circulating biomarkers could theoretically augment imaging-based response monitoring. This systematic review aimed to present and evaluate evidence for differential expression and diagnostic accuracy of circulating biomarkers with respect to outcomes of tumor response, progression, stable disease, and treatment effects (pseudoprogression, radionecrosis, pseudoresponse, and pseudolesions) in patients undergoing treatment for World Health Organization grades II–IV diffuse astrocytic and oligodendroglial tumors. MEDLINE, EMBASE, Web Of Science, and SCOPUS databases were searched until August 18, 2019, for observational or diagnostic studies on multiple circulating biomarker types: extracellular vesicles, circulating nucleic acids, circulating tumor cells, circulating proteins, and metabolites, angiogenesis related cells, immune cells, and other cell lines. Methodological quality of included studies was assessed using an adapted Quality Assessment of Diagnostic Accuracy Studies-2 tool, and level of evidence (IA–IVD) for individual biomarkers was evaluated using an adapted framework from the National Comprehensive Cancer Network guidelines on evaluating tumor marker utility. Of 13,202 unique records, 58 studies met the inclusion criteria. One hundred thirty-three distinct biomarkers were identified in a total of 1,853 patients across various treatment modalities. Fifteen markers for response, progression, or stable disease and five markers for pseudoprogression or radionecrosis reached level IB. No biomarkers reached level IA. Only five studies contained data for diagnostic accuracy measures. Overall methodological quality of included studies was low. While extensive data on biomarker dysregulation in varying response categories were reported, no biomarkers ready for clinical application were identified. Further assay refinement and evaluation in larger cohorts with diagnostic accuracy study designs are required.PROSPERO Registration: CRD42018110658.

The animal’s blood is the most complicated and important biological liquid for veterinary medicine. In addition to standard methods that are always in use, recent technologies such as dynamic tensiometry (DT) of blood serum and PCR analysis of particular markers are in progress. The standard and modern biochemical tests are commonly used for general screening and, finally, complete diagnosis of animal health. Interpretation of major biochemical parameters is similar across animal species, but there are a few peculiarities in each case, especially well-known for cattle. The following directions are discussed here: hematological indicators; “total protein” and its fractions; some enzymes; major low-molecular metabolites (glucose, lipids, bilirubin, etc.); cations and anions. As example, the numerous correlations between DT data and biochemical parameters of cattle serum have been obtained and discussed. Changes in the cell-free nucleic acids (cfDNA) circulating in the blood have been studied and analyzed in a variety of conditions; for example, pregnancy, infectious and chronic diseases, and cancer. CfDNA can easily be detected using standard molecular biological techniques like DNA amplification and next-generation sequencing. The application of digital PCR even allows exact quantification of copy number variations which are for example important in prenatal diagnosis of chromosomal aberrations.

Circulating mitochondrial DNA (mtDNA), widely studied as a disease biomarker, comprises of mtDNA located within mitochondria, indicative of mitochondrial function, and cell-free (cf) mtDNA linked to inflammation. The purpose of this study was to determine the ranges of, and relationship between, cellular and cf mtDNA in human blood. Whole blood from 23 controls (HC) and 20 patients with diabetes was separated into peripheral blood mononuclear cells (PBMCs), plasma, and serum. Total DNA was isolated and mtDNA copy numbers were determined using absolute quantification. Cellular mtDNA content in PBMCs was higher than in peripheral blood and a surprisingly high level of cf mtDNA was present in serum and plasma of HC, with no direct relationship between cellular and cf mtDNA content within individuals. Diabetes patients had similar levels of cellular mtDNA compared to healthy participants but a significantly higher cf mtDNA content. Furthermore, only in patients with diabetes, we observed a correlation between whole blood and plasma mtDNA levels, indicating that the relationship between cellular and cf mtDNA content is affected by disease status. In conclusion, when evaluating mtDNA in human blood as a biomarker of mitochondrial dysfunction, it is important to measure both cellular and cf mtDNA. [ABSTRACT FROM AUTHOR]

