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Rapid and sensitive nucleic acid detection is vital in disease diagnosis and therapeutic assessment. Herein, we propose a co-freezing localized CRISPR-Cas12a (CL-Cas12a) strategy for sensitive nucleic acid detection. The CL-Cas12a was obtained through a 15-minute co-freezing process, allowing the Cas12a/crRNA complex and hairpin reporter confined on the AuNPs surface with high load efficiency, for rapid sensing of nucleic acid with superior performance to other localized Cas12a strategies. This CL-Cas12a based platform could quantitatively detect targets down to 98 aM in 30 min with excellent specificity. Furthermore, the CL-Cas12a successful applied to detect human papillomavirus infection and human lung cancer-associated single-nucleotide mutations. We also achieved powerful signal amplification for imaging Survivin mRNA in living cells. These findings highlight the potential of CL-Cas12a as an effective tool for nucleic acid diagnostics and disease monitoring. [ABSTRACT FROM AUTHOR]

Introduction Personalized and tumor-informed circulating tumor DNA (ctDNA) testing is feasible and allows for molecular residual disease (MRD) identification in patients with pancreatic ductal adenocarcinoma (PDAC). Methods In this retrospective analysis of commercial cases from multiple US institutions, personalized, tumor-informed, whole-exome sequenced, and germline-controlled ctDNA levels were quantified and analyzed in patients with PDAC. Plasma samples (n  = 1329) from 298 clinically validated patients were collected at diagnosis, perioperatively (MRD-window; within 2-12 weeks after surgery, before therapy), and during surveillance (>12 weeks post-surgery if no ACT or starting 4 weeks post-ACT) from November 2019 to March 2023. Results Of the initially diagnosed patients with stages I-III PDAC who went for resection, the median follow-up time from surgery was 13 months (range 0.1-214). Positive ctDNA detection rates were 29% (29/100) and 29.6% (45/152) during the MRD and surveillance windows, respectively. Positive ctDNA detection was significantly associated with shorter DFS within the MRD window (median DFS of 6.37 months for ctDNA-positive vs 33.31 months for ctDNA-negative patients; HR: 5.45, P  < .0001) as well as during the surveillance period (median DFS: 11.40 months for ctDNA-positive vs NR for ctDNA-negative; HR: 12.38, P  < .0001). Additionally, DFS was significantly better with KRAS wildtype status followed by KRAS G12R (HR: 0.99, P  = .97), KRAS G12D (HR: 1.42, P  = .194), and worse with KRAS G12V (HR: 2.19, P  = .002) status. In multivariate analysis, ctDNA detection at surveillance was found to be the most significant prognostic factor for recurrence (HR: 24.28, P  < .001). Conclusions Perioperative tumor-informed ctDNA detection in PDAC is feasible across all stages and is associated with patient survival outcomes. [ABSTRACT FROM AUTHOR]

Simple Summary: Peripheral blood analyses can offer a minimally invasive view into systemic immunity during cancer and can lead to the identification of biomarkers for cancer screening and therapeutic management. While a limited number of studies have reported blood transcriptome in breast cancer (BC) using RNA-seq analysis, our study is the first that aimed to identify potential BC biomarkers by analyzing transcriptome at an isoform level in peripheral blood mononuclear cells (PBMCs) from BC patients and healthy women. Our approach has led to the identification of an isoform of the RASGEF1A gene, the ENST00000374459 transcriptional variant, as a promising blood mRNA biomarker for distinguishing BC and healthy subjects. Additionally, our association analysis with clinicopathological characteristics revealed that lower ENST00000374459 expression in PBMCs of breast cancer patients was associated with higher proliferation and circulating tumor DNA (ctDNA) shedding, thereby linking expression of this isoform in blood immune cells to cancer progression and spreading. Background: Breast cancer (BC) comprises multiple subtypes with distinct molecular features, which differ in their interplay with host immunity, prognosis, and treatment. Non-invasive blood analyses can provide valuable insights into systemic immunity during cancer. The aim of this study was to analyze the expression of transcriptional isoforms in peripheral blood mononuclear cells (PBMCs) from BC patients and healthy women to identify potential BC immune biomarkers. Methods: RNA sequencing and isoform-level bioinformatics were performed on PBMCs from 12 triple-negative and 13 luminal A patients. Isoform expression validation by qRT-PCR and clinicopathological correlations were performed in a larger cohort (156 BC patients and 32 healthy women). Results: Transcriptional analyses showed a significant (p < 0.001) decrease in the ENST00000374459 RASGEF1A isoform in PBMCs of BC compared to healthy subjects, indicating disease-related expression changes. The decrease was associated with higher ctDNA and Ki-67 values. Conclusions: The levels of the RASGEF1A transcriptional isoform ENST00000374459 may have the potential to distinguish between BC and healthy subjects. The downregulation of ENST00000374459 in breast cancer is associated with higher proliferation and ctDNA shedding. Specialized bioinformatics analyses such as isoform analyses hold significant promise in the detection of biomarkers, since standard RNA sequencing analyses may overlook specific transcriptional changes that may be disease-associated and biologically important. [ABSTRACT FROM AUTHOR]

Simple Summary: Liquid biopsy based on the detection of circulating tumor DNA (ctDNA) is a well-consolidated tool to guide treatment decision-making and monitor patients with cancers other than uveal melanoma. The aim of this study was to explore the current technical possibilities of liquid biopsy to detect ctDNA in patients with uveal melanoma, with a particular focus on the clinical setting, to provide an overview of the current use of this technique. Uveal melanoma (UM) is the most common primary malignant intraocular tumor in adults. Distant metastasis is common, affecting around 50% of patients. Prognostic accuracy relies on molecular characterization of tumor tissue. In these patients, however, conventional biopsy can be challenging due to the difficulty of obtaining sufficient tissue for the analysis due to the small tumor size and/or post-brachytherapy shrinkage. An alternative approach is liquid biopsy, a non-invasive technique that allows for real-time monitoring of tumor dynamics. Liquid biopsy plays an increasingly prominent role in precision medicine, providing valuable information on the molecular profile of the tumor and treatment response. Liquid biopsy can facilitate early detection and can be used to monitor progression and recurrence. ctDNA-based tests are particularly promising due to their ease of integration into clinical practice. In this review, we discuss the application of ctDNA in liquid biopsies for UM. More specifically, we explore the emerging technologies in this field and the advantages and disadvantages of using different bodily fluids for liquid biopsy. Finally, we discuss the current barriers to routine clinical use of this technique. [ABSTRACT FROM AUTHOR]

