Sodium alginate hydrogels co-encapsulated with cell free fat extract-loaded core-shell nanofibers and menstrual blood stem cells derived exosomes for acceleration of articular cartilage regeneration.

This study presents a novel scaffold system comprising sodium alginate hydrogels (SAh) co-encapsulated with cell-free fat extract (CEFFE)-loaded core-shell nanofibers (NFs) and menstrual blood stem cell-derived exosomes (EXOs). The scaffold integrates the regenerative potential of EXOs and CFFFE, offering a multifaceted strategy for promoting articular cartilage repair. Coaxially electrospun core-shell NFs exhibited successful encapsulation of CEFFE and seamless integration into the SAh matrix. Structural modifications induced by the incorporation of CEFFE-NFs enhanced hydrogel porosity, mechanical strength, and degradation kinetics, facilitating cell adhesion, proliferation, and tissue ingrowth. The release kinetics of growth factors from the composite scaffold demonstrated sustained and controlled release profiles, essential for optimal tissue regeneration. In vitro studies revealed high cell viability, enhanced chondrocyte proliferation, and migration in the presence of EXOs/CEFFE-NFs@SAh composite scaffolds. Additionally, in vivo experiments demonstrated significant cartilage regeneration, with the composite scaffold outperforming controls in promoting hyaline cartilage formation and defect bridging. Overall, this study underscores the potential of EXOs and CEFFE-NFs integrated into SAh matrices for enhancing chondrocyte viability, proliferation, migration, and ultimately, articular cartilage regeneration. Future research directions may focus on elucidating underlying mechanisms and conducting long-term in vivo studies to validate clinical applicability and scalability.

Detection of circulating tumor DNA by tumor-informed whole-genome sequencing enables prediction of recurrence in stage III colorectal cancer patients.

INTRODUCTION: Circulating tumor (ctDNA) can be used to detect residual disease after cancer treatment. Detecting low-level ctDNA is challenging, due to the limited number of recoverable ctDNA fragments at any target loci. In response, we applied tumor-informed whole-genome sequencing (WGS), leveraging thousands of mutations for ctDNA detection. METHODS: Performance was evaluated in serial plasma samples (n = 1283) from 144 stage III colorectal cancer patients. Tumor/normal WGS was used to establish a patient-specific mutational fingerprint, which was searched for in 20x WGS plasma profiles. For reproducibility, paired aliquots of 172 plasma samples were analyzed in two independent laboratories. De novo variant calling was performed for serial plasma samples with a ctDNA level > 10 % (n = 17) to explore genomic evolution. RESULTS: WGS-based ctDNA detection was prognostic of recurrence: post-operation (Hazard ratio [HR] 6.75, 95 %CI 3.18-14.3, p < 0.001), post-adjuvant chemotherapy (HR 28.9, 95 %CI 10.1-82.8; p < 0.001), and during surveillance (HR 22.8, 95 %CI 13.7-37.9, p < 0.0001). The 3-year cumulative incidence of ctDNA detection in recurrence patients was 95 %. ctDNA was detected a median of 8.7 months before radiological recurrence. The independently analyzed plasma aliquots showed excellent agreement (Cohens Kappa=0.9, r = 0.99). Genomic characterization of serial plasma revealed significant evolution in mutations and copy number alterations, and the timing of mutational processes, such as 5-fluorouracil-induced mutations. CONCLUSION: Our study supports the use of WGS for sensitive ctDNA detection and demonstrates that post-treatment ctDNA detection is highly prognostic of recurrence. Furthermore, plasma WGS can identify genomic differences distinguishing the primary tumor and relapsing metastasis, and monitor treatment-induced genomic changes.

Plasma Cell-Free DNA Concentration and Fragmentomes Predict Neoadjuvant Chemotherapy Response in Cervical Cancer Patients.