Imaging-based monitoring of disease burden in glioma patients is frequently confounded by treatment effects. Circulating biomarkers could theoretically augment imaging-based response monitoring. This systematic review aimed to present and evaluate evidence for differential expression and diagnostic accuracy of circulating biomarkers with respect to outcomes of tumor response, progression, stable disease, and treatment effects (pseudoprogression, radionecrosis, pseudoresponse, and pseudolesions) in patients undergoing treatment for World Health Organization grades II–IV diffuse astrocytic and oligodendroglial tumors. MEDLINE, EMBASE, Web Of Science, and SCOPUS databases were searched until August 18, 2019, for observational or diagnostic studies on multiple circulating biomarker types: extracellular vesicles, circulating nucleic acids, circulating tumor cells, circulating proteins, and metabolites, angiogenesis related cells, immune cells, and other cell lines. Methodological quality of included studies was assessed using an adapted Quality Assessment of Diagnostic Accuracy Studies-2 tool, and level of evidence (IA–IVD) for individual biomarkers was evaluated using an adapted framework from the National Comprehensive Cancer Network guidelines on evaluating tumor marker utility. Of 13,202 unique records, 58 studies met the inclusion criteria. One hundred thirty-three distinct biomarkers were identified in a total of 1,853 patients across various treatment modalities. Fifteen markers for response, progression, or stable disease and five markers for pseudoprogression or radionecrosis reached level IB. No biomarkers reached level IA. Only five studies contained data for diagnostic accuracy measures. Overall methodological quality of included studies was low. While extensive data on biomarker dysregulation in varying response categories were reported, no biomarkers ready for clinical application were identified. Further assay refinement and evaluation in larger cohorts with diagnostic accuracy study designs are required. PROSPERO Registration : CRD42018110658. [ABSTRACT FROM AUTHOR]

The animal’s blood is the most complicated and important biological liquid for veterinary medicine. In addition to standard methods that are always in use, recent technologies such as dynamic tensiometry (DT) of blood serum and PCR analysis of particular markers are in progress. The standard and modern biochemical tests are commonly used for general screening and, finally, complete diagnosis of animal health. Interpretation of major biochemical parameters is similar across animal species, but there are a few peculiarities in each case, especially well-known for cattle. The following directions are discussed here: hematological indicators; “total protein” and its fractions; some enzymes; major low-molecular metabolites (glucose, lipids, bilirubin, etc.); cations and anions. As example, the numerous correlations between DT data and biochemical parameters of cattle serum have been obtained and discussed. Changes in the cell-free nucleic acids (cfDNA) circulating in the blood have been studied and analyzed in a variety of conditions; for example, pregnancy, infectious and chronic diseases, and cancer. CfDNA can easily be detected using standard molecular biological techniques like DNA amplification and next-generation sequencing. The application of digital PCR even allows exact quantification of copy number variations which are for example important in prenatal diagnosis of chromosomal aberrations. [ABSTRACT FROM AUTHOR]

• The distribution of circulating cell-free DNA coverage over the respective reference genome is highly non-uniform. • Retrotransposable elements and non-telomeric satellite DNA are consistently overrepresented in human plasma and bovine serum. • Telomeric satellite DNA is underrepresented. • DNA from retrotransposable elements and non-telomeric satellite DNA is potentially actively secreted from cells, most likely via exosomes. • Sepsis patients have elevated fractions of DNA from retrotransposable elements. Circulating cell-free DNAs (cfDNAs) are DNA fragments which can be isolated from mammalian blood serum or plasma. In order to gain deeper insight into their origin(s), we have characterized the composition of human and cattle cfDNA via large-scale analyses of high-throughput sequencing data. We observed significant differences between the composition of cfDNA in serum/plasma and the corresponding DNA sequence composition of the human genome. Retrotransposable elements and non-telomeric satellite DNA were particularly overrepresented in the cfDNA population, while telomeric satellite DNA was underrepresented. This was consistently observed for human plasma, bovine serum and for the supernatant of human cancer cell cultures. Our results suggest that reverse transcription of retrotransposable elements and secondary-structure formation during the replication of satellite DNA are contributing to the composition of the cfDNA molecules in the mammalian blood stream. We believe that our work is an important step towards the understanding of the biogenesis of cfDNAs and thus may also facilitate the future exploitation of their diagnostic potential. [ABSTRACT FROM AUTHOR]