Simple Summary: Mitochondria are important organelles in the human body, and they act as a source of chemical energy for different cellular processes. Multiple copies of mitochondria are normally detected in cells, and their copy number has also been reported to be aberrated in cancers, including liver cancer. Target-panel sequencing is frequently used in oncology studies, but the optimal pooling ratio of nuclear and mitochondrial probes has remained elusive. Therefore, it is necessary to determine the physiological mitochondrial DNA copy number in non-liver cancer subjects, which can serve as a useful reference for deciding the desirable ratio of nuclear and mitochondrial probes for a less biased target enrichment of nuclear and mitochondrial genomes and hence their even representation. In addition, it can also be used as a physiological basis that can distinguish normal physiological variations from the pathological conditions in liver cancer. Mitochondria generate energy to support cells. They are important organelles that engage in key biological pathways. The dysfunction of mitochondria can be linked to hepatocarcinogenesis, which has been actively explored in recent years. To investigate the mitochondrial dysfunction caused by genetic variations, target-panel sequencing is a flexible and promising strategy. However, the copy number of mitochondria generally exceeds nuclear DNA, which raises a concern that uneven target enrichment of mitochondrial DNA (mtDNA) and nuclear DNA (ncDNA) in target-panel sequencing would lead to an undesirably biased representation of them. To resolve this issue, we evaluated the optimal pooling of mtDNA probes and ncDNA probes by a series of dilutions of mtDNA probes in both genomic DNA (gDNA) and cell-free DNA (cfDNA) samples. The evaluation was based on read count, average sequencing depth and coverage of targeted regions. We determined that an mtDNA:ncDNA probe ratio of around 1:10 would offer a good balance of sequencing performance and cost effectiveness. Moreover, we estimated the median physiological mtDNA:ncDNA copy ratio as 38.1 and 2.9 in cfDNA and gDNA samples of non-liver cancer subjects, respectively, whereas they were 20.0 and 2.1 in the liver cancer patients. Taken together, this study revealed the appropriate pooling strategy of mtDNA probes and ncDNA probes in target-panel sequencing and suggested the normal range of physiological variation of the mtDNA:ncDNA copy ratio in non-liver cancer individuals. This can serve as a useful reference for future target-panel sequencing investigations of the mitochondrial genome in liver cancer. [ABSTRACT FROM AUTHOR]

In this article, we defined comprehensive recommendations for the clinical follow-up of pregnant women with a malignancy-suspicious NIPT result, on the basis of the vast experience with population-based NIPT screening programs in two European countries complemented with published large data sets. These recommendations provide a tool for classifying NIPT results as malignancy-suspicious, and guide health care professionals in structured clinical decision making for the diagnostic process of pregnant women who receive such a malignancy-suspicious NIPT result. [ABSTRACT FROM AUTHOR]

Abstract Rapid and sensitive nucleic acid detection is vital in disease diagnosis and therapeutic assessment. Herein, we propose a co-freezing localized CRISPR-Cas12a (CL-Cas12a) strategy for sensitive nucleic acid detection. The CL-Cas12a was obtained through a 15-minute co-freezing process, allowing the Cas12a/crRNA complex and hairpin reporter confined on the AuNPs surface with high load efficiency, for rapid sensing of nucleic acid with superior performance to other localized Cas12a strategies. This CL-Cas12a based platform could quantitatively detect targets down to 98 aM in 30 min with excellent specificity. Furthermore, the CL-Cas12a successful applied to detect human papillomavirus infection and human lung cancer-associated single-nucleotide mutations. We also achieved powerful signal amplification for imaging Survivin mRNA in living cells. These findings highlight the potential of CL-Cas12a as an effective tool for nucleic acid diagnostics and disease monitoring. Graphical abstract

Simple Summary: Circulating tumor DNA (ctDNA) is a non-invasive method of identifying and monitoring genitourinary cancers, including prostate, bladder, and renal cell carcinoma, via blood or urine samples. CtDNA introduces a potential method for cancer screening. If detected, ctDNA may reveal genetic alterations that have prognostic value. As treatment options for genitourinary cancers progress towards accounting for tumor genetic profiles, ctDNA may predict which patients have improved responses to therapeutic targets. CtDNA may play an important role in surveillance after tumor resection and may be used to reveal mechanisms of treatment resistance. The clinical utility of ctDNA has yet to be established. Significantly more research is needed to understand the utility ctDNA has in clinical practice. CtDNA is emerging as a non-invasive clinical detection method for several cancers, including genitourinary (GU) cancers such as prostate cancer, bladder cancer, and renal cell carcinoma (RCC). CtDNA assays have shown promise in early detection of GU cancers, providing prognostic information, assessing real-time treatment response, and detecting residual disease and relapse. The ease of obtaining a "liquid biopsy" from blood or urine in GU cancers enhances its potential to be used as a biomarker. Interrogating these "liquid biopsies" for ctDNA can then be used to detect common cancer mutations, novel genomic alterations, or epigenetic modifications. CtDNA has undergone investigation in numerous clinical trials, which could address clinical needs in GU cancers, for instance, earlier detection in RCC, therapeutic response prediction in castration-resistant prostate cancer, and monitoring for recurrence in bladder cancers. The utilization of liquid biopsy for ctDNA analysis provides a promising method of advancing precision medicine within the field of GU cancers. [ABSTRACT FROM AUTHOR]

Recent progress in adjuvant immunotherapy offers hope for improving disease-free survival in high-risk bladder cancer (BC) and renal cell carcinoma (RCC). This review focuses on key trials such as CheckMate 274 and KEYNOTE-564, which show promising results with nivolumab in BC and pembrolizumab in RCC, including a 30% reduction in progression risk. Pembrolizumab also demonstrated overall survival (OS) benefit in RCC. The review also explores the potential of circulating tumor DNA (ctDNA) as a biomarker for better therapy selection and patient stratification. It emphasizes the need for ongoing research to establish survival benefits and suggests integrating biomarkers and risk stratification to optimize adjuvant immunotherapy in BC and RCC. [ABSTRACT FROM AUTHOR]

Simple Summary: Predicting which patients will respond to therapy or experience disease relapse can help clinicians select treatments that could slow down or prevent the spread of cancer. Clinicians have routinely used imaging to measure the size of the tumor to assess whether or not it is responding to treatment. A recent development for monitoring tumors involves a test to detect mutated DNA shed by tumors into the blood, called circulating tumor DNA (ctDNA). The authors searched the scientific literature to find studies that have combined imaging and ctDNA to build tools that can predict treatment response or patient survival. The authors noted that only a few studies have been reported, indicating that this field is new and needs further exploration. These early studies, however, showed that combining these two clinical tests (imaging + ctDNA) may improve the prediction of tumors' response to therapy and the return of cancer. While promising, these tools need to be refined to improve the accuracy of the predictions and the results confirmed in more extensive studies. Biomarkers for evaluating tumor response to therapy and estimating the risk of disease relapse represent tremendous areas of clinical need. To evaluate treatment efficacy, tumor response is routinely assessed using different imaging modalities like positron emission tomography/computed tomography or magnetic resonance imaging. More recently, the development of circulating tumor DNA detection assays has provided a minimally invasive approach to evaluate tumor response and prognosis through a blood test (liquid biopsy). Integrating imaging- and circulating tumor DNA-based biomarkers may lead to improvements in the prediction of patient outcomes. For this mini-review, we searched the scientific literature to find original articles that combined quantitative imaging and circulating tumor DNA biomarkers to build prediction models. Seven studies reported building prognostic models to predict distant recurrence-free, progression-free, or overall survival. Three discussed building models to predict treatment response using tumor volume, pathologic complete response, or objective response as endpoints. The limited number of articles and the modest cohort sizes reported in these studies attest to the infancy of this field of study. Nonetheless, these studies demonstrate the feasibility of developing multivariable response-predictive and prognostic models using regression and machine learning approaches. Larger studies are warranted to facilitate the building of highly accurate response-predictive and prognostic models that are generalizable to other datasets and clinical settings. [ABSTRACT FROM AUTHOR]

