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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.

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Diagnosis of pulmonary tuberculosis (TB) relies on a sputum sample, which cannot be obtained from all symptomatic individuals. Mycobacterium tuberculosis (Mtb) transrenal DNA (trDNA) has been detected in urine, an easily obtainable, noninvasive, alternative sample type. However, reported sensitivities have been variable and likely depend on collection and assay procedures and aspects of trDNA biology. We analyzed three serial urine samples from each of 75 adults with culture-confirmed pulmonary TB disease in Lima, Peru for detection of trDNA using short-fragment real-time PCR. Additionally, we examined host, urine, and sampling factors associated with detection. Overall per-sample sensitivity was 38 % (95 % Confidence Interval [CI] 30-45 %). On an individual level (i.e., any of the three samples positive), sensitivity was 73 % (95 % CI: 62-83 %). Sensitivity was highest among samples from patients with smear-positive TB, 92 % (95 % CI: 62-100 %). Specificity from a single sample from each of 10 healthy controls was 100 % (95 % CI: 69-100 %). Adjusting our assay positivity threshold increased individual-level sensitivity to 88 % (95 % CI: 78-94 %) overall without affecting the specificity. We did not find associations between Mtb trDNA detection and individual characteristics or urine sample characteristics. Overall, our results support the potential of trDNA detection for TB diagnosis.

Asunto(s)

ADN Bacteriano , Mycobacterium tuberculosis , Tuberculosis Pulmonar , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Adulto , Femenino , Perú/epidemiología , Masculino , ADN Bacteriano/orina , ADN Bacteriano/genética , Tuberculosis Pulmonar/orina , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/microbiología , Persona de Mediana Edad , Adulto Joven , Reacción en Cadena en Tiempo Real de la Polimerasa , Valor Predictivo de las Pruebas , Urinálisis/métodos , Estudios de Casos y Controles , Reproducibilidad de los Resultados , Anciano

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Mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) have been increasingly investigated for cancer therapy and drug delivery, and they offer an advanced cell-free therapeutic option. However, their overall effects and efficacy depend on various factors, including the MSC source and cargo content. In this study, we isolated EVs from the conditioned medium of human immature dental pulp stem cells (hIDPSC-EVs) and investigated their effects on two papillary thyroid cancer (PTC) cell lines (BCPAP and TPC1). We observed efficient uptake of hIDPSC-EVs by both PTC cell lines, with a notable impact on gene regulation, particularly in the Wnt signaling pathway in BCPAP cells. However, no significant effects on cell proliferation were observed. Conversely, hIDPSC-EVs significantly reduced the invasive capacity of both PTC cell lines after 120 h of treatment. These in vitro findings suggest the therapeutic potential of hIDPSC-EVs in cancer management and emphasize the need for further research to develop novel and effective treatment strategies. Furthermore, the successful internalization of hIDPSC-EVs by PTC cell lines underscores their potential use as nanocarriers for anti-cancer agents.

Asunto(s)

Proliferación Celular , Pulpa Dental , Vesículas Extracelulares , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides , Humanos , Pulpa Dental/citología , Vesículas Extracelulares/metabolismo , Cáncer Papilar Tiroideo/terapia , Cáncer Papilar Tiroideo/patología , Cáncer Papilar Tiroideo/metabolismo , Neoplasias de la Tiroides/terapia , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Línea Celular Tumoral , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Vía de Señalización Wnt , Medios de Cultivo Condicionados/farmacología

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INTRODUCTION: Despite thromboprophylaxis, women with antiphospholipid syndrome (APS) face high-risk pregnancies due to proinflammatory and prothrombotic states. This highlights the need for new monitoring and prognostic tools. Recent insights into the pathophysiological role of neutrophil activation and extracellular trap (NET) formation in this syndrome led to the exploration of plasma cell-free DNA (cfDNA), a derivative of NETosis, as a promising biomarker. MATERIALS AND METHODS: cfDNA was isolated and quantified from plasma samples of healthy pregnant women (control group, HC) and women with APS (APS group). We assessed the physiological variability of cfDNA across the three trimesters in HC. Levels of cfDNA were compared between APS and HC by gestational trimester. ROC curve analysis was performed to evaluate the efficacy of cfDNA levels for classifying APS patients. Furthermore, cfDNA levels in pregnant women with APS with obstetric complications were compared to those from uncomplicated pregnancies. RESULTS: Among HC, cfDNA significantly increased in the third trimester compared to the first and second. Elevated cfDNA levels in APS compared to HC were observed in the first and second trimesters. First-trimester cfDNA levels demonstrated the highest classification ability to discriminate between APS and HC patients (AUC: 0.906). Among APS, those with complicated pregnancies (fetal growth restriction, preeclampsia, placenta accreta) exhibited significantly elevated cfDNA levels in the second trimester. CONCLUSIONS: Elevated levels of cfDNA in pregnant women with APS, particularly among those with obstetric complications, supports further investigation into the potential of cfDNA as a valuable tool in the obstetric management of women with APS.