• Pre-analytical factors strongly influence the analysis of CTCs, cfNAs and exosomes. • The pre-analytical phase is defined as all the steps before the examination process. • The pre-analytical workflow for LB analysis is a complex and multistep process. • Different pre-analytical workflows for each component of the LB are required. • Standardization of the pre-analytical phase of LB is a challenging and unsolved issue. The term 'liquid biopsy', introduced in 2013 in reference to the analysis of circulating tumour cells (CTCs) in cancer patients, was extended to cell-free nucleic acids (cfNAs) circulating in blood and other body fluids. CTCs and cfNAs are now considered diagnostic and prognostic markers, used as surrogate materials for the molecular characterisation of solid tumours, in particular for research on tumour-specific or actionable somatic mutations. Molecular characterisation of cfNAs and CTCs (especially at the single cell level) is technically challenging, requiring highly sensitive and specific methods and/or multi-step processes. The analysis of the liquid biopsy relies on a plethora of methods whose standardisation cannot be accomplished without disclosing criticisms related to the pre-analytical phase. Thus, pre-analytical factors potentially influencing downstream cellular and molecular analyses must be considered in order to translate the liquid biopsy approach into clinical practice. The present review summarises the most recent reports in this field, discussing the main pre-analytical aspects related to CTCs, cfNAs and exosomes in blood samples for liquid biopsy analysis. A short discussion on non-blood liquid biopsy samples is also included. [ABSTRACT FROM AUTHOR]

• Assay is based on host-response, NOT on pathogen detection • Assay detects sepsis 2–3 days before the first clinical signs • Assay is robust (DNA-based) • Assay uses serum or plasma without further isolation/purification steps We have identified 24 molecular markers, based on circulating nucleic acids (CNA) originating from the human genome, which in combination can be used in a quantitative real-time PCR (qPCR) assay to identify the presence of human sepsis, starting two to three days before the first clinical signs develop and including patients who meet the SEPSIS-3 criteria. The accuracy was more than 87 % inside of the same patient cohort for which the markers were developed and up to 81 % in blind studies of patient cohorts which were not included in the marker development. As our markers are host-based, they can be used to capture bacterial as well as fungal sepsis, unlike the current PCR-based tests, which require species-specific primer sets for each organism causing human sepsis. Our assay directly uses an aliquot of cell-free blood as the substrate for the PCR reaction, thus allowing to obtain the diagnostic results in three to four hours after the collection of the blood samples. [ABSTRACT FROM AUTHOR]

Circulating tumour nucleic acids (ctNAs) are released from tumours cells and can be detected in blood samples, providing a way to track tumors without requiring a tissue sample. This "liquid biopsy" approach has the potential to replace invasive, painful, and costly tissue biopsies in cancer diagnosis and management. However, a very sensitive and specific approach is required to detect relatively low amounts of mutant sequences linked to cancer because they are masked by the high levels of wild‐type sequences. This review discusses high‐performance nucleic acid biosensors for ctNA analysis in patient samples. We compare sequencing‐ and amplification‐based methods to next‐generation sensors for ctDNA and ctRNA (including microRNA) profiling, such as electrochemical methods, surface plasmon resonance, Raman spectroscopy, and microfluidics and dielectrophoresis‐based assays. We present an overview of the analytical sensitivity and accuracy of these methods as well as the biological and technical challenges they present. [ABSTRACT FROM AUTHOR]

Cell-free DNA (cf. DNA) is a powerful noninvasive biomarker for cancer and prenatal testing, and it circulates in plasma as short fragments. To elucidate the biology of cf. DNA fragmentation, we explored the roles of deoxyribonuclease 1 (DNASE1), deoxyribonuclease 1 like 3 (DNASE1L3), and DNA fragmentation factor subunit beta (DFFB) with mice deficient in each of these nucleases. By analyzing the ends of cf. DNA fragments in each type of nuclease-deficient mice with those in wild-type mice, we show that each nuclease has a specific cutting preference that reveals the stepwise process of cf. DNA fragmentation. Essentially, we demonstrate that cf. DNA is generated first intracellularly with DFFB, intracellular DNASE1L3, and other nucleases. Then, cf. DNA fragmentation continues extracellularly with circulating DNASE1L3 and DNASE1. With the use of heparin to disrupt the nucleosomal structure, we also show that the 10 bp periodicity originates from the cutting of DNA within an intact nucleosomal structure. Altogether, this work establishes a model of cf. DNA fragmentation. [ABSTRACT FROM AUTHOR]

Objective: To characterize circulating oestrogen receptor (ER) mutants and splice variants in men with advanced prostate cancer. Materials and Methods: Sequential blood samples were obtained from men with advanced prostate cancer, and from healthy controls. Blood‐derived RNA samples were analysed using droplet digital PCR for the presence of six ERα mutations (E380Q, L536Q, Y537C, Y537S, Y537N and D538G), and six ERα and ERβ splice variants (ERα‐66, ERα‐36, ERβ1, ERβ2, ERβ4 & ERβ5). Results: A total of 94 samples were collected from 42 men with advanced prostate cancer. Four mutations (E380Q, L536Q, Y537S and D538G) and all six splice variants were detected in patient samples. Splice variants were detectable in non‐cancer control samples. The presence of ER mutations was associated with bone metastases and castration resistance. ERβ splice variant concentrations decreased after successive lines of treatment. Conclusions: The ER mutations were detectable in plasma from patients with advanced prostate cancer. ER splice variants were frequently detected in both men with and without prostate cancer. [ABSTRACT FROM AUTHOR]