Context: CytoPath®Easy kit (DiaPath S.p.A.) offers a major advantage compared to other commercially available kits available for the screening of cervical cancer, as it does not require additional equipment for sample processing. Using this methodology, collected epithelial cells are immersed in a preservative liquid before setting as a thin layer on a slide via gravity sedimentation. Aims: To evaluate the suitability of the CytoPath®Easy kit for the processing of cervical samples, detection of pre-neoplastic lesions, and nucleic preservation and extraction for HR-HPV diagnosis. Materials and Methods: A total of 242 self-sampled cervical specimens were utilized, with 192 collected in CytoPath®Easy vials and 50 collected and processed using the ThinPrep™ for comparative analysis. The samples underwent processing, Papanicolaou staining, and microscopic evaluation for morphological parameters. The extracted nucleic acids were assessed for purity and integrity, and the detection of high-risk human papillomavirus (HR-HPV) was carried out using the Alinitym HR HPV system kit (Abbott Laboratórios Lda). Results: Both methods demonstrated effective performance, enabling the morphological assessment of the cervical epithelium. Statistical analysis indicated that ThinPrepTM yielded significantly better results in terms of cellularity. Conversely, CytoPath®Easy exhibited superior performance in terms of the quantity of extracted DNA and its degree of purification. Concerning the time consumed during processing, both methods were comparable, with the CytoPath®Easy methodology standing out for its cost-effectiveness, as it does not necessitate additional instruments and consumables. Conclusions: The novel CytoPath®Easy methodology proves effective in preserving both nucleic acids and cell morphology characteristics, two crucial features for cervical cancer screening. [ABSTRACT FROM AUTHOR]

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Simple Summary: In this literature review, we explore the milestone events from the discovery of BRAF mutations to present-day clinical intervention strategies. We delve into the role of the BRAF gene in various cancer types such as melanoma, non-small-cell lung cancer, colorectal cancer, and thyroid cancer. Additionally, we reviewed clinical trials that led to the FDA approval of therapeutic regimens as monotherapy or, more recently, as combinatorial approaches to treat cancer types harboring BRAF hotspot mutations. Since their discovery in 2002, BRAF mutations have been identified as clear drivers of oncogenesis in several cancer types. Currently, their incidence rate is nearly 7% of all solid tumors with BRAF V600E constituting approximately 90% of these diagnoses. In melanoma, thyroid cancer, and histiocytic neoplasms, BRAF hotspot mutations are found at a rate of about 50%, while in lung and colorectal cancers they range from 3% to 10% of reported cases. Though present in other malignancies such as breast and ovarian cancers, they constitute a small portion of diagnoses (<1%). Given their frequency along with advancements in screening technologies, various methods are used for the detection of BRAF-mutant cancers. Among these are targeted next-generation sequencing (NGS) on tumor tissue or circulating tumor DNA (ctDNA) and immunohistochemistry (IHC)-based assays. With advancements in detection technologies, several approaches to the treatment of BRAF-mutant cancers have been taken. In this review, we retrace the milestones that led to the clinical development of targeted therapies currently available for these tumors. [ABSTRACT FROM AUTHOR]

AbstractMicroRNA (miRNA) has emerged as a promising biomarker for disease diagnosis and a potential therapeutic targets for drug development. The detection of miRNA can serve as a noninvasive tool in diseases diagnosis and predicting diseases prognosis. CRISPR/Cas12a system has great potential in nucleic acid detection due to its high sensitivity and specificity, which has been developed to be a versatile tool for nucleic acid-based detection of targets in various fields. However, conversion from RNA to DNA with or without amplification operation is necessary for miRNA detection based on CRISPR/Cas12a system, because dsDNA containing PAM sequence or ssDNA is traditionally considered as the activator of Cas12a. Until recently, direct detection of miRNA by CRISPR/Cas12a system has been reported. In this review, we provide an overview of the evolution of biosensors based on CRISPR/Cas12a for miRNA detection from indirect to direct, which would be beneficial to the development of CRISPR/Cas12a-based sensors with better performance for direct detection of miRNA. [ABSTRACT FROM AUTHOR]

Accurate pathogenic detection in wastewater is critical for safeguarding public health and the environment. However, the presence of free nucleic acids in wastewater samples poses significant challenges to molecular detection accuracy. This comprehensive review explores the current status and future potential of pretreatment methods to remove free nucleic acids from wastewater samples. The study contributes a comprehensive analysis of the mechanisms, strengths, and limitations of various pretreatment approaches, including physical, chemical, and enzymatic processes. The effect of various factors on the removal efficiency of these pretreatment methods is also discussed. This review enhances our comprehension of pretreatment techniques and their vital role in achieving precise pathogenic detection in complex wastewater matrices. Furthermore, it outlines future perspectives and developments for improving the speed and effectiveness of pathogenic detection, contributing significantly to disease surveillance, early warning systems, and environmental protection. [ABSTRACT FROM AUTHOR]

Simple Summary: A liquid biopsy is a minimally invasive strategy that provides useful information for the management of cancer patients. Cell-free DNA (cfDNA) extraction and quantification could be easily integrated into standard care protocols. This study analyzes cfDNA concentration as a biomarker in 256 lymphoma cases, including unexplored subtypes. The findings reveal that all lymphoma subtypes release higher cfDNA levels than controls, and these levels correlate with subtype, aggressiveness, and prognostic markers like the International Prognostic Index (IPI) and Ann Arbor Stage. Additionally, the study includes 49 large B-cell lymphomas (LBCL) cases, in which pre- and post-treatment levels of cfDNA were assessed, demonstrating that cfDNA dynamics correlate with treatment response and can, in some cases, improve PET-based response assessment. The study also includes NGS genetic characterization in tissue and cfDNA in a subgroup of LBCL. Overall, the study highlights cfDNA's potential as a biomarker for lymphoma management, enabling its integration in the near future into routine clinical practice. Background: Cell-free DNA (cfDNA) analysis has become a promising tool for the diagnosis, prognosis, and monitoring of lymphoma cases. Until now, research in this area has mainly focused on aggressive lymphomas, with scanty information from other lymphoma subtypes. Methods: We selected 256 patients diagnosed with lymphomas, including a large variety of B-cell and T-cell non-Hodgkin and Hodgkin lymphomas, and quantified cfDNA from plasma at the time of diagnosis. We further selected 49 large B-cell lymphomas (LBCL) and analyzed cfDNA levels at diagnosis (pre-therapy) and after therapy. In addition, we performed NGS on cfDNA and tissue in this cohort of LBCL. Results: Lymphoma patients showed a statistically significant higher cfDNA concentration than healthy controls (mean 53.0 ng/mL vs. 5.6 ng/mL, p < 0.001). The cfDNA concentration was correlated with lymphoma subtype, lactate dehydrogenase, the International Prognostic Index (IPI) score, Ann Arbor (AA), and B-symptoms. In 49 LBCL cases, the cfDNA concentration decreased after therapy in cases who achieved complete response (CR) and increased in non-responders. The median cfDNA at diagnosis of patients who achieved CR and later relapsed was higher (81.5 ng/mL) compared with levels of those who did not (38.6 ng/mL). A concordance of 84% was observed between NGS results in tumor and cfDNA samples. Higher VAF in cfDNA is correlated with advanced stage and bulky disease. Conclusions: cfDNA analysis can be easily performed in almost all lymphoma cases. The cfDNA concentration correlated with the characteristics of the aggressiveness of the lymphomas and, in LBCL, with the response achieved after therapy. These results support the utility of cfDNA analysis as a complementary tool in the management of lymphoma patients. [ABSTRACT FROM AUTHOR]