Asunto(s)

Síndrome Antifosfolípido , Ácidos Nucleicos Libres de Células , Embarazo de Alto Riesgo , Humanos , Femenino , Embarazo , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/complicaciones , Ácidos Nucleicos Libres de Células/sangre , Adulto , Embarazo de Alto Riesgo/sangre , Biomarcadores/sangre , Complicaciones del Embarazo/sangre

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Mesenchymal stem/stromal cells (MSC) play a pivotal role in regenerative therapies. Recent studies show that factors secreted by MSC can replicate their biological activity, driving the emergence of cell-free therapy, likely to surpass stem cell therapy. Patents are an objective measure of R&D and innovation activities, and patent mapping allows us to verify the state of the art and technology, anticipate trends, and identify emerging lines of research. This review performed a search on Derwent World Patents Index™ and retrieved 269 patent families related to the MSC-derived cell-free products. Analysis reveals an exponential increase in patents from the mid-2010s, primarily focusing on exosomes. The patent's contents offer a great diversity of applications and associated technologies by using the products as medicinal agents or drug delivery systems. Nevertheless, numerous application branches remain unexplored, suggesting vast potential for cell-free technologies alone or combined with other approaches.

Asunto(s)

Células Madre Mesenquimatosas , Patentes como Asunto , Células Madre Mesenquimatosas/citología , Humanos , Exosomas , Sistema Libre de Células , Medicina Regenerativa/métodos , Animales

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Using whey, a by-product of the cheese-making process, is important for maximizing resource efficiency and promoting sustainable practices in the food industry. Reusing whey can help minimize environmental impact and produce bio-preservatives for foods with high bacterial loads, such as Mexican-style fresh cheeses. This research aims to evaluate the antimicrobial and physicochemical effect of CFS from Lactobacillus casei 21/1 produced in a conventional culture medium (MRS broth) and another medium using whey (WB medium) when applied in Mexican-style fresh cheese inoculated with several indicator bacteria (Escherichia coli, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Listeria monocytogenes). The CFSs (MRS or WB) were characterized for organic acids concentration, pH, and titratable acidity. By surface spreading, CFSs were tested on indicator bacteria inoculated in fresh cheese. Microbial counts were performed on inoculated cheeses during and after seven days of storage at 4 ± 1.0 °C. Moreover, pH and color were determined in cheeses with CFS treatment. Lactic and acetic acid were identified as the primary antimicrobial metabolites produced by the Lb. casei 21/1 fermentation in the food application. A longer storage time (7 days) led to significant reductions (p < 0.05) in the microbial population of the indicator bacteria inoculated in the cheese when it was treated with the CFSs (MRS or WB). S. enterica serovar Typhimurium was the most sensitive bacteria, decreasing 1.60 ± 0.04 log10 CFU/g with MRS-CFS, whereas WB-CFS reduced the microbial population of L. monocytogenes to 1.67 log10 CFU/g. E. coli and S. aureus were the most resistant at the end of storage. The cheese's pH with CFSs (MRS or WB) showed a significant reduction (p < 0.05) after CFS treatment, while the application of WB-CFS did not show greater differences in color (ΔE) compared with MRS-CFS. This study highlights the potential of CFS from Lb. casei 21/1 in the WB medium as an ecological bio-preservative for Mexican-style fresh cheese, aligning with the objectives of sustainable food production and guaranteeing food safety.