Cervical cancer remains one of the most lethal gynecological malignancies. However, biomarkers for more precise patient care are an unmet need. Herein, the concentration of 285 plasma cell-free DNA (cfDNA) samples are analyzed from 84 cervical patients and the clinical significance of cfDNA fragmentomic characteristics across the neoadjuvant chemotherapy (NACT) treatment. Patients with poor NACT response exhibit a significantly greater escalation in cfDNA levels following the initial cycle of treatment, in comparison to patients with a favorable response. Distinctive end motif profiles and promoter coverages of cfDNA in initial plasma are observed between patients with differing NACT responses. Notably, the DNASE1L3 analysis further demonstrates the intrinsic association between cfDNA characteristics and chemotherapy resistance. The cfDNA and motif ratios show a good discriminative capacity for predicting non-responders from responders (area under the curve (AUC) > 0.8). In addition, transcriptional start sites (TSS) coverages around promoters discern the alteration of biological processes associated with chemotherapy resistance and reflect the potential value in predicting chemotherapy response. These findings in predictive biomarkers may optimize treatment selection, minimize unnecessary treatment, and assist in establishing personalized treatment strategies for cervical cancer patients.

Lactobacilli and Bifidobacteria: A Parapostbiotic Approach to Study and Explain Their Mutual Bioactive Influence.

Three strains of Lactiplantibacillus plantarum and three bifidobacteria (Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, and Bifidobacterium subtile) were used as target strains; in addition, for each microorganism, the cell-free supernatant (CFS) was produced and used as an ingredient of the growth medium. Namely CFSs from lactobacilli were used on bifidobacteria and CFSs from bifidobacteria were used on lactobacilli. The viable count was assessed, and the data were modelled through a reparametrized Gompertz equation cast both in the positive and negative form to evaluate the parameters t-7log, which is the time after which the viable count was 7 log CFU/mL, and the t-7log*, which is the time after which the viable count was below 7 log CFU/mL; the difference between the t-7log* and t-7log defines the stability time. Statistics through a multiparametric ANOVA (analysis of variance) provided evidence for the presence of a bifidogenic and/or bioactive factor produced by bifidobacteria and active on lactobacilli, and vice versa (bioactive factor of lactobacilli with a functional effect on bifidobacteria), although further studies are required to better explain the mechanisms beyond the positive effects. In addition, the influence on the target strains can be found during the growth phase (stimulation), as well as during senescence and death phase (protective effect), with a strong strain/species dependence on both CFS production and target strain.

Dellaglioa Algida Cell-Free Supernatant Inhibits Pseudomonas Fluorescence and Pseudomonas Fragi by Destroying Cell Membranes.

The aim of this study was to examine the components of the cell-free supernatant (CFS) derived from a novel strain of psychrophilic Lactobacillus, Dellaglioa algida, and to further elucidate the impact of this CFS on various cellular processes. Specifically, we sought to understand its effects on the cell membrane, protein and DNA release, protease activity, and metabolites of Pseudomonas fluorescens and Pseudomonas fragi, thereby clarifying the antibacterial mechanism involved. The CFS components were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS), the Coomassie Brilliant Blue method, and the phenol-sulfuric acid method. The inhibitory effect of the CFS on Pseudomonas fluorescens and Pseudomonas fragi was assessed using the ethidium bromide (EB) assay, Oxford cup assay, and ultramicroassay. Additionally, we analyzed the metabolites produced by Pseudomonas fluorescens and Pseudomonas fragi when treated with the CFS. The findings reveal that the CFS of Dellaglioa algida contains 94 volatile components, with protein and sugar concentrations of 32.857 ± 0.9705 mg/mL and 98.250 ± 4.210 mg/L, respectively. The CFS induces varying degrees of damage to the cell membranes of both Pseudomonas fluorescens and Pseudomonas fragi, leading to the release of intracellular proteins and DNA. Furthermore, the CFS reduced the protease activity and metabolic capacity of Pseudomonas fluorescens and Pseudomonas fragi. These results enhance our understanding of the mechanism by which psychrophilic Dellaglioa algida inhibits Pseudomonas fluorescens and Pseudomonas fragi, confirming that its inhibitory effect predominantly occurs through damage to the biological cell membranes of Pseudomonas. Dellaglioa algida is a newly identified cold-adapted inhibitor of Pseudomonas, indicating that its CFS is an effective microbial inhibitor in cold environments. This discovery suggests potential applications in inhibiting the growth and reproduction of Pseudomonas fluorescens and Pseudomonas fragi in food, pharmaceuticals, perfumes, and other chemicals, providing a valuable new reference for industrial preservation.