Simple Summary: Colorectal cancer (CRC) is the second leading cause of cancer-related death globally. Developing liquid biopsies that aid in the detection and monitoring of recurrent CRC may improve clinical management and patient outcomes. In this study, we investigated a serially sampled blood assay of methylated DNA markers (MDMs) in combination with carcinoembryonic antigen as a clinical tool to surveil and monitor recurrent cancer in a prospective cohort of patients who completed curative-intent therapy for CRC. We demonstrated that the MDMs detected recurrent CRC before the clinical or radiographic detection of recurrence. In a small number of patients, we further showed that the MDMs may correlate with the tumor burden. These data highlight the importance of incorporating epigenetic signatures into liquid biopsies and support further research to validate the correlations of circulating tumor DNA with the tumor burden. Background: Radiographic surveillance of colorectal cancer (CRC) after curative-intent therapy is costly and unreliable. Methylated DNA markers (MDMs) detected primary CRC and metastatic recurrence with high sensitivity and specificity in cross-sectional studies. This study evaluated using serial MDMs to detect recurrence and monitor the treatment response to anti-cancer therapies. Methods: A nested case-control study was drawn from a prospective cohort of patients with CRC who completed curative-intent therapy for CRC of all stages. Plasma MDMs were assayed vis target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, and analyzed in combination with serum carcinoembryonic antigen to yield an MDM score. Clinical information, including treatment and radiographic measurements of the tumor burden, were longitudinally collected. Results: Of the 35 patients, 18 had recurrence and 17 had no evidence of disease during the study period. The MDM score was positive in 16 out of 18 patients who recurred and only 2 of the 17 patients without recurrence. The MDM score detected recurrence in 12 patients preceding clinical or radiographic detection of recurrent CRC by a median of 106 days (range 90–232 days). Conclusions: Plasma MDMs can detect recurrent CRC prior to radiographic detection; this tumor-agnostic liquid biopsy approach may assist cancer surveillance and monitoring. [ABSTRACT FROM AUTHOR]

Background: Circulating tumor DNA (ctDNA) is increasingly used as a biomarker for metastatic rectal cancer and has recently shown promising results in the early detection of recurrence risk. Methods: We conducted a systematic review and meta-analysis to explore the prognostic value of ctDNA detection in LARC patients undergoing neoadjuvant chemoradiotherapy (nCRT). We systematically searched electronic databases for observational or interventional studies that included LARC patients undergoing nCRT. Study selection according to the PRISMA guidelines and quality assessment of the REMARK tool for biomarker studies. The primary endpoint was the impact of ctDNA detection at different time points (baseline, post-nCRT, post-surgery) on relapse-free survival (RFS) and overall survival (OS). The secondary endpoint was to study the association between ctDNA detection and pathological complete response(pCR) at different time points. Results: After further review and analysis of the 625 articles initially retrieved, we finally included 10 eligible studies. We found no significant correlation between ctDNA detection at baseline and long-term survival outcomes or the probability of achieving a pCR. However, the presence of ctDNA at post-nCRT was associated with worse RFS (HR = 9.16, 95% CI, 5.48-15.32), worse OS (HR = 8.49, 95% CI, 2.20-32.72), and worse pCR results (OR = 0.40, 95%CI, 0.18-0.89). The correlation between the presence of ctDNA at post-surgery and worse RFS was more obvious (HR = 14.94; 95% CI, 7.48-9.83). Conclusions: Our results suggest that ctDNA detection is a promising biomarker for the evaluation of response and prognosis in LARC patients undergoing nCRT, which merits further evaluation in the following prospective trials. Circulating tumor DNA (ctDNA) is increasingly used as a biomarker for metastatic rectal cancer and has shown promising results in the early detection of recurrence risk. This review explores the prognostic value of ctDNA detection in patients with locally advanced rectal cancer undergoing neoadjuvant chemoradiotherapy. [ABSTRACT FROM AUTHOR]

Nucleic acid (DNA and RNA) detection and quantification methods play vital roles in molecular biology. With the development of molecular biology, isothermal amplification of DNA/RNA, as a new molecular biology technology, can be amplified under isothermal condition, it has the advantages of high sensitivity, high specificity, and high efficiency, and has been applied in various fields of biotechnology, including disease diagnosis, pathogen detection, food hygiene and safety detection and so on. This paper introduces the progress of isothermal amplification technology, including rolling circle amplification (RCA), nucleic acid sequence-dependent amplification (NASBA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), cross-priming amplification (CPA), and its principle, advantages and disadvantages, and application development are briefly summarized. [ABSTRACT FROM AUTHOR]

Methylsulfonylmethane (MSM), a natural organosulfur compound, is a popular dietary supplement sold both as a single product and as a constituent of multi-ingredient products. It has been postulated that MSM may serve as a donor for methyl groups for various cellular processes; however, studies have yet to demonstrate this. Therefore, the goal of this study was to determine whether or not MSM, supplemented to fully differentiated human HepaRG cells at physiologically-relevant concentrations, can serve as a donor for methyl groups for DNA methylation. For this purpose, methyl groups in the MSM molecule were labeled with deuterium (deuterated) and incorporation of the labeled 5-methylcytosine into the HepaRG cell DNA was evaluated using liquid chromatography/mass spectrometry (LC-MS/MS). We report that MSM supplementation resulted in significant incorporation of deuterated product into DNA in a time- and dose-dependent fashion. These changes were not associated with increased 5-methylcytosine content, did not result in changes of DNA methylation or re-distribution of DNA methylation patterns between the retrotransposons LINE-1 and HERV18, and were not associated with cytotoxicity. In conclusion, short-term supplementation with MSM in vitro demonstrates that MSM can serve as a donor of methyl groups for methylation of DNA, but does not affect the levels of DNA methylation globally and does not lead to redistribution of the DNA methylation patterns within the most abundant repetitive elements. Future studies will be needed to validate these findings in vivo and to investigate whether or not MSM can restore normal DNA methylation patterns within the hypomethylated phenotype. [ABSTRACT FROM AUTHOR]

In digital microfluidics, discrete liquid droplets move on a dielectric surface with the help of AC or DC voltage. Digital microfluidics system has extended the scope of microfluidics by providing automated, precise flow control for point-of-care devices capable of medical diagnostics, environmental, and chemical sensing platforms. In this paper, we introduce a digital microfluidics platform based on electrowetting on a dielectric using PCB substrate designed on Easy EDA software. The platform's performance was analyzed at various DC voltages between 200 V and 400 V for droplet actuation. PCB fabrication, the utilization of easily accessible and cheap materials such as cooking oil and grafting tape as a dielectric layer, allowed the fabrication to be affordable and simple. The results indicate that droplet actuation takes place at 220 V. The droplet velocity obtained was 2.6 mm/s and 1.51 mm/s using silicon oil and cooking oil, respectively, at 400 V. Our proposed digital microfluidics platform will play a vital role in droplet actuation and can be used for diagnostic applications such as DNA analysis and cell culture due to its simple fabrication and cost-effective technique. [ABSTRACT FROM AUTHOR]

Simple Summary: Colonoscopies are effective in the prevention of colorectal cancer (CRC) but considered burdensome. An alternative for or addition to this procedure might be found in circulating tumor DNA (ctDNA) analysis. However, to date, it is not clear which analysis is most suitable for such a ctDNA-based blood test for CRC. Therefore, we assessed this in ten patients with colonoscopies for Lynch syndrome or in the context of the Dutch national screening program who were diagnosed with CRC or its precursor lesion (advanced adenoma). The results of this proof-of-principle study could form the foundation for subsequent studies on ctDNA-based blood test development for CRC screening and management, specifically in carriers of Lynch syndrome. Colorectal cancer (CRC) colonoscopic surveillance is effective but burdensome. Circulating tumor DNA (ctDNA) analysis has emerged as a promising, minimally invasive tool for disease detection and management. Here, we assessed which ctDNA assay might be most suitable for a ctDNA-based CRC screening/surveillance blood test. In this prospective, proof-of-concept study, patients with colonoscopies for Lynch surveillance or the National Colorectal Cancer screening program were included between 7 July 2019 and 3 June 2022. Blood was drawn, and if advanced neoplasia (adenoma with villous component, high-grade dysplasia, ≥10 mm, or CRC) was detected, it was analyzed for chromosomal copy number variations, single nucleotide variants, and genome-wide methylation (MeD-seq). Outcomes were compared with corresponding patients' tissues and the MeD-seq results of healthy blood donors. Two Lynch carriers and eight screening program patients were included: five with CRC and five with advanced adenomas. cfDNA showed copy number variations and single nucleotide variants in one patient with CRC and liver metastases. Eight patients analyzed with MeD-seq showed clustering of Lynch-associated and sporadic microsatellite instable lesions separate from microsatellite stable lesions, as did healthy blood donors. In conclusion, whereas copy number changes and single nucleotide variants were only detected in one patient, cfDNA methylation profiles could discriminate all microsatellite instable advanced neoplasia, rendering this tool particularly promising for LS surveillance. Larger studies are warranted to validate these findings. [ABSTRACT FROM AUTHOR]