Asunto(s)

Queso , Lacticaseibacillus casei , Suero Lácteo , Queso/microbiología , Queso/análisis , Lacticaseibacillus casei/metabolismo , Suero Lácteo/química , Suero Lácteo/microbiología , Microbiología de Alimentos , Concentración de Iones de Hidrógeno , Conservación de Alimentos/métodos , México , Fermentación

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PURPOSE: Circulating cell-free DNA (cfDNA) is a promising biomarker for predicting treatment response and disease outcomes in Breast Cancer (BC) patients undergoing neoadjuvant chemotherapy (NAC). To determine if cfDNA originates from tumors, matching tumor and cfDNA gene mutations are necessary, often requiring tumor DNA sequencing. We assessed plasma cfDNA integrity by measuring concentrations and ratios of larger-to-smaller Alu DNA fractions as a potential biomarker, eliminating the need for prior tumor sequencing. METHODS: We included patients with localized and/or locally advanced BC receiving standard NAC alone or in combination with immunotherapy and/or anti-HER2 targeted therapy. Blood samples were collected before treatment, every 2 weeks during treatment, and before surgery. RESULTS: Of the 38 evaluated patients, only 28 completed the protocol and underwent surgery after NAC. Seven patients (25%) achieved a pathologic complete response (pCR). We found that cfDNA integrity (cfDNAI) levels at 15 days after starting NAC were significantly higher in patients who achieved pCR (p = 0.045) and correlated significantly with Disease-Free Survival (DFS) in univariate analysis (p = 0.0371). CONCLUSIONS: Evaluation of cfDNAI 2 weeks after NAC initiation appears to be an early biomarker for tumor pCR and DFS. Measuring Alu fragments of different lengths may replace techniques requiring prior tumor sequencing to measure ctDNA, reducing costs and complexity of cfDNA serial measurements in BC patients undergoing NAC.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica , Biomarcadores de Tumor , Neoplasias de la Mama , Ácidos Nucleicos Libres de Células , Terapia Neoadyuvante , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Terapia Neoadyuvante/métodos , Biomarcadores de Tumor/sangre , Persona de Mediana Edad , Proyectos Piloto , Adulto , Anciano , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , ADN Tumoral Circulante/sangre , ADN Tumoral Circulante/genética , Resultado del Tratamiento , Pronóstico , Quimioterapia Adyuvante/métodos

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Sepsis and acute kidney injury (AKI) are two major public health concerns that contribute significantly to illness and death worldwide. Early diagnosis and prompt treatment are essential for achieving the best possible outcomes. To date, there are no specific clinical, imaging, or biochemical indicators available to diagnose sepsis, and diagnosis of AKI based on the KDIGO criterion has limitations. To improve the diagnostic process for sepsis and AKI, it is essential to continually evolve our understanding of these conditions. Delays in diagnosis and appropriate treatment can have serious consequences. Sepsis and AKI often occur together, and patients with kidney dysfunction are more prone to developing sepsis. Therefore, identifying potential biomarkers for both conditions is crucial. In this review, we talk about the main biomarkers that evolve the diagnostic of sepsis and AKI, namely neutrophil gelatinase-associated lipocalin (NGAL), proenkephalin (PENK), and cell-free DNA.

RESUMEN

BACKGROUND: The objective of this study is to evaluate the diagnostic accuracy of plasma-based liquid biopsy for the detection of the BRAF V600E mutation in circulating cell-free DNA from patients with ameloblastoma. METHODS: This is a prospective diagnostic accuracy study conducted based on the Standards for Reporting Diagnostic Accuracy recommendations. The index test was the plasma-based liquid biopsy, whereas the reference standard was the conventional tissue biopsy. The target condition was the detection of BRAF V600E mutation. The study population consisted of individuals with ameloblastoma recruited from three tertiary hospitals from Brazil. A negative control group composed of three individuals with confirmed wild-type BRAF lesions were included. The participants underwent plasma circulating cell-free DNA and tumor tissue DNA isolation, and both were submitted to using competitive allele-specific TaqMan™ real-time polymerase chain reaction technology mutation detection assays. Sensitivity and specificity measures and positive and negative predictive values were calculated. RESULTS: Twelve patients with conventional ameloblastoma were included. BRAF V600E mutation was detected in 11/12 (91.66%) ameloblastoma tissue samples. However, the mutation was not detected in any of the plasma-based liquid biopsy circulating cell-free DNA samples in both ameloblastomas and negative control group. The sensitivity and specificity of plasma-based liquid biopsy for the detection of the BRAF V600E mutation in circulating cell-free DNA was 0.0 and 1.0, respectively. The agreement between index test and reference standard results was 26.66%. CONCLUSION: Plasma-based liquid biopsy does not seem to be an accurate method for the detection of the BRAF V600E mutation in circulating circulating cell-free DNA from patients with ameloblastoma, regardless of tumor size, anatomic location, recurrence status, and other clinicopathological features.