Cell-Free DNA Hydroxymethylation in Cancer: Current and Emerging Detection Methods and Clinical Applications.

In the era of precision oncology, identifying abnormal genetic and epigenetic alterations has transformed the way cancer is diagnosed, managed, and treated. 5-hydroxymethylcytosine (5hmC) is an emerging epigenetic modification formed through the oxidation of 5-methylcytosine (5mC) by ten-eleven translocase (TET) enzymes. DNA hydroxymethylation exhibits tissue- and cancer-specific patterns and is essential in DNA demethylation and gene regulation. Recent advancements in 5hmC detection methods and the discovery of 5hmC in cell-free DNA (cfDNA) have highlighted the potential for cell-free 5hmC as a cancer biomarker. This review explores the current and emerging techniques and applications of DNA hydroxymethylation in cancer, particularly in the context of cfDNA.

Analytical and Diagnostic Performance of a Dual-Target Blood Detection Test for Hepatocellular Carcinoma.

BACKGROUND: Surveillance approaches with high sensitivity and specificity for hepatocellular carcinoma (HCC) are still urgently needed. Previous studies have shown that methylation of GNB4 and Riplet can effectively diagnose HCC. AIMS: This study plan to analyze the performance of a blood test for detecting HCC using GNB4 and Riplet methylation. METHODS AND RESULTS: This study mainly investigated the analytical performance of the dual-target HCC blood test (DT-HBT), including cut-off value, limit of detection (LOD), precision, analytical specificity, and coincidence rate. In addition, the detection performance for HCC was validated in 1030 clinical plasma samples (214 HCC and 816 non-HCC). Plasma samples from 25 HCC patients after hepatectomy were collected to assess the feasibility of the kit for postoperative recurrence monitoring. All analytical performance of the DT-HBT met prespecified requirements. The LOD for GNB4, Riplet, and ß-actin was 1% methylation/100 copies/µL with cut-offs of 43, 43, and 35, respectively. The DT-HBT showed excellent precision, within 5% CV. It had a specificity of 91.5% for detecting other cancers, and 100% for breast, lung, and bladder cancer. No cross-reactions were observed with 9 potential interfering substances. The DT-HBT achieved a 100% coincidence rate in detecting reference and clinical samples. The clinical performance study found that the kit showed a sensitivity of 81.7% for stage I HCC, and an overall sensitivity and specificity of 87.4% and 92.3%, respectively. The detection sensitivity for postoperative recurrent patients was 95.8%, with a specificity of 100%. CONCLUSION: The analytical performance of the DT-HBT met prespecified criteria. It provided HCC patients with a reliable and high-performing new blood test for the HCC diagnosis and surveillance. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT05685524.

GWAS shows the genetics behind cell-free DNA and highlights the importance of p.Arg206Cys in DNASE1L3 for non-invasive testing.

The properties of cell-free DNA (cfDNA) are intensely studied for their potential as non-invasive biomarkers. We explored the effect of common genetic variants on the concentration and fragmentation properties of cfDNA using a genome-wide association study (GWAS) based on low-coverage whole-genome sequencing data of 140,000 Dutch non-invasive prenatal tests (NIPTs). Our GWAS detects many genome-wide significant loci, functional enrichments for phagocytes, liver, adipose tissue, and macrophages, and genetic correlations with autoimmune and cardiovascular disease. A common (7%) missense variant in DNASE1L3 (p.Arg206Cys) strongly affects all cfDNA properties. It increases the size of fragments, lowers cfDNA concentrations, affects the distribution of cleave-site motifs, and increases the fraction of circulating fetal DNA during pregnancy. For the application of NIPT, and potentially other cfDNA-based tests, this variant has direct clinical consequences, as it increases the odds of inconclusive results and impairs the sensitivity of NIPT by causing predictors to overestimate the fetal fraction.

Molecular basis of sepsis: A New insight into the role of mitochondrial DNA as a damage-associated molecular pattern.

