PI3K/AKT signaling alters glucose metabolism in uterine microenvironment of women with idiopathic recurrent spontaneous miscarriage.

BACKGROUND: This study aims to identify metabolomic signatures in uterine fluid of women with idiopathic recurrent spontaneous miscarriage (IRSM) during window of implantation (WOI). Also, glucose transporters GLUT3 and GLUT4 and proteins of PI3K-Akt signaling pathway in endometrial tissue are assessed. METHODS: Paired uterine fluid and endometrial biopsies were collected during WOI from women with IRSM (n = 24) and healthy women with azoospermic male partners as controls (n = 15). NMR metabolomics was used to identify the dysregulated metabolites in uterine fluid of IRSM women. Additionally, proteins and glucose transporters were investigated in the endometrial tissue using immunohistochemistry (IHC) and western blotting. RESULTS: Uterine fluid metabolomics indicated eleven metabolites to be significantly downregulated in IRSM. While expression levels of PI3K (p85), PI3K (p110), p-Akt (Thr308), p-Akt (Ser473), GLUT3 and GLUT4 were significantly downregulated in endometrial tissue of these women, p-IKK α/ß (Ser176/180) and p-NFkBp65 (Ser536) were significantly increased. CONCLUSION: Our findings suggest that dysregulation of PI3K/Akt pathway in the uterine microenvironment could be a likely cause of endometrial dysfunction, thereby affecting implantation. Further studies on the downstream effects of the Akt signaling pathway in-vitro for improved understanding of the Akt-mediated cellular responses in IRSM is, therefore, warranted.

Understanding pulmonary hypertension: the need for an integrative metabolomics and transcriptomics approach.

Pulmonary hypertension (PH), characterised by mean pulmonary arterial pressure (mPAP) >20 mm Hg at rest, is a complex pathophysiological disorder associated with multiple clinical conditions. The high prevalence of the disease along with increased mortality and morbidity makes it a global health burden. Despite major advances in understanding the disease pathophysiology, much of the underlying complex molecular mechanism remains to be elucidated. Lack of a robust diagnostic test and specific therapeutic targets also poses major challenges. This review provides a comprehensive update on the dysregulated pathways and promising candidate markers identified in PH patients using the transcriptomics and metabolomics approach. The review also highlights the need of using an integrative multi-omics approach for obtaining insight into the disease at a molecular level. The integrative multi-omics/pan-omics approach envisaged to help in bridging the gap from genotype to phenotype is outlined. Finally, the challenges commonly encountered while conducting omics-driven studies are also discussed.

Bioinformatics analysis of hypoxia associated genes and inflammatory cytokine profiling in COPD-PH.

Pulmonary hypertension (PH) in chronic obstructive pulmonary disease (COPD) is associated with worse clinical outcomes and decreased survival rates. In absence of disease specific diagnostic/therapeutic targets and unclear pathophysiology, there is an urgent need for the identification of potential genetic/molecular markers and disease associated pathways. The present study aims to use a bioinformatics approach to identify and validate hypoxia-associated gene signatures in COPD-PH patients. Additionally, hypoxia-related inflammatory profile is also explored in these patients. Microarray dataset obtained from the Gene Expression Omnibus repository was used to identify differentially expressed genes (DEGs) in a hypoxic PH mice model. The top three hub genes identified were further validated in COPD-PH patients, with chemokine (C-X-C motif) ligand 9 (CXCL9) and CXCL12 showing significant changes in comparison to healthy controls. Furthermore, multiplexed analysis of 10 inflammatory cytokines, tumor necrosis factor alpha (TNF-α), transforming growth factor ß (TGF-ß), interleukin 1-beta (IL-1ß), IL-4, IL-5, IL-6, IL-13, IL-17, IL-18 and IL-21 was also performed. These markers showed significant changes in COPD-PH patients as compared to controls. They also exhibited the ability to differentially diagnose COPD-PH patients in comparison to COPD. Additionally, IL-6 and IL-17 showed significant positive correlation with systolic pulmonary artery pressure (sPAP). This study is the first report to assess the levels of CXCL9 and CXCL12 in COPD-PH patients and also explores their link with the inflammatory profile of these patients. Our findings could be extended to better understand the underlying disease mechanism and possibly used for tailoring therapies exclusive for the disease.