Asunto(s)

Ameloblastoma , Ácidos Nucleicos Libres de Células , Humanos , Ameloblastoma/diagnóstico , Ameloblastoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Estudios Prospectivos , Mutación , Ácidos Nucleicos Libres de Células/genética

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BACKGROUND: Cell-free DNA (cfDNA) is a source for liquid biopsy used for cancer diagnosis, therapy selection, and disease monitoring due to its non-invasive nature and ease of extraction. However, cfDNA also participates in cancer development and progression by horizontal transfer. In humans, cfDNA circulates complexed with extracellular vesicles (EV) and macromolecular complexes such as nucleosomes, lipids, and serum proteins. The present study aimed to demonstrate whether cfDNA not associated with EV induces cell transformation and tumorigenesis. METHODS: Supernatant of the SW480 human colon cancer cell line was processed by ultracentrifugation to obtain a soluble fraction (SF) and a fraction associated with EV (EVF). Primary murine embryonic fibroblast cells (NIH3T3) underwent passive transfection with these fractions, and cell proliferation, cell cycle, apoptosis, cell transformation, and tumorigenic assays were performed. Next, cfDNA was analyzed by electronic microscopy, and horizontal transfer was assessed by human mutant KRAS in recipient cells via PCR and recipient cell internalization via fluorescence microscopy. RESULTS: The results showed that the SF but not the EVF of cfDNA induced proliferative and antiapoptotic effects, cell transformation, and tumorigenesis in nude mice, which were reduced by digestion with DNAse I and proteinase K. These effects were associated with horizontal DNA transfer and cfDNA internalization into recipient cells. CONCLUSIONS: The results suggest pro-tumorigenic effects of cfDNA in the SF that can be offset by enzyme treatment. Further exploration of the horizontal tumor progression phenomenon mediated by cfDNA is needed to determine whether its manipulation may play a role in cancer therapy.

Asunto(s)

Ácidos Nucleicos Libres de Células , Humanos , Animales , Ratones , Ácidos Nucleicos Libres de Células/genética , Ratones Desnudos , Células 3T3 NIH , Carcinogénesis , ADN

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Ameloblastoma , Proteínas Proto-Oncogénicas B-raf , Adulto , Femenino , Humanos , Masculino , Ameloblastoma/genética , Ácidos Nucleicos Libres de Células/genética , Neoplasias Maxilomandibulares/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética

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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores ErbB/genética , Mutación/genética

RESUMEN

The use of plasma cell-free DNA (cfDNA) as a useful biomarker in obstetric clinical practice has been delayed due to the lack of reliable quantification protocols. We developed a protocol to quantify plasma cfDNA using an internal standard strategy to overcome difficulties posed by low levels and high fragmentation of cfDNA. cfDNA was isolated from plasma samples of non-pregnant (NP, n = 26) and pregnant (P, n = 26) women using a commercial kit and several elution volumes were evaluated. qPCR parameters were optimized for cfDNA quantification, and several quantities of a recombinant standard were evaluated as internal standard. Absolute quantification was performed using a standard curve and the quality of the complete method was evaluated. cfDNA was eluted in a 50-µl volume, actin-ß (ACTB) was selected as the target gene, and qPCR parameters were optimized. The ACTB standard was constructed and 1000 copies were selected as internal standard. The standard curve showed R2 = 0.993 and E = 109.7%, and the linear dynamic range was defined between 102 and 106 ACTB copies/tube. Repeatability and reproducibility in terms of CV were 19% and up to 49.5% for ACTB copies per milliliter of plasma, respectively. The range of cfDNA levels was 428-18,851 copies/mL in NP women and 4031-2,019,363 copies/mL in P women, showing significant differences between the groups. We recommend the application of internal standard strategy for a reliable plasma cfDNA quantification. This methodology holds great potential for a future application in the obstetric field.