Sepsis remains a critical challenge in the field of medicine, claiming countless lives each year. Despite significant advances in medical science, the molecular mechanisms underlying sepsis pathogenesis remain elusive. Understanding molecular sequelae is gaining deeper insights into the roles played by various damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) in disease pathogenesis. Among the known DAMPs, circulating cell-free mitochondrial DNA (mtDNA) garners increasing attention as a key player in the immune response during sepsis and other diseases. Mounting evidence highlights numerous connections between circulating cell-free mtDNA and inflammation, a pivotal state of sepsis, characterized by heightened inflammatory activity. In this review, we aim to provide an overview of the molecular basis of sepsis, particularly emphasizing the role of circulating cell-free mtDNA as a DAMP. We discuss the mechanisms of mtDNA release, its interaction with pattern recognition receptors (PRRs), and the subsequent immunological responses that contribute to sepsis progression. Furthermore, we discuss the forms of cell-free mtDNA; detection techniques of circulating cell-free mtDNA in various biological fluids; and the diagnostic, prognostic, and therapeutic implications offering insights into the potential for innovative interventions in sepsis management.

Differences in cognitive impairment and its correlation with circulating cell-free mitochondrial DNA in medication-free depression and bipolar depression patients.

OBJECTIVE: This study aimed to investigate whether there are differences in cognitive impairment between medication-free patients with bipolar depression (BD) and major depressive disorder (MDD) and whether these differences are related to circulating cell-free mtDNA (ccf-mtDNA). METHODS: For this cross-sectional study, 76 outpatients with BD, 86 outpatients with MDD and 70 healthy controls (HCs) were enrolled. Sociodemographic and clinical data were collected. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and the Stroop Color-Word Test were used to assess cognitive function. Plasma ccf-mtDNA levels were measured via qPCR. RESULTS: BD and MDD patients had similar scores for immediate memory, language, attention, delayed memory, the RBANS total score, Stroop color, Stroop word, and Stroop total score, which were significantly lower than the HCs. The visuospatial/constructive scores of the BD patients were significantly lower than those of the HCs (p < 0.001) and MDD patients (p = 0.008), but there was no difference between the HCs and MDD patients. The ccf-mtDNA levels in the BD and MDD patient groups were significantly higher than those in the HC group, and those in the MDD group were higher than those in the BD group (p = 0.016). Multiple stepwise regression analysis showed that ccf-mtDNA was negatively correlated with language in patients with depression (t = -2.11, p = 0.039). CONCLUSION: There were differences in specific cognitive dimensions between patients with BD and MDD. Increased ccf-mtDNA levels were found in BD and MDD patients, suggesting ccf-mtDNA may be involved in the pathophysiology of MDD and BD.

Characterizing the Cell-Free Transcriptome in a Humanized Diffuse Large B-Cell Lymphoma Patient-Derived Tumor Xenograft Model for RNA-Based Liquid Biopsy in a Preclinical Setting.