Cell Penetrability of a γ-Crystallin Peptide Fragment from the Discarded Cataractous Eye Emulsion.

The efficiency of the intracellular transport of medication and target specificity is frequently hampered by biological obstacles. The potential for therapeutic use of peptide fragments from naturally occurring proteins is promising, as peptides exhibit high selectivity due to several possibilities of interaction with their target. Certain peptide sequences, often referred to as cell-penetrating peptides (CPPs), are those that can penetrate cell membranes. Our goal is to find these sequences in the discarded postcataractery surgery emulsion known as the cataractous eye protein isolate (CEPI). One peptide fragment from this discarded protein has been identified to be a potential CPP based on the similarities with other well-known CPPs. Cell membrane penetrability and cytotoxicity of the peptide have been investigated. Fibroblast cells were incubated with the fluorescently labeled peptide and were observed under fluorescence as well as under confocal microscopy. It was found that the peptide possesses a cell-penetrating ability.

On paper characterisation of droplet and evaporation study using impedance spectroscopy.

The development of paper-based devices has drawn a significant amount of attention, ranging from the creation of paper electronics to microfluidic devices. The flow of fluids through the paper substrate can be controlled by establishing a variety of barriers, which can be accomplished by either cutting or producing layers that are hydrophobic. Through the utilisation of this feature, a number of investigations, including mixing, modifying, and analytical studies, have been carried out on the paper substrate. However, because of the difficulties associated with its wettability, it is seldom investigated for the purpose of conducting evaporation studies of droplets. Traditionally, evaporation studies are carried out on a solid substrate like glass or silicon. Here we report a paper chip employing an impedance method to determine the characteristics of the droplet. It is also possible to determine the identity of the droplet by utilising the dielectric property of the liquid on a paper chip. A comparison is made between the traditional method of evaporation and the usage of the paper chip for the purpose of studying the evaporation of various liquids, ranging from ionic chemicals to volatile compounds. A subsequent step involves the utilisation of an electrical equivalent circuit in order to acquire the complex system attribute of the evaporation of the cellulose fibres. Finally, this reveals that paper chips have a significant amount of promise for use in scientific applications regarding evaporation analysis.

Biochemical and immunomodulatory insights of extracellular matrix from decellularized human whole cervix: recellularization andin vivoECM remodeling interplay.

Extracellular matrix (ECM) rich whole organ bio-scaffolds, preserving structural integrity and essential growth factors, has potential towards regeneration and reconstruction. Women with cervical anomalies or trauma can benefit from clinical cervicovaginal repair using constructs rich in site specific ECM. In this study, complete human cervix decellularization was achieved using a modified perfusion-based stir bench top decellularization method. This was followed by physico-chemical processes including perfusion of ionic agents, enzymatic treatment and washing using detergent solutions for a duration of 10-12 d. Histopathological analysis, as well as DNA quantification confirmed the efficacy of the decellularization process. Tissue ultrastructure integrity was preserved and the same was validated via scanning electron microscopy and transmission electron microscopy studies. Biochemical analysis and structural characterizations like Fourier transform infrared, Raman spectroscopy of decellularized tissues demonstrated preservation of important proteins, crucial growth factors, collagen, and glycosaminoglycans.In vitrostudies, using THP-1 and human umbilical vein endothelial cell (HUVEC) cells, demonstrated macrophage polarization from M1 to M2 and vascular functional genes enhancement, respectively, when treated with decellularized human cervical matrix (DHCp). Crosslinked DHC scaffolds were recellularized with site specific human cervical epithelial cells and HUVEC, showing non-cytotoxic cell viability and enhanced proliferation. Furthermore, DHC scaffolds showed immunomodulatory effectsin vivoon small rodent model via upregulation of M2 macrophage genes as compared to decellularized rat cervix matrix scaffolds (DRC). DHC scaffolds underwent neo-vascularization followed by ECM remodeling with enhanced tissue integration.

Artificial Intelligence Assisted Multi-modal Photoacoustic-Ultrasound Imaging for Studying Renal Tissue Function and Hemodynamics.