The emergence of immunotherapy in oncology requires the discovery, validation and subsequent adoption of robust, sensitive and specific predictive and prognostic biomarkers for daily practice. Until now, anti-PD-L1 immunohistochemistry (IHC) on tissue sections has been the only validated companion diagnostic test for first-line immunotherapy for advanced and metastatic cancer, notably non-small-cell lung cancer (NSCLC). However, detection of this biomarker presents limitations that have stimulated the development of other biomarkers and other approaches. Within this context, the use of a liquid biopsy (LB) could provide an important complementary or alternative added value to PD-L1 IHC. In this review, we discuss how LBs have been used in the field of immuno-oncology (I-O) to predict response, relapse or adverse advents for patients undergoing immune-checkpoint inhibitor (ICI) therapy (anti-PD-1/PD-L1 and CTLA-4) and we highlight recent developments. Circulating tumor cells (CTCs), cell-free DNA (cfDNA), proteins and cytokines detected in plasma as well as circulating T-lymphocytes are discussed as potential sources for developing new I-O biomarkers. The quantification of cfDNA as a predictive biomarker, as well as its sequencing for the determination of tumor mutational burden, is already well advanced. Additionally, the quantification of PD-L1 from CTCs, bound on exosomes or free in plasma, as well as the determination of cytokines, are also being actively investigated with promising results having recently been published. Lastly, analysis of T-lymphocytes, especially by analyzing the T-cell receptor, has recently emerged as a valuable biomarker that might become relevant for the prediction of response to ICIs. While LBs have not yet been implemented in routine I-O clinical practice, recent promising data and rapidly advancing technologies indicate that this approach has the potential to soon personalize the clinical management of cancer patients receiving ICIs. [ABSTRACT FROM AUTHOR]

Previous studies report detection of high concentrations of circulating nucleic acids (CNAs), which are likely related to cell apoptosis, in the plasma of patients with cancers, stroke, trauma, and relapsing-remitting multiple sclerosis. However, the relationship between Alzheimer's disease (AD) and CNAs is unclear. A total of 36 adult participants (9 non-demented controls and 27 patients with AD) and patients with mild AD, who met the criteria for probable AD, were enrolled in the present study, which was conducted at the Department of Neurology of National Cheng Kung University Hospital. The CNA levels were increased in the plasma of patients with AD, culture medium of amyloid-β-treated SH-SY5Y cells, and plasma from a mouse model of AD. The CNA concentrations in the plasma were positively correlated with the cognitive scores. Further, CNAs in patients with AD contained neuronal tissue-specific methylated LHX2, at CpG sites 1 and 5. These results showed that the increased levels of plasma CNAs could be related to neuronal cell death that was induced by β-amyloid toxicity. Thus, the results suggested that the levels of plasma CNAs and LHX2 methylation might serve as potential biomarkers for the diagnosis of AD, particularly during the early stages of the disease. [ABSTRACT FROM AUTHOR]

Circulating free nucleic acids; cell free DNA and circulating micro-RNA, are found in the plasma of patients with hematologic and solid malignancies at levels higher than that of healthy individuals. In patients with hematologic malignancy cell free DNA reflects the underlying tumor mutational profile, whilst micro-RNAs reflect genetic interference mechanisms within a tumor and potentially the surrounding microenvironment and immune effector cells. These circulating nucleic acids offer a potentially simple, non-invasive, repeatable analysis that can aid in diagnosis, prognosis and therapeutic decisions in cancer treatment.

Graphene-based materials are intriguing nanomaterials with applications ranging from nanotechnology-related devices to drug delivery systems and biosensing. Multifunctional graphene platforms were proposed for the detection of several typical biomarkers (i.e., circulating tumor cells, exosomes, circulating nucleic acids, etc.) in liquid biopsy, and numerous methods, including optical, electrochemical, surface-enhanced Raman scattering (SERS), etc., have been developed for their detection. Due to the massive advancements in biology, material chemistry, and analytical technology, it is necessary to review the progress in this field from both medical and chemical sides. Liquid biopsy is considered a revolutionary technique that is opening unexpected perspectives in the early diagnosis and, in therapy monitoring, severe diseases, including cancer, metabolic syndrome, autoimmune, and neurodegenerative disorders. Although nanotechnology based on graphene has been poorly applied for the rapid diagnosis of viral diseases, the extraordinary properties of graphene (i.e., high electronic conductivity, large specific area, and surface functionalization) can be also exploited for the diagnosis of emerging viral diseases, such as the coronavirus disease 2019 (COVID-19). This review aimed to provide a comprehensive and in-depth summarization of the contribution of graphene-based nanomaterials in liquid biopsy, discussing the remaining challenges and the future trend; moreover, the paper gave the first look at the potentiality of graphene in COVID-19 diagnosis.