The potential of RNA-based liquid biopsy is increasingly being recognized in diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma. This study explores the cell-free transcriptome in a humanized DLBCL patient-derived tumor xenograft (PDTX) model. Blood plasma samples (n = 171) derived from a DLBCL PDTX model, including 27 humanized (HIS) PDTX, 8 HIS non-PDTX, and 21 non-HIS PDTX non-obese diabetic (NOD)-scid IL2Rgnull (NSG) mice were collected during humanization, xenografting, treatment, and sacrifice. The mice were treated with either rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), CD20-targeted human IFNα2-based AcTaferon combined with CHOP (huCD20-Fc-AFN-CHOP), or phosphate-buffered saline (PBS). RNA was extracted using the miRNeasy serum/plasma kit and sequenced on the NovaSeq 6000 platform. RNA sequencing data of the formalin-fixed paraffin-embedded (FFPE) tissue and blood plasma samples of the original patient were included. Flow cytometry was performed on immune cells isolated from whole blood, spleen, and bone marrow. Bulk deconvolution was performed using the Tabula Sapiens v1 basis matrix. Both R-CHOP and huCD20-Fc-AFN-CHOP were able to control tumor growth in most mice. Xenograft tumor volume was strongly associated with circulating tumor RNA (ctRNA) concentration (p < 0.001, R = 0.89), as well as with the number of detected human genes (p < 0.001, R = 0.79). Abundance analysis identified tumor-specific biomarkers that were dynamically tracked during tumor growth or treatment. An 8-gene signature demonstrated high accuracy for assessing therapy response (AUC 0.92). The tumoral gene detectability in the ctRNA of the PDTX-derived plasma was associated with RNA abundance levels in the patient's tumor tissue and blood plasma (p < 0.001), confirming that tumoral gene abundance contributes to the cell-free RNA (cfRNA) profile. Decomposing the transcriptome, however, revealed high inter- and intra-mouse variability, which was lower in the HIS PDTX mice, indicating an impact of human engraftment on the stability and profile of cfRNA. Immunochemotherapy resulted in B cell depletion, and tumor clearance was reflected by a decrease in the fraction of human CD45+ cells. Lastly, bulk deconvolution provided complementary biological insights into the composition of the tumor and circulating immune system. In conclusion, the blood plasma-derived transcriptome serves as a biomarker source in a preclinical PDTX model, enables the assessment of biological pathways, and enhances the understanding of cfRNA dynamics.

Liquid biopsy: paving a new avenue for cancer research.

The current constraints associated with cancer diagnosis and molecular profiling, which rely on invasive tissue biopsies or clinical imaging, have spurred the emergence of the liquid biopsy field. Liquid biopsy involves the extraction of circulating tumor cells (CTCs), circulating free or circulating tumor DNA (cfDNA or ctDNA), circulating cell-free RNA (cfRNA), extracellular vesicles (EVs), and tumor-educated platelets (TEPs) from bodily fluid samples. Subsequently, these components undergo molecular characterization to identify biomarkers that are critical for early cancer detection, prognosis, therapeutic assessment, and post-treatment monitoring. These innovative biosources exhibit characteristics analogous to those of the primary tumor from which they originate or interact. This review comprehensively explores the diverse technologies and methodologies employed for processing these biosources, along with their principal clinical applications.

Plasma cell-free RNA signatures of inflammatory syndromes in children.

Inflammatory syndromes, including those caused by infection, are a major cause of hospital admissions among children and are often misdiagnosed because of a lack of advanced molecular diagnostic tools. In this study, we explored the utility of circulating cell-free RNA (cfRNA) in plasma as an analyte for the differential diagnosis and characterization of pediatric inflammatory syndromes. We profiled cfRNA in 370 plasma samples from pediatric patients with a range of inflammatory conditions, including Kawasaki disease (KD), multisystem inflammatory syndrome in children (MIS-C), viral infections, and bacterial infections. We developed machine learning models based on these cfRNA profiles, which effectively differentiated KD from MIS-C-two conditions presenting with overlapping symptoms-with high performance [test area under the curve = 0.98]. We further extended this methodology into a multiclass machine learning framework that achieved 80% accuracy in distinguishing among KD, MIS-C, viral, and bacterial infections. We further demonstrated that cfRNA profiles can be used to quantify injury to specific tissues and organs, including the liver, heart, endothelium, nervous system, and the upper respiratory tract. Overall, this study identified cfRNA as a versatile analyte for the differential diagnosis and characterization of a wide range of pediatric inflammatory syndromes.

Minimal residual disease testing for classical Hodgkin lymphoma: A comprehensive review.

Classical Hodgkin lymphoma (cHL) is a common lymphoma that affects young patients. Fortunately, the disease is highly curable as it is susceptible to the currently available treatment modalities. Disease monitoring with Positron Emission Tomography and Computed Tomography (PET/ CT) is an integral part of managing these patients. PET guided protocols are currently used to adjust treatment according to the response. The pivotal idea behind the use of response-adapted approaches is to preserve efficacy while decreasing the toxicity. It also helps to intensify therapy in patients in need because of suboptimal response. However, imaging techniques are limited by their sensitivity and specificity. Minimal Residual Disease (MRD) assessment is a newly emerging concept in many hematologic malignancies. It utilizes various molecular techniques such as polymerase chain reaction (PCR), and next-generation sequencing (NGS) as well as flow cytometry, to detect disease traces. This review looks into MRD detection techniques, its current applications, and the evidence in the literature for its use in cHL.