Combined functional-anatomic imaging modalities, which integrate the benefits of visualizing gross anatomy along with the functional or metabolic information of tissue has revolutionized the world of medical imaging. However, such existing imaging modalities are very costly. An alternative option could be a hybrid modality combining contrast-enhanced ultrasound, doppler and photoacoustic imaging. In the current study, we propose an artificial intelligence assisted multi-modal imaging platform where we have used U-net model for segmenting the anatomical features from the ultrasound images obtained from an animal model study. The neural network has performed accurately for three different cases, each with a high dice score. The model was co-validated with doppler images. Further, blood perfusion and tissue oxygenation information from the predicted anatomical structures were also studied. The present findings confirm the feasibility of using this multimodal imaging modality facilitated by artificial intelligence for better understanding of the hemodynamics of the kidney.Clinical Relevance-A multi-modal imaging technique has been proposed which would provide anatomical and functional information to the clinicians for early detection and tracking of the disease prognosis. Unlike existing imaging modalities like PET-CT (Positron Emission Tomography- Computed Tomography), the proposed modality is much more costeffective and radiation free (non-ionizing nature).

Paper Sensor Modified with MoS2 for Detection of Dopamine Using a Machine-Intelligent Web App Interface.

The sensing behavior of a MoS2-functionalized paper sensor towards dopamine was explored through a combinatorial approach of theoretical analysis, subsequent experimental validation, and machine-learning-driven predictive modeling of the measured electrochemical outputs. The suitability of the chosen 2D material for efficient detection of dopamine was confirmed using density functional theory. The physisorption behavior along with electrostatic interaction due to the incorporation of dopamine on MoS2 was unraveled under the purview of theoretically estimated noncovalent interaction and charge density difference plot. The theoretical Löwdin population analysis elucidates the alteration in oxidation potential of dopamine, as observed in electrochemical experiments. The electrochemical responses of the developed sensor with the spiked serum samples showed an average accuracy of more than 96% with a limit of detection of 10 nM. Furthermore, implementation of a machine-intelligent interactive web app interface improved the resolution of the sensing platform significantly with an enhanced accuracy of nearly 99%.

Biodegradable Multi-layered Silk Fibroin-PCL Stent for the Management of Cervical Atresia: In Vitro Cytocompatibility and Extracellular Matrix Remodeling In Vivo.

Cervical atresia is a rare congenital Müllerian duct anomaly that manifests as the absence or deformed nonfunctional presence of the cervix. Herein, a multi-layered biodegradable stent is fabricated using a homogeneous blend of silk fibroin with polycaprolactone using hexafluoroisopropanol as a common solution. Briefly, a concentric cylinder of 3D honeycomb layer is sandwiched within electrospun sheets for fixing at the cervico-uterine junction to pave the way of cervical reconstruction. An average length of 40 mm with 3 mm diameter is fabricated for the hybrid stent design. SEM evidences an evenly distributed pore architecture of the electrospun layer, and mechanical characterization of stent reveals a tensile strength of 1.7 ± 0.2 MPa, with a Young's modulus of 5.9 ± 0.1 MPa. Physico-chemical characterization confirms the presence of silk fibroin and poly caprolactone within the engineered stent. Following 14 days of pepsin enzymatic degradation, 18% degradation and a contact angle measurement of 97° are observed. In vitro cytocompatibility studies are performed using site-specific primary human cervical squamous, columnar epithelial cells, and human endometrial stromal cells. The study demonstrates non-cytotoxic cells' viability (no significant toxicity), improved cell anchoring, adherence among the stent layers, and proliferation in the 3D microenvironment. Furthermore, in vivo subcutaneous studies in the rodent model indicate that the implanted stent undergoes constructive remodeling, neo-tissue creation, neo-vasculature formation, and re-epithelialization while maintaining patency for 2 months.

Discovery of novel metabolic signatures for early identification of women at risk of developing gestational hypertension.