Asunto(s)

Ácidos Nucleicos Libres de Células , Mujeres Embarazadas , Humanos , Femenino , Embarazo , Reproducibilidad de los Resultados , Ácidos Nucleicos Libres de Células/genética , Biomarcadores

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ABSTRACT BACKGROUND: The human telomerase reverse transcriptase (hTERT) enzyme, encoded by the hTERT gene, synthesizes protective telomeric sequences on chromosomes and plays a fundamental role in cancer formation. Methylation of the hTERT gene has an upregulatory effect, increasing hTERT enzyme synthesis and allowing continuous tumor cell division. OBJECTIVE: In a group of patients with breast cancer, we aimed to analyze the methylation status of hTERT in the tumor, surrounding tissue, and circulating free deoxyribonucleic acid (cfDNA) of blood collected on the day of mastectomy and then approximately one year later. DESIGN AND SETTING: A prospective study was conducted at a university hospital in Rio de Janeiro, Brazil. METHODS: Samples were collected from 15 women with breast cancer on the day of mastectomy and approximately one year postoperatively. cfDNA was analyzed by sodium bisulfite conversion, followed by polymerase chain reaction, electrophoresis, and silver nitrate staining. RESULTS: Methylation of hTERT was detected in the tumors and surrounding tissues of all 15 patients. Five patients displayed hTERT methylation in the cfDNA from the blood of the first collection. Of the ten patients who returned for the second collection, three showed methylation. Two patients with methylation in the first collection did not display methylation in the second collection. One patient with no methylation in the first collection displayed methylation in the second collection, and one patient had a diminished level of methylation in the second collection. CONCLUSION: Only one-third of patients displayed methylation in their cfDNA, which may be related to the success of chemotherapy.

RESUMEN

Mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) have been increasingly investigated for cancer therapy and drug delivery, and they offer an advanced cell-free therapeutic option. However, their overall effects and efficacy depend on various factors, including the MSC source and cargo content. In this study, we isolated EVs from the conditioned medium of human immature dental pulp stem cells (hIDPSC-EVs) and investigated their effects on two papillary thyroid cancer (PTC) cell lines (BCPAP and TPC1). We observed efficient uptake of hIDPSC-EVs by both PTC cell lines, with a notable impact on gene regulation, particularly in the Wnt signaling pathway in BCPAP cells. However, no significant effects on cell proliferation were observed. Conversely, hIDPSC-EVs significantly reduced the invasive capacity of both PTC cell lines after 120 h of treatment. These in vitro findings suggest the therapeutic potential of hIDPSC-EVs in cancer management and emphasize the need for further research to develop novel and effective treatment strategies. Furthermore, the successful internalization of hIDPSC-EVs by PTC cell lines underscores their potential use as nanocarriers for anti-cancer agents.

RESUMEN

Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver (JAK2/CALR/MPL) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1ß and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets.

Asunto(s)

Ácidos Nucleicos Libres de Células , Mielofibrosis Primaria , Humanos , Inflamasomas/metabolismo , Citocinas/metabolismo , Interleucina-18/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Mielofibrosis Primaria/genética , Inflamación/inducido químicamente , ADN , Progresión de la Enfermedad

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Toehold switches are biosensors useful for the detection of endogenous and environmental RNAs. They have been successfully engineered to detect virus RNAs in cell-free gene expression reactions. Their inherent sequence programmability makes engineering a fast and predictable process. Despite improvements in the design, toehold switches suffer from leaky translation in the OFF state, which compromises the fold change and sensitivity of the biosensor. To address this, we constructed and tested signal amplification circuits for three toehold switches triggered by Dengue and SARS-CoV-2 RNAs and an artificial RNA. The serine integrase circuit efficiently contained leakage, boosted the expression fold change from OFF to ON, and decreased the detection limit of the switches by 3-4 orders of magnitude. Ultimately, the integrase circuit converted the analog switches' signals into digital-like output. The circuit is broadly useful for biosensors and eliminates the hard work of designing and testing multiple switches to find the best possible performer.