CRISPR/Cas systems have found widespread applications in gene editing due to their high accuracy, high programmability, ease of use, and affordability. Benefiting from the cleavage properties (trans- or cis-) of Cas enzymes, the scope of CRISPR/Cas systems has expanded beyond gene editing and they have been utilized in various fields, particularly in live-cell imaging and bioanalysis. In this review, we summarize some fundamental working mechanisms and concepts of the CRISPR/Cas systems, describe the recent advances and design principles of CRISPR/Cas mediated techniques employed in live-cell imaging and bioanalysis, highlight the main applications in the imaging and biosensing of a wide range of molecular targets, and discuss the challenges and prospects of CRISPR/Cas systems in live-cell imaging and biosensing. By illustrating the imaging and bio-sensing processes, we hope this review will guide the best use of the CRISPR/Cas in imaging and quantifying biological and clinical elements and inspire new ideas for better tool design in live-cell imaging and bioanalysis. [ABSTRACT FROM AUTHOR]

The utilization of cell-free DNA (cfDNA) screening has expanded rapidly across the age spectrum of pregnant persons. With cfDNA's widespread adoption, genetic fetal sex is now often known before a phenotypic assessment on anatomic survey. CfDNA detects sex discordance in 1/1500 to 2000 pregnancies. Upon detection of sex discordance, lab error or other factors should first be assessed. Once other causes have been ruled out, this may indicate an underlying disorder/difference in sex development. A multidisciplinary team should coordinate diagnosis, treatment, and support for the family. This review discusses the diagnostic workup, emphasizing the multidisciplinary counseling and management of disorder/differences in sex development. [ABSTRACT FROM AUTHOR]

Single-gene disorders (SGDs), also known as monogenic disorders, are caused by pathogenic variants at individual loci. Prenatal cell-free DNA screening for SGDs has been investigated for decades. Detecting paternal and de novo variants may be somewhat straightforward, whereas detecting maternally inherited variants poses a significant challenge. Although commercially available in both high-risk and low-risk patients, many limitations exist. The use of SGDs is not supported by professional medical societies. [ABSTRACT FROM AUTHOR]

Considering the diagnostic limitations of cfDNA-based noninvasive prenatal testing (NIPT), scientists have long been interested in isolating and analyzing rare intact fetal and trophoblast cells from maternal blood or endocervical samples to diagnose fetal genetic conditions. These cells may be scarce and difficult to isolate, but they are a direct source of pure fetal genetic material. In this review, we summarize the history of cell-based NIPT, present an updated review on its current developments, evaluate its genetic diagnostic potential, and discuss its future prospects for clinical use. [ABSTRACT FROM AUTHOR]

Simple Summary: In our study, we found genetic mutations in the DNA of cancer cells obtained from the blood of many patients with metastatic prostate cancer. These changes were more common in patients whose cancer was progressing and aggressive in nature, often unresponsive to conventional treatment strategies. Additionally, these genetic mutation findings were more sensitive than the conventional method of checking the prostate-specific antigen (PSA) level in the blood for detecting prostate cancer progression. This means a simple blood test could help us track genetic mutations, detect aggressive cancer subtypes, determine the progression of the disease, and manage advanced prostate cancer more effectively. The positivity rate of circulating tumor DNA (ctDNA) next-generation sequencing (NGS) varies among patients with metastatic prostate cancer (mPC), complicating its incorporation into regular practice. This retrospective study analyzed the ctDNA sequencing results of 100 mPC patients from May 2021 to March 2023 to identify the factors associated with positive ctDNA. Three custom gene panels were used for sequencing. Overall, 63% of the patients exhibited tier I/II somatic alterations, while 12% had pathogenic/likely pathogenic germline alterations. The key genes that were altered included AR, TP53, RB1, PTEN, and APC. Mutations in BRCA1/2, either germline or somatic, were observed in 21% of the patients. Among the metastatic castration-resistant prostate cancer (mCRPC) patients, the ctDNA-positive samples generally showed higher median prostate-specific antigen (PSA) levels and were more likely to be at the radiographic and clinical progressive disease stages, although they were not significantly associated with PSA progression. Our results suggest that ctDNA analysis could detect meaningful genetic changes in mPC patients, especially during disease progression. [ABSTRACT FROM AUTHOR]

Simple Summary: Glioblastoma is a highly aggressive brain cancer, and early detection and accurate diagnosis are crucial for effective treatment. Traditional diagnostic methods have limitations, and liquid biopsies offer a non-invasive and dynamic approach to detecting and monitoring glioblastoma. This review provides a comprehensive overview of various cancer biomarkers, including circulating tumor cells, cell-free DNA, and RNA, such as microRNA, as well as extracellular vesicles. It highlights their clinical utility in glioblastoma detection, monitoring, and prognosis. In addition, challenges and limitations in implementing liquid biopsy strategies in clinical practice are addressed. Glioblastoma (GBM) is a highly aggressive and lethal primary brain cancer that necessitates early detection and accurate diagnosis for effective treatment and improved patient outcomes. Traditional diagnostic methods, such as imaging techniques and tissue biopsies, have limitations in providing real-time information and distinguishing treatment-related changes from tumor progression. Liquid biopsies, used to analyze biomarkers in body fluids, offer a non-invasive and dynamic approach to detecting and monitoring GBM. This article provides an overview of GBM biomarkers in body fluids, including circulating tumor cells (CTCs), cell-free DNA (cfDNA), cell-free RNA (cfRNA), microRNA (miRNA), and extracellular vesicles. It explores the clinical utility of these biomarkers for GBM detection, monitoring, and prognosis. Challenges and limitations in implementing liquid biopsy strategies in clinical practice are also discussed. The article highlights the potential of liquid biopsies as valuable tools for personalized GBM management but underscores the need for standardized protocols and further research to optimize their clinical utility. [ABSTRACT FROM AUTHOR]