INTRODUCTION: Gestational hypertension (GH) is defined as the presence of systolic blood pressure (BP) ≥ 140 mm Hg and/or diastolic BP ≥ 90 mm Hg, measured at least 4 h apart after 20 weeks of gestation. Early identification of women at high-risk of developing GH could contribute significantly towards improved maternal and fetal outcomes. OBJECTIVES: To determine early metabolic biomarkers in women with GH as compared with normotensive women. METHODS: Serum samples were collected from subjects during three stages of their pregnancy: 8-12 weeks, 18-20 weeks and after 28 weeks (< 36 weeks) of gestation and studied using nuclear magnetic resonance (NMR) metabolomics approach. Multivariate and univariate analyses were performed to determine the significantly altered metabolites in GH women. RESULTS: A total of 10 metabolites, including isoleucine, glutamine, lysine, proline, histidine, phenylalanine, alanine, carnitine, N-acetyl glycoprotein and lactic acid were observed to be significantly downregulated during all pregnancy stages in women with GH as compared with controls. Furthermore, expression of 5 metabolites in the first trimester i.e., phenylalanine [area under the curve (AUC) = 0.745], histidine [AUC = 0.729], proline [AUC = 0.722], lactic acid [AUC = 0.722], and carnitine [AUC = 0.714] exhibited highest potential in discriminating GH from normotensive women. CONCLUSION: The present study is the first of its kind to identify significantly altered metabolites that have the potential to discriminate between women at risk of developing GH and normotensive women across three trimesters of pregnancy. This opens up the possibility of exploring these metabolites as potential early predictive markers of GH.

Identification of Common Dysregulated Genes in COVID-19 and Hypersensitivity Pneumonitis: A Systems Biology and Machine Learning Approach.

A comprehensive knowledge on systems biology of severe acute respiratory syndrome coronavirus 2 is crucial for differential diagnosis of COVID-19. Interestingly, the radiological and pathological features of COVID-19 mimic that of hypersensitivity pneumonitis (HP), another pulmonary fibrotic phenotype. This motivated us to explore the overlapping pathophysiology of COVID-19 and HP, if any, and using a systems biology approach. Two datasets were obtained from the Gene Expression Omnibus database (GSE147507 and GSE150910) and common differentially expressed genes (DEGs) for both diseases identified. Fourteen common DEGs, significantly altered in both diseases, were found to be implicated in complement activation and growth factor activity. A total of five microRNAs (hsa-miR-1-3p, hsa-miR-20a-5p, hsa-miR-107, hsa-miR-16-5p, and hsa-miR-34b-5p) and five transcription factors (KLF6, ZBTB7A, ELF1, NFIL3, and ZBT33) exhibited highest interaction with these common genes. Next, C3, CFB, MMP-9, and IL1A were identified as common hub genes for both COVID-19 and HP. Finally, these top-ranked genes (hub genes) were evaluated using random forest classifier to discriminate between the disease and control group (coronavirus disease 2019 [COVID-19] vs. controls, and HP vs. controls). This supervised machine learning approach demonstrated 100% and 87.6% accuracy in differentiating COVID-19 from controls, and HP from controls, respectively. These findings provide new molecular leads that inform COVID-19 and HP diagnostics and therapeutics research and innovation.

Metabolomics of asthma, COPD, and asthma-COPD overlap: an overview.

The two common progressive lung diseases, asthma and chronic obstructive pulmonary disease (COPD), are the leading causes of morbidity and mortality worldwide. Asthma-COPD overlap, referred to as ACO, is another complex pulmonary disease that manifests itself with features of both asthma and COPD. The disease has no clear diagnostic or therapeutic guidelines, thereby making both diagnosis and treatment challenging. Though a number of studies on ACO have been documented, gaps in knowledge regarding the pathophysiologic mechanism of this disorder exist. Addressing this issue is an urgent need for improved diagnostic and therapeutic management of the disease. Metabolomics, an increasingly popular technique, reveals the pathogenesis of complex diseases and holds promise in biomarker discovery. This comprehensive narrative review, comprising 99 original research articles in the last five years (2017-2022), summarizes the scientific advances in terms of metabolic alterations in patients with asthma, COPD, and ACO. The analytical tools, nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS), commonly used to study the expression of the metabolome, are discussed. Challenges frequently encountered during metabolite identification and quality assessment are highlighted. Bridging the gap between phenotype and metabotype is envisioned in the future.

Transition metallo-curcumin complexes: a new hope for endometriosis?

Endometriosis is a debilitating gynecological disorder in women of reproductive age. Laparoscopy, a minimally invasive surgical procedure, provides a definitive diagnosis of the disease. Current treatments, including hormonal therapy and pain medication, are often associated with undesirable side effects limiting their long-term usage. This calls for exploring newer diagnostic and therapeutic options with minimal side effects. Curcumin is an established anti-endometriotic agent with inherent fluorescent properties; however, poor bioavailability limits its clinical utility. To address this shortcoming, various transition metals were conjugated with curcumin to improve its stability, specificity and pharmacological properties. The chemical stability, hemocompatibility and ability of the synthesized metallo-curcumin complexes (MCCs) to ameliorate endometriotic lesions were investigated. While all of the MCCs exhibited low hemolytic activity, their chemical and biological activities were largely dependent on the nature of the metal ion conjugated to the curcumin molecule. Copper-curcumin and nickel-curcumin complexes demonstrated superior therapeutic efficacy evidenced by enhanced antioxidant activity, selective cytotoxicity and increased accumulation in endometriotic cells mediated by an energy-dependent active transport process.