Engineering a PbrR-Based Biosensor for Cell-Free Detection of Lead at the Legal Limit.

Industrialization and failing infrastructure have led to a growing number of irreversible health conditions resulting from chronic lead exposure. While state-of-the-art analytical chemistry methods provide accurate and sensitive detection of lead, they are too slow, expensive, and centralized to be accessible to many. Cell-free biosensors based on allosteric transcription factors (aTFs) can address the need for accessible, on-demand lead detection at the point of use. However, known aTFs, such as PbrR, are unable to detect lead at concentrations regulated by the Environmental Protection Agency (24-72 nM). Here, we develop a rapid cell-free platform for engineering aTF biosensors with improved sensitivity, selectivity, and dynamic range characteristics. We apply this platform to engineer PbrR mutants for a shift in limit of detection from 10 µM to 50 nM lead and demonstrate use of PbrR as a cell-free biosensor. We envision that our workflow could be applied to engineer any aTF.

Multi-scale reactor designs extend the physical limits of CO 2 fixation.

CO 2 valorization is a promising strategy for climate adaptation and transitioning towards a circular carbon economy. Here, we present a multi-scale, integrated systems approach for designing biomanufacturing systems that can utilize CO 2 as a feedstock, focusing on the Wood-Ljungdahl and reductive glycine pathways. This approach relies on first principles, coupling the optimization of pathway and process variables. We examine the CO 2 -fixation capacity of both pathways in single- and multi-compartment reactor systems, demonstrating that the reductive glycine pathway has the potential to fix CO 2 at significantly higher rates than photosynthetic organisms. We show that small differences in the energy-dissipative and stoichiometric structures of carbon-fixation pathways could significantly impact optimal designs and feasible design spaces. Our first-principle, systems-level approach quantifies these differences and uncovers strategies to expand the design space and extend the physical limits of carbon fixation, offering insights into pathway selection and process configurations for efficient biomanufacturing.

Implementing Massive Parallel Sequencing into Biliary Samples Obtained through Endoscopic Retrograde Cholangiopancreatography for Diagnosing Malignant Bile Duct Strictures.

Despite advancements in radiologic, laboratory, and pathological evaluations, differentiating between benign and malignant bile duct strictures remains a diagnostic challenge. Recent developments in massive parallel sequencing (MPS) have introduced new opportunities for early cancer detection and management, but these techniques have not yet been rigorously applied to biliary samples. We prospectively evaluated the Oncomine Comprehensive Assay (OCA) and the Oncomine Pan-Cancer Cell-Free Assay (OPCCFA) using biliary brush cytology and bile fluid obtained via endoscopic retrograde cholangiopancreatography from patients with bile duct strictures. The diagnostic performance of MPS testing was assessed and compared to the pathological findings of biliary brush cytology and primary tissue. Mutations in TP53, BRAF, CTNNB1, SMAD4, and K-/N-RAS identified in biliary brush cytology samples were also detected in the corresponding bile fluid samples from patients with extrahepatic cholangiocarcinoma. These mutations were also identified in the bile fluid samples, but with variant allele frequencies lower than those in the corresponding biliary brush cytology samples. In control patients diagnosed with gallstones, neither the biliary brush cytology samples nor the bile fluid samples showed any pathogenic mutations classified as tier 1 or 2. Our study represents a prospective investigation into the role of MPS-based molecular testing in evaluating bile duct strictures. MPS-based molecular testing shows promise in identifying actionable genomic alterations, potentially enabling the stratification of patients for targeted chemotherapeutic treatments. Future research should focus on integrating OCA and OPCCFA testing, as well as similar MPS-based assays, into existing surveillance and management protocols for patients with bile duct strictures.

Donor-derived post-transplant lymphoproliferative disease detection by donor-derived cell free DNA.