PURPOSE In patients with metastatic lung adenocarcinoma, evidence-based first-line treatment decisions require analysis of tumors for genomic alterations (GAs). Optimizing the genotyping paradigm may improve the delivery of precision oncology care. Actionable GAs can be identified by analyzing tumor tissue or circulating tumor DNAusing liquid biopsy. Consensus guidelines for when to use liquid biopsy have not been established. We evaluated the routine use of liquid biopsy performed simultaneously with tissue testing in patients with newly diagnosed, stage IV lung adenocarcinoma. METHODS We performed a retrospective study comparing patients who underwent tissue genotyping alone (standard biopsy group) with patients who had simultaneous liquid and tissue genotyping (combined biopsy group). We examined the time to reach a final diagnosis, the need for repeat biopsies, and diagnostic accuracy. RESULTS Forty two patients in the combined biopsy group and 78 in the standard biopsy group met the inclusion criteria. The standard group had a mean time to diagnosis of 33.5 days, compared with 20.6 days in the combined group (P < .001 by two-tailed t-test). In the combined group, 14 patients did not have sufficient tissue for molecular analysis (30%); however, in 11 (79%) of these patients, liquid biopsy identified a GA that eliminated the need for a second tissue biopsy. In patients who completed both tests, each test found actionable GAs missed by the other. CONCLUSION Performing liquid biopsy simultaneously with tissue genotyping is feasible in an academic community medical center. Potential advantages of simultaneous liquid and tissue biopsies include shorter time to obtain a definitive molecular diagnosis, reduced need for a repeat biopsy, and improved detection of actionable mutations, although a sequential strategy that saves costs by beginning with a liquid biopsy may be ideal. [ABSTRACT FROM AUTHOR]

Purpose: The aim was to describe the prevalence, molecular epidemiology and clinical manifestations of human bocavirus (HBoV) in patients attended at a tertiary hospital in Barcelona, Spain. Methods: From October 2014 to May 2017, respiratory specimens from paediatric patients were collected for respiratory viruses' laboratory-confirmation. Phylogenetic analyses from partial VP1 sequences were performed from all HBoV laboratory-confirmed specimens. Clinical features were retrospectively studied. Results: 178/10271 cases were HBoV laboratory-confirmed. The median age was 1.53 (IQR 1.0–2.3). Co-detection was highly reported (136; 76%). All viruses belonged into HBoV1 genotype but one into HBoV2. Non-reported mutations were observed and two sites were suggestive to be under negative selection. 61% (109/178) cases had lower RTI (LRTI), of whom 84 had co-detections (77%) and 76 had comorbidities (70%). LRTI was the cause of hospitalization in 85 out of 109 cases (78%), and no differences were found regarding severity factors during hospitalization between co- and single-detections, except for median length of respiratory support, which was longer in cases with co-detections. Conclusions: Close monitoring of predominant HBoV1 showed a high similarity between viruses. The presence of comorbidities might explain the high prevalence of LRTI. Symptomatology in HBoV single-detected cases suggest that HBoV is a true pathogen. [ABSTRACT FROM AUTHOR]

The RNA contained in exosomes plays a crucial role in information transfer between cells in various life activities. The accurate detection of low-abundance exosome RNA (exRNA) is of great significance for cell function studies and the early diagnosis of diseases. However, their intrinsic properties, such as their short length and high sequence homology, represent great challenges for exRNA detection. In this paper, we developed a dual-signal isothermal amplification method based on rolling circle amplification (RCA) coupled with DNAzyme (RCA–DNAzyme). The sensitive detection of low-abundance exRNA, the specific recognition of their targets and the amplification of the detection signal were studied and explored. By designing padlock probes to specifically bind to the target exRNA, while relying on the ligation reaction to enhance recognition, the precise targeting of exosome RNA was realized. The combination of RCA and DNAzyme could achieve a twice-as-large isothermal amplification of the signal compared to RCA alone. This RCA–DNAzyme assay could sensitively detect a target exRNA at a concentration as low as 527 fM and could effectively distinguish the target from other miRNA sequences. In addition, this technology was successfully proven to be effective for the quantitative detection of miR-21 by spike recovery, providing a new research approach for the accurate detection of low-abundance exRNA and the exploration of unknown exRNA functions. [ABSTRACT FROM AUTHOR]

Electrochemiluminescence (ECL) is the chemiluminescence triggered by electrochemical reactions. Due to the unique excitation mode and inherent low background, ECL has been a powerful analytical technique to be widely used in biosensing and imaging. As an emerging ECL luminophore, semiconductor quantum dots (QDs) have apparent advantages over traditional molecular luminophores in terms of luminescence efficiency and signal modulation ability. Therefore, the development of an efficient ECL system with QDs as luminophores is of great significance to improve the sensitivity and detection flux of ECL biosensors. In this review, we give a comprehensive summary of recent advances in ECL using semiconductor QDs as luminophores. The luminescence process and ECL mechanism of semiconductor QDs with various coreactants are discussed first. Specifically, the influence of surface defects on ECL performance of semiconductor QDs is emphasized and several typical ECL enhancement strategies are summarized. Then, the applications of semiconductor QDs in ECL biosensing are overviewed, including immunoassay, nucleic acid analysis and the detection of small molecules. Finally, the challenges and prospects of semiconductor QDs as ECL luminophores in biosensing are featured. [ABSTRACT FROM AUTHOR]

The discovery of cell-free fetal DNA (cffDNA) in maternal blood and the rapid development of massively parallel sequencing have revolutionized prenatal testing from invasive to noninvasive. Noninvasive prenatal screening (NIPS) based on cffDNA enables the detection of fetal trisomy through sequencing, comparison, and bioassays. Its accuracy is better than that of traditional screening methods, and it is the most advanced clinical application of high-throughput sequencing technologies. However, the existing sequencing methods are limited by high costs and complex sequencing procedures. These limitations restrict the availability of NIPS for pregnant women. Many amplification methods have been developed to overcome the limitations of sequencing methods. The rapid development of non-sequencing methods has not been accompanied by reviews to summarize them. In this review, we initially describe the detection principles for sequencing-based NIPS. We summarize the rapidly evolving amplification technologies, focusing on the need to reduce costs and simplify the procedures. To ensure that the testing systems are feasible and that the testing processes are reliable, we expand our vision to the clinic. We evaluate the clinical validity of NIPS in terms of sensitivity, specificity, and positive predictive value. Finally, we summarize the application guidelines and discuss the corresponding quality control methods for NIPS. In addition to cffDNA, extracellular vesicle DNA, RNA, protein/peptide, and fetal cells can also be detected as biomarkers of NIPS. With the development of prenatal testing, NIPS has become increasingly important. Notably, NIPS is a screening test instead of a diagnostic test. The testing methods and procedures used in the NIPS process require standardization. [ABSTRACT FROM AUTHOR]

In this paper, a stochastic COVID-19 model with large-scale nucleic acid detection and isolation measures is proposed. Firstly, the existence and uniqueness of the global positive solution is obtained. Secondly, threshold criteria for the stochastic extinction and persistence in the mean with probability one are established. Moreover, a sufficient condition for the existence of unique ergodic stationary distribution for any positive solution is also established. Finally, numerical simulations are carried out in combination with real COVID-19 data from Urumqi, China and the theoretical results are verified. [ABSTRACT FROM AUTHOR]

Simple Summary: ESR1 mutations in breast cancer are one of the mechanisms of resistance to aromatase inhibitors (AI). These mutations are common in metastatic breast cancer (MBC). In past reports, mutations in primary tumors were so rare that they were thought to occur de novo with AI therapy. Conversely, previous studies using droplet digital polymerase chain reaction (ddPCR) have suggested the existence of ESR1-mutant minor clones with low allele frequencies; however, no large-scale studies have been conducted. In this study, we attempted to detect ultra-rare ESR1 mutations in primary breast cancer tumors using locked nucleic acid (LNA)-clamp ddPCR, and 28 ESR1 mutations were found in 27 patients. Most of these mutations were minor clones with a variant allele frequency of <0.1% which may have been overlooked by conventional methods. LNA-clamp ddPCR can also be applied to detect other gene mutations, which would be very useful. ESR1 mutations in breast cancer are one of the mechanisms of resistance to aromatase inhibitors. These mutations are common in metastatic breast cancer; however, these are rare in primary breast cancer. However, these data have been analyzed mainly in formalin-fixed, paraffin-embedded tissue; thus, rare mutations that may be present in primary breast cancer may be overlooked. In this study, we developed a highly sensitive mutation detection method called locked nucleic acid (LNA)-clamp droplet digital PCR (ddPCR) and validated it. The mutation detection sensitivity was substantiated to 0.003%. Then, we used this method to analyze ESR1 mutations in fresh-frozen (FF) tissues of primary breast cancer. cDNA extracted from the FF tissues of 212 patients with primary breast cancers were measured. Twenty-eight ESR1 mutations were found in twenty-seven (12.7%) patients. Sixteen (7.5%) patients had Y537S mutations and twelve (5.7%) had D538G mutations. Two mutations with a variant allele frequency (VAF) of ≥0.1% and twenty-six mutations with a VAF of <0.1% were found. By using this LNA-clamp ddPCR, this study demonstrated the presence of minor clones with a VAF of <0.1% in primary breast cancer. [ABSTRACT FROM AUTHOR]