Processing of semolina, a wonder resource for resistant starch production: In vitro digestibility and biochemical evaluation.

With the advent of modern technology, the utilization of residues obtained after food processing are being largely explored for commercialization. Semolina, a starch rich food ingredient is one of such byproducts of food processing that has not been yet vividly studied, although it is profusely used as an important ingredient in Indian cuisines. Rapid digestibility of most starch rich foods boosts up the blood glucose level. Thus, the present study put forward an attempt to curtail the rapid digestibility of starch rich semolina flour by increasing its resistant starch content through enzymatic process. The enzymatically modified semolina flour (MS) was compared with its native counterpart (NS) on grounds of their digestibility pattern, biochemical and functional properties. A rise in resistant starch content by 9.3 ± 1.6 %, amylose content by 10.9 ± 1.2 %, crystallinity by 10.4 % and the drop in readily digestible starch by 11.9 ± 1.4 % and oil absorption by 2.1 ± 0.3 g/g were observed in MS. These initial findings of the present study are interesting as the results showed elevated potential of the modified semolina flour to be used as functional ingredient in cuisines worldwide.

Risk factors associated with mortality in hypersensitivity pneumonitis: a meta-analysis.

BACKGROUND: Hypersensitivity pneumonitis (HP) related deaths have increased substantially in recent years. It is important to identify the risk factors of HP significantly associated with mortality to ensure close patient monitoring and assess disease progression. RESEARCH DESIGN AND METHODS: Extensive literature search was conducted in accordance with the PRISMA checklist. Literature search of PubMed, Embase, and Cochrane Library database between January 2009 and April 2021 using the terms 'hypersensitivity pneumonitis', 'hazard ratio', and 'mortality' identified 325 articles. A total of 22 independent original studies focusing on mortality of HP patients were assessed. RESULTS: This systematic review and meta-analysis suggests that increased age, male sex, honeycombing, and traction bronchiectasis patterns on high-resolution computed tomography (HRCT) images are the major mortality-related risk factors of patients with HP. In case of chronic HP, antigen exposure appeared to be an additional risk factor. CONCLUSIONS: The clinico-radiological risk factors of mortality identified for HP will enable effective and close monitoring of patients, prognostication, and guide toward appropriate management decisions. However, association between the type of antigen and mortality remains to be explored.

Machine learning based urinary pH sensing using polyaniline deposited paper device and integration of smart web app interface: Theory to application.

The present study employs density functional theory-based first principle calculation to investigate the electron transport properties of polyaniline following exposure to acidic and alkaline pH. In-situ deposited polyaniline-based paper device maintains emeraldine salt form while it is exposed to acidic pH and converts to emeraldine base when it is subjected to alkaline pH solutions. These structural changes at acidic and alkaline pH are validated experimentally by Raman spectra. Furthermore, the Raman spectra computed from density functional theory are validated with the experimental spectra. The changes in the theoretical energy band gap of polyaniline obtained from first principle calculations were correlated with the changes in the experimental impedimetric response of the sensor after exposure to acidic and alkaline solutions. Finally, the impedimetric responses were used to predict urine pH through a machine learning based smart and interactive web application. Different machine learning based regression models were implemented to acquire the best possible outcome. Gradient Boosting Regressor with least square loss model was selected as it showed lowest mean square, mean absolute, and root mean square error than other models. The smart sensing platform successfully predicts the unknown pH of urine samples with an average accuracy of more than 98%. The locally deployed smart web app can be accessed within a local area network by the end-user, which holds promise towards effective detection of urinary pH.

Nanoencapsulation as a Promising Platform for the Delivery of the Morin-Cu(II) Complex: Antibacterial and Anticancer Potential.