Post-transplant lymphoproliferative disorder (PTLD) is a life-threatening complication of organ transplantation, commonly diagnosed after patients present with nonspecific constitutional symptoms and/or transplant organ dysfunction. Here we report a case of a kidney transplant recipient who was found to have highly elevated circulating donor-derived cell free DNA (dd-cfDNA) levels on routine serum surveillance for allograft rejection, initially without organ dysfunction or evidence of allograft rejection on biopsy. Later for cause imaging revealed retroperitoneal lymphadenopathy and an allograft hilar mass, which was biopsied to show post-transplant lymphoproliferative disorder/diffuse large B-cell lymphoma (DLBCL). The elevated circulating dd-cfDNA levels in this patient prompted targeted next-generation sequencing of the same 266 single-nucleotide polymorphisms used to detect dd-cfDNA on the DLBCL, which identified it as donor-derived. The patient achieved complete remission with retained allograft kidney function after reduced immunosuppression and 6 cycles of immunochemotherapy. This case suggests that dd-cfDNA may be an early detection tool in rare but potentially life-threatening cases of donor-derived malignancy, such as donor-derived PTLD.

Analysis and identification of mRNAsi­related expression signatures via RNA sequencing in lung cancer.

High stemness index scores are associated with poor survival in patients with lung cancer. Studies on the mRNA expression-based stemness index (mRNAsi) are typically conducted using tumor tissues; however, mRNAsi-related expression signatures based on cell-free RNA (cfRNA) are yet to be comprehensively investigated. The present study aimed to elucidate the gene expression profiles of tumor stemness in lung cancer tissues and corresponding cfRNAs in blood, and to assess their links with immune infiltration. Tumor tissue, paracancerous tissue, peripheral blood and lymph node samples were collected from patients with stage I-III non-small cell lung cancer and RNA sequencing was performed. The TCGAbiolinks package was used to calculate the mRNAsi for each of these four types of sample. Weighted gene co-expression network analysis and differentially expressed gene analyses were performed to investigate mRNAsi-related genes, and pathway enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology-based annotation system. In addition, the STAR-Fusion tool was used to detect fusion variants, and CIBERSORT was used to analyze the correlations of stemness signatures in tissues and blood with immune cell infiltration. The mRNAsi values in peripheral blood and lymph nodes were found to be higher than those in cancer tissues. 'Hematopoietic cell lineage' was the only KEGG pathway enriched in mRNAsi-related genes in both lung cancer tissues and peripheral blood. In addition, the protein tyrosine phosphatase receptor type C associated protein gene was the only gene commonly associated with the mRNAsi in these two types of sample. The expression of mRNAsi-related genes was increased in the dendritic and Treg cells in tumor tissues, but was elevated in Treg and CD8 cells in the blood. In conclusion, cfRNAs in the blood exhibit unique stemness signatures that have potential for use in the diagnosis of lung cancer.

Circulating Cell-Free Mitochondrial DNA in Plasma of Individuals with Schizophrenia and Cognitive Deficit in Mexican Population.

Purpose: Cognitive domains are affected in patients with schizophrenia. Mitochondrial dysfunction has been proposed as a possible origin of these symptoms. Cell-free mitochondrial DNA (cf-mtDNA) is an indicator of cellular stress, and it can be identified in individuals with age-associated disorders, this study aimed to explore the presence of cf-mtDNA in plasma of schizophrenia patients and its association with cognitive deficit. Patients and Methods: Ninety-nine subjects were clinically evaluated; the case group included 60 patients diagnosed with schizophrenia and 39 randomly-individuals without psychiatric disorders were included in the comparison group. Cognitive status (MoCA scale) and cell-free mtDNA in blood plasma were assessed and quantified in both groups. Results: From the original sample, cf-mtDNA was identified in 43 subjects, 40 patients with schizophrenia and 3 controls (Χ2 = 31.10, p-value < 0.0001). Thirty-nine out of forty patients with schizophrenia had a cognitive deficit. Conclusion: According to our findings, cognitive impairment and presence of cf-mtDNA were related in subjects with schizophrenia. Thus, while the cognitive deficit might reflect an accelerated aging process, the cf-mtDNA plays a role as a potential biomarker in this mechanism.