Lung cancer is the most frequently occurring malignancy and the leading cause of cancer-related death for men in our country. The only recommended screening method is clinic based low-dose computed tomography (also called a low-dose CT scan, or LDCT). However, the effect of LDCT on overall mortality observed in lung cancer patients is not statistically significant. Over-diagnosis, excessive cost, risks associated with radiation exposure, false positive results and delay in the commencement of the treatment procedure questions the use of LDCT as a reliable technique for population-based screening. Therefore, identification of minimal-invasive biomarkers able to detect malignancies at an early stage might be useful to reduce the disease burden. Circulating nucleic acids are emerging as important source of information for several chronic pathologies including lung cancer. Of these, circulating cell free miRNAs are reported to be closely associated with the clinical outcome of lung cancer patients. Smaller size, sequence homology between species, low concentration and stability are some of the major challenges involved in characterization and specific detection of miRNAs. To circumvent these problems, synthesis of a quantum dot based nano-biosensor might assist in sensitive, specific and cost-effective detection of differentially regulated miRNAs. The wide excitation and narrow emission spectra of these nanoparticles result in excellent fluorescent quantum yields with a broader color spectrum which make them ideal bio-entities for fluorescence resonance energy transfer (FRET) based detection for sequential or simultaneous study of multiple targets. In addition, photo-resistance and higher stability of these nanoparticles allows extensive exposure and offer state-of-the art sensitivity for miRNA targeting. A major obstacle for integrating QDs into clinical application is the QD-associated toxicity. However, the use of non-toxic shells along with surface modification not only overcomes the toxicity issues, but also increases the ability of QDs to quickly detect circulating cell free miRNAs in a non-invasive mode. The present review illustrates the importance of circulating miRNAs in lung cancer diagnosis and highlights the translational prospects of developing QD-based nano-biosensor for rapid early disease detection.

Darwin's gemmules were supposed to be "thrown off" by cells and were "inconceivably minute and numerous as the stars in heaven." They were capable of self-propagation and diffusion from cell to cell, and circulation through the system. The word "gene" coined by Wilhelm Johannsen, was derived from de Vries's term "pangen," itself a substitute for "gemmule" in Darwin's Pangenesis. Johannsen resisted the "morphological" conception of genes as particles with a certain structure. Morgan's genes were considered to be stable entities arranged in an orderly linear pattern on chromosomes, like beads on a string. In the late 1940s, McClintock challenged the concept of the stability of the gene when she discovered that some genes could move within a chromosome and between chromosomes. In 1948, Mandel and Metais reported the presence of cell-free nucleic acids in human blood for the first time. Over the past several decades, it has been universally accepted that almost all types of cells not only shed molecules such as cell-free DNA (including genomic DNA, tumor DNA and fetal DNA), RNAs (including mRNA and small RNAs) and prions, but also release into the extracellular environment diverse types of membrane vesicles (known as extracellular vesicles) containing DNA, RNA and proteins. Thus Darwin's speculative gemmules of the 19th century have become the experimentally demonstrated circulating cell-free DNA, mobile RNAs, prions and extracellular vesicles. [ABSTRACT FROM AUTHOR]

Mobile genetic elements play a major role in shaping biotic genomes and bringing about evolutionary transformations. Herein, a new class of mobile genetic elements is proposed in the form of circulating nucleic acids (CNAs) derived from the billions of cells that die in the body every day due to normal physiology and that act intra-corporeally. A recent study shows that CNAs can freely enter into healthy cells, integrate into their genomes by a unique mechanism and cause damage to their DNA. Being ubiquitous and continuously arising, CNA-induced DNA damage may be the underlying cause of ageing, ageing-related disabilities and the ultimate demise of the organism. Thus, DNA seems to act in the paradoxical roles of both preserver and destroyer of life. This new class of mobile genetic element may be relevant not only to multi-cellular organisms with established circulatory systems, but also to other multi-cellular organisms in which intra-corporeal mobility of nucleic acids may be mediated via the medium of extra-cellular fluid.