Nanoencapsulation has emerged as a promising approach for the effective delivery of poorly aqueous soluble compounds. The current study focuses on the preparation of human serum albumin (HSA)-based nanoparticles (NPs) and poly lactic-co-glycolic acid (PLGA)-based nanoparticles for effective delivery of the morin-Cu(II) complex. The NPs were analyzed based on different parameters such as particle size, surface charge, morphology, encapsulation efficiency, and in vitro release properties. The average particle sizes were found to be 214 ± 6 nm for Mor-Cu-HSA-NPs and 185 ± 7.5 nm for Mor-Cu-PLGA-NPs. The release of the morin-Cu(II) complex from both the NPs (Mor-Cu-HSA-NPs and Mor-Cu-PLGA-NPs) followed a biphasic behavior, which comprises an early burst release followed by a sustained and controlled release. The resulting NPs also exhibit free radical scavenging activity confirmed by a standard antioxidant assay. The antibacterial activities of the NPs were investigated using a disk diffusion technique, and it was observed that both the NPs showed better antibacterial activity than morin and the morin-Cu(II) complex. The anticancer activities of the prepared NPs were examined on MDA-MB-468 breast cancer cell lines using a cytotoxicity assay, and the mode of cell death was visualized using fluorescence microscopy. Our results revealed that NPs kill the cancer cells with greater efficiency than free morin and the morin-Cu(II) complex. Thus, both HSA-based NPs and PLGA-based NPs can act as promising delivery systems for the morin-Cu(II) complex and can be utilized for further biomedical applications.

Therapeutic potential of curcumin in endometrial disorders: Current status and future perspectives.

Endometrial disorders collectively encompass a broad spectrum of pathologies, including but not limited to endometriosis, endometrial cancer and endometritis. The current therapeutic management of these diseases is associated with several limitations. This has prompted interest in the use of plant-based bioactive compounds as alternative strategies to achieve high therapeutic efficacy and avoid adverse effects. In this context, curcumin, a polyphenol abundantly present in turmeric, is gaining increasing attention for its therapeutic potential to restore homeostasis in endometrial dysfunctionality. We comprehensively review the multifaceted role of curcumin, discussing mechanistic insights in various endometrial pathologies. We also provide an in-depth analysis of the concerns and challenges associated with the role of curcumin in endometrial research and outline a road map for future investigations.

Immunological profiling and development of a sensing device for detection of IL-13 in COPD and asthma.

Chronic obstructive pulmonary disease (COPD) and asthma are the two most common obstructive lung diseases which affects millions worldwide and impose an enormous burden on global healthcare. The overlapping features shared by these two diseases often make differential diagnosis difficult to achieve, leading to misdiagnosis of these patients. Both asthma and COPD are associated with chronic inflammation of the airways which is perpetuated by the interplay between immunological mediators. The crucial role played by these mediators make them attractive targets for disease diagnosis. The present study investigates the immunological mediator profile in these patients as compared with controls. Further, a potential biomarker for the development of a sensing platform is identified. Multiplexed analysis of 8 commonly studied immunological markers (IL-4, IL-5, IL-6, IL-13, TGF-ß, IFN-γ, MCP-1 and NGAL) in serum showed distinct dysregulation pattern, with IL-13 showing the highest potential for differential diagnosis. An impedimetric self-assembled monolayer (SAM) based sensor for detecting IL-13 is developed to distinguish between asthma and COPD. The device shows reliable output with high accuracy and sensitivity towards the detection of IL-13.

A photonic dual nano-hybrid assay for detection of cell-free circulating mitochondrial DNA.

Circulating cell free mitochondrial DNA (ccf-mtDNA) has emerged as a potential marker for diagnosis and prognosis of different chronic and age associated non-communicable diseases. Therefore, owing to its biomarker potential, we herein assessed a novel nano-photonic dual hybrid assay system for rapid and specific detection of ccf-mtDNA. The assay comprised of two systems, i.e. a capture and screen facet containing aminopyrene tethered carbon quantum dots for effective screening of circulating cell free nucleic acids (ccf-NAs) and a quantum dot conjugated probe for precise detection of ccf-mtDNA in the screened ccf-NAs. Our observations suggested that the developed dual-assay system possesses high feasibility and selectivity in screening of ccf-NAs and estimation of ccfmtDNA in a given sample. It also offers high versatility of measurement in different analytical platforms, indicating the translational potential of the method for possible disease risk assessment in control and field settings.