The utility of cell-free tumor deoxyribonucleic acid analysis is currently being evaluated in a wide range of clinical studies. The validity of cell-free tumor deoxyribonucleic acid analysis methods used for screening and detecting malignant diseases, monitoring the effectiveness of treatment and disease progression, and identifying potential relapse is tested. Molecular technologies used for cell-free tumor deoxyribonucleic acid analysis include targeted polymerase chain reaction assays and next-generation sequencing approaches along with newly introduced epigenetic analysis methods such as methylation-specific polymerase chain reaction. The aim of this review was to compare the methods, pitfalls, and advantages of tests developed for the analysis of cell-free tumor deoxyribonucleic acid in the diagnosis and treatment of pediatric solid tumors. For this purpose, the PubMed database was searched for articles published in the last 10 years, in English, that investigated a human cohort aged 0 to 18 years. A total of 272 references were analyzed. A total of 33 studies were included in the review. Cell-free tumor deoxyribonucleic acid analysis has emerged as a promising novel approach that could potentially bring a significant improvement in the field of pediatric oncology, but the implementation of cell-free tumor deoxyribonucleic acid in clinical practice is largely hindered by the lack of standardized methods for processing and analysis. [ABSTRACT FROM AUTHOR]

Simple Summary: Head and Neck Squamous Cell Carcinomas (HNSCCs) are cancers that originate from cells of the head and neck region, including the mouth, nose, and throat. The diversity of these cell types is also mirrored by the high number of different mutations that promote cancer development and progression. For the clinical management of this disease, it is important to identify biomarkers that allow early detection or predict relapse and resistance to therapy. A non-invasive way to monitor these markers over time are so called liquid biopsies, which mostly refers to the detection and analysis of tumor cells or cell-free DNA (cfDNA) in the blood of patients. This review summarizes our current understanding of HNSCC genetics and discusses how the detection of genetic variation in the cfDNA of HNSCC patients can be used to monitor disease and guide therapy. Tumors shed cell-free DNA (cfDNA) into the plasma. "Liquid biopsies" are a diagnostic test to analyze cfDNA in order to detect minimal residual cancer, profile the genomic tumor landscape, and monitor cancers non-invasively over time. This technique may be useful in patients with head and neck squamous cell carcinoma (HNSCC) due to genetic tumor heterogeneity and limitations in imaging sensitivity. However, there are technical challenges that need to be overcome for the widespread use of liquid biopsy in the clinical management of these patients. In this review, we discuss our current understanding of HNSCC genetics and the role of cfDNA genomic analyses as an emerging precision diagnostic tool. [ABSTRACT FROM AUTHOR]

Summary With its candybar form factor and low initial investment cost, the MinION brought affordable portable nucleic acid analysis within reach. However, translating the electrical signal it outputs into a sequence of bases still requires mid-tier computer hardware, which remains a caveat when aiming for deployment of many devices at once or usage in remote areas. For applications focusing on detection of a target sequence, such as infectious disease monitoring or species identification, the computational cost of analysis may be reduced by directly detecting the target sequence in the electrical signal instead. Here, we present baseLess, a computational tool that enables such target-detection-only analysis. BaseLess makes use of an array of small neural networks, each of which efficiently detects a fixed-size subsequence of the target sequence directly from the electrical signal. We show that baseLess can accurately determine the identity of reads between three closely related fish species and can classify sequences in mixtures of 20 bacterial species, on an inexpensive single-board computer. Availability and implementation baseLess and all code used in data preparation and validation are available on Github at https://github.com/cvdelannoy/baseLess , under an MIT license. Used validation data and scripts can be found at https://doi.org/10.4121/20261392 , under an MIT license. Supplementary information Supplementary data are available at Bioinformatics Advances online. [ABSTRACT FROM AUTHOR]

Simple Summary: In breast cancer management, neoadjuvant chemotherapy is well established as therapeutic choice for selected high-risk early or locally advanced breast cancer. However, besides there being few clinical genomic classifiers, there is no technology that can predict for certain whether breast cancer patients will benefit from neoadjuvant chemotherapy in terms of pathological complete response and disease-free survival. The analysis of miRNAs from biological fluids at the beginning of therapy is simple and may aid in the identification of patients who will receive the greatest benefit. On the other hand, monitoring circulating miRNA levels during treatment could allow the early identification of patients who will not benefit from it (avoiding unnecessary treatments and related side effects). Therefore, there is an urgent clinical need for non-invasive biomarkers in the neoadjuvant setting, and circulating miRNAs could theoretically meet this need, but there is still a long way to go until their use in clinical practice can be established. Recently, circulating microRNAs (miRNAs) have emerged as potential non-invasive biomarkers for breast cancer (BC) management. In the context of BC patients undergoing neoadjuvant chemotherapy (NAC), the possibility of obtaining repeated, non-invasive biological samples from patients before, during, and after treatment is incredibly convenient and provides the opportunity to investigate circulating miRNAs as diagnostic, predictive, and prognostic tools. The present review aims to summarize major findings in this setting, thus highlighting their potential applicability in daily clinical practice and their possible limitations. In all the contexts (diagnostic, predictive, and prognostic), circulating miR-21-5p and miR-34a-5p have emerged as the most promising non-invasive biomarkers for BC patients undergoing NAC. Specifically, their high baseline level could discriminate between BC patients and healthy controls. On the other hand, in predictive and prognostic investigations, low circulating miR-21-5p and miR-34a-5p levels may identify patients with better outcomes, in terms of both treatment response and invasive disease-free survival. However, the findings in this field have been very heterogeneous. Indeed, pre-analytical and analytical variables, as well as factors related to patients, may explain the inconsistency among different study results. Thus, further clinical trials, with more precise patient inclusion criteria and more standardized methodological approaches, are definitely needed to better define the potential role of these promising non-invasive biomarkers. [ABSTRACT FROM AUTHOR]

Normalized interventions were implemented in different cities in China to contain the outbreak of COVID-19 before December 2022. However, the differences in the intensity and timeliness of the implementations lead to differences in final size of the infections. Taking the outbreak of COVID-19 in three representative cities Xi'an, Zhengzhou and Yuzhou in January 2022, as examples, we develop a compartmental model to describe the spread of novel coronavirus and implementation of interventions to assess concretely the effectiveness of Chinese interventions and explore their impact on epidemic patterns. After applying reported human confirmed cases to verify the rationality of the model, we apply the model to speculate transmission trend and length of concealed period at the initial spread phase of the epidemic (they are estimated as 10.5, 7.8, 8.2 days, respectively), to estimate the range of basic reproduction number (2.9, 0.7, 1.6), and to define two indexes (transmission rate vt and controlled rate vc) to evaluate the overall effect of the interventions. It is shown that for Zhengzhou, vc is always more than vt with regular interventions, and Xi'an take 8 days to achieve vc > vt twice as long as Yuzhou, which can interpret the fact that the epidemic situation in Xi'an was more severe. By carrying out parameter values, it is concluded that in the early stage, strengthening the precision of close contact tracking and frequency of large-scale nucleic acid testing of non-quarantined population are the most effective on controlling the outbreaks and reducing final size. And, if the close contact tracking strategy is sufficiently implemented, at the late stage largescale nucleic acid testing of non-quarantined population is not essential. [ABSTRACT FROM AUTHOR]

Simple Summary: In recent years, the genetic landscape of hepatocellular carcinoma (HCC) has been explored, identifying TERT promoter, CTNNB1, and TP53 as the most frequent mutated genes. Therapies directed against specific targetable genomic alterations are the basis of personalized medicine and represent the cornerstone of systemic treatment for many malignancies, but are not yet available in HCC. Tools such as liquid biopsy and, in particular, circulating tumor DNA (ctDNA) may help in identifying biomarkers of response or resistance to treatment, and their role in HCC is an active field of research. In this review, we summarize the available evidence on the HCC genomic landscape and the potential role of ctDNA in clinical practice. Hepatocellular carcinoma (HCC) accounts for 90% of primary hepatic malignancies and is one of the major causes of cancer-related death. Over the last 15 years, the molecular landscape of HCC has been deciphered, with the identification of the main driver genes of liver carcinogenesis that belong to six major biological pathways, such as telomere maintenance, Wnt/b-catenin, P53/cell cycle regulation, oxidative stress, epigenetic modifiers, AKT/mTOR and MAP kinase. The combination of genetic and transcriptomic data composed various HCC subclasses strongly related to risk factors, pathological features and prognosis. However, translation into clinical practice is not achieved, mainly because the most frequently mutated genes are undruggable. Moreover, the results derived from the analysis of a single tissue sample may not adequately catch the intra- and intertumor heterogeneity. The analysis of circulating tumor DNA (ctDNA) is broadly developed in other types of cancer for early diagnosis, prognosis and monitoring under systemic treatment in order to identify primary and secondary mechanisms of resistance. The aim of this review is to describe recent data about the HCC molecular landscape and to discuss how ctDNA could be used in the future for HCC detection and management. [ABSTRACT FROM AUTHOR]

Background The diagnosis of infective endocarditis (IE) can be difficult, particularly if blood cultures fail to yield a pathogen. This study evaluates the potential utility of microbial cell-free DNA (mcfDNA) as a tool to identify the microbial etiology of IE. Methods Blood samples from patients with suspected IE were serially collected. mcfDNA was extracted from plasma and underwent next-generation sequencing. Reads were aligned against a library containing DNA sequences belonging to >1400 different pathogens. mcfDNA from organisms present above a statistical threshold were reported and quantified in molecules per milliliter (MPM). Additional mcfDNA was collected on each subject every 2–3 days for a total of 7 collections or until discharge. Results Of 30 enrolled patients with suspected IE, 23 had definite IE, 2 had possible IE, and IE was rejected in 5 patients by modified Duke Criteria. Only the 23 patients with definite IE were included for analysis. Both mcfDNA and blood cultures achieved a sensitivity of 87%. The median duration of positivity from antibiotic treatment initiation was estimated to be approximately 38.1 days for mcfDNA versus 3.7 days for blood culture (proportional odds, 2.952; P =.02771), using a semiparametric survival analysis. mcfDNA (log10) levels significantly declined (−0.3 MPM log10 units, 95% credible interval −0.45 to −0.14) after surgical source control was performed (pre- vs postprocedure, posterior probability >0.99). Conclusion mcfDNA accurately identifies the microbial etiology of IE. Sequential mcfDNA levels may ultimately help to individualize therapy by estimating a patient's burden of infection and response to treatment. [ABSTRACT FROM AUTHOR]

Background and Aim: Several methods have been employed to identify Coxiella burnetii isolates based on the specific C. burnetii QpH1 plasmid to distinguish the acute form from the chronic form of Q fever disease in humans and animals owing to the presence of unique gene sequences in this plasmid. Therefore, the present study aimed to investigate the panel of nucleic acid fragments resulting from the enzymatic cleavage in the QpH1 plasmid isolated from cow and buffalo milk by nested polymerase chain reaction (Nested-PCR). Materials and Methods: A total of 86 isolates of C. burnetii QpH1 plasmid, which was confirmed by the Nested-PCR method in 2018, were used to determine the RFLP panel of the QpH1 plasmid. Plasmids were first extracted with the kit and were then affected by the Hph1 restriction enzyme. Additionally, 4 nucleic acid samples were sent to Pishgam Company for sequencing with the IS1111 gene primer. Results: Based on the results of the PCR-RFLP test, all plasmid samples showed a similar two-fragment pattern under the influence of Hph1. The results of the nucleic acid sequencing of all 4 samples indicated that they had a C. burnetii type (Nine Mile RSA493 strain). Conclusion: RFLP patterns exhibited no difference on the C. burnetii QpH1 plasmid isolated from cow and buffalo milk. Hence, all isolates were genetically identical, and the infection in animals could originate from one C. burnetii strain (Nine Mile RSA493 strain). [ABSTRACT FROM AUTHOR]

Objective: Escherichia coli O157:H7 (E. coli O157:H7) can induce severe diseases in animals and humans that result in significant public health problems. Therefore, the development of rapid and visual detection methods to diagnose E. coli O157:H7 infections and monitor its prevalence is critical for the prevention and control purposes. Methods: A colorimetric loop-mediated isothermal amplification (LAMP) assay was utilized to detect E. coli O157:H7. A DNA extraction kit and Flinders Technology Associates (FTA) cards were used to extract nucleic acid in conjunction with colorimetric LAMP detection. Results: The method developed effectively distinguished E. coli O157:H7 from other pathogens with a detection limit of 25 CFU/mL in spiked stool samples. In addition, the nucleic acid of these samples was easily extracted and transported with an FTA card at room temperature. The entire detection process was completed within 35 min using simple constant-temperature equipment. Conclusion: The colorimetric LAMP method with FTA card-based nucleic acid purification was shown to rapidly detect E. coli O157:H7 with sensitivity and specificity. This visual method is expected to be widely used to control E. coli O157:H7 infections, particularly in resource-limited settings. [ABSTRACT FROM AUTHOR]

Objective: Solid-phase reversible immobilization (SPRI) beads are widely used for high-throughput sequencing library construction to purify and recover nucleic acids. This research was aimed at investigating the effects of SPRI bead ratio, incubation time, and elution time on nucleic acid recovery during fulllength 16S rDNA high-throughput sequencing library construction. Methods: The effects of different SPRI bead ratios, incubation times, and elution times were compared for three different initial sample amounts. An L9(3³) orthogonal experiment was designed to determine the optimal combination of these factors. Results: The incubation time of three factors including SPRI beads ratio, incubation time, and elution time had a statistically significant effect on the recovery rate for the initial sample amount of 1500 ng and 3000 ng. The orthogonal experiment results indicated that incubation time had the greatest impact among the three factors. Conclusion: Incubation time significantly influences recovery rate in full-length 16S rDNA high-throughput sequencing library construction. The use of 0.8× SPRI beads, 15 minutes of incubation, and 10 minutes of elution resulted in the highest recovery rate. SPRI beads offer a viable method for recovering fulllength 16S rDNA amplicons. [ABSTRACT FROM