Asunto(s)

Técnicas Biosensibles , COVID-19 , Humanos , SARS-CoV-2/genética , ARN , Integrasas

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Diagnosis of tuberculosis (TB) relies on a sputum sample, which cannot be obtained from all symptomatic patients. Mycobacterium tuberculosis (Mtb) transrenal DNA (trDNA) has been detected in urine, an easily obtainable, noninvasive, alternative sample type. However, reported sensitivities have been variable and likely depend on collection/assay procedures and aspects of trDNA biology. We analyzed three serial urine samples from each of 75 adults with culture-confirmed pulmonary TB disease in Lima, Peru for detection of trDNA using short-fragment real-time PCR. Additionally, we examined host, urine, and sampling factors associated with detection. Overall sample sensitivity was 38% (95% Confidence Interval [CI] 30-45%). On a patient level (i.e., any of three samples positive), sensitivity was 73% (95% CI: 62-83%). Sensitivity was highest among samples from patients with smear-positive TB, 92% (95% CI: 62-100%). Specificity from a single sample from each of 10 healthy controls was 100% (95% CI: 69-100%). Adjusting our assay positivity threshold increased patient-level sensitivity to 88% (95% CI: 78-94%) overall without affecting the specificity. We did not find associations between Mtb trDNA detection and either patient characteristics or urine sample characteristics. Overall, our results support the potential of trDNA detection for TB diagnosis.

RESUMEN

The liver is the world's sixth most common primary tumor site, responsible for approximately 5% of all cancers and over 8% of cancer-related deaths. Hepatocellular carcinoma (HCC) is the predominant type of liver cancer, accounting for approximately 75% of all primary liver tumors. A major therapeutic tool for this disease is liver transplantation. Two of the most significant issues in treating HCC are tumor recurrence and graft rejection. Currently, the detection and monitoring of HCC recurrence and graft rejection mainly consist of imaging methods, tissue biopsies, and alpha-fetoprotein (AFP) follow-up. However, they have limited accuracy and precision. One of the many possible components of cfDNA is circulating tumor DNA (ctDNA), which is cfDNA derived from tumor cells. Another important component in transplantation is donor-derived cfDNA (dd-cfDNA), derived from donor tissue. All the components of cfDNA can be analyzed in blood samples as liquid biopsies. These can play a role in determining prognosis, tumor recurrence, and graft rejection, assisting in an overall manner in clinical decision-making in the treatment of HCC.

RESUMEN

BACKGROUND: Colorectal cancer (CRC) screening can help to reduce its incidence and mortality. Noninvasive strategies, such as plasma analysis of epigenetic alterations, can constitute important biomarkers of CRC detection. OBJECTIVE: This study aimed to evaluate the plasma methylation status of SEPT9 and BMP3 promoters as biomarkers for detection of CRC and its precursor lesions in a Brazilian population. METHODS: Plasma samples from 262 participants of the CRC screening program of Barretos Cancer Hospital who had a positive fecal occult blood test and underwent colonoscopy and cancer patients were analyzed. Participants were grouped according to the worst lesion detected in the colonoscopy. Cell-free circulating DNA (cfDNA) was bisulfite treated followed by the analysis of SEPT9 and BMP3 methylation status using a droplet digital PCR system (ddPCR). The best methylation cutoff value for group discrimination was calculated by receiver operating characteristic (ROC) curve analysis. RESULTS: Among the 262 participants, 38 were diagnosed with CRC, 46 with advanced adenomas 119 with nonadvanced adenomas, three with sessile serrated lesions, and 13 with hyperplastic polyps. In 43 participants, no lesion was detected in the colonoscopy and were used as controls. The CRC group showed the highest cfDNA concentration (10.4 ng/mL). For the SEPT9 gene, a cutoff of 2.5% (AUC = 0.681) that discriminates between CRC and the control group resulted in CRC sensitivity and specificity of 50% and 90%, respectively. Concerning the BMP3 gene, a cutoff of 2.3% (AUC = 0.576) showed 40% and 90% of sensitivity and specificity for CRC detection, respectively. Combining SEPT9, BMP3 status, and age over 60 years resulted in a better performance for detecting CRC (AUC = 0.845) than the individual gene models, yielding 80% and 81% of sensitivity and specificity, respectively. CONCLUSION: The present study suggests that a combination of SEPT9 and BMP3 plasma methylation, along with age over 60 years, showed the highest performance in detecting CRC in a Brazilian population. These noninvasive biomarkers can potentially serve as useful tools for CRC screening programs.

Asunto(s)

Adenoma , Ácidos Nucleicos Libres de Células , Neoplasias Colorrectales , Humanos , Persona de Mediana Edad , Detección Precoz del Cáncer , Brasil/epidemiología , Metilación de ADN , Septinas/genética , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , Sensibilidad y Especificidad , Adenoma/diagnóstico , Adenoma/genética , Biomarcadores de Tumor/genética , Proteína Morfogenética Ósea 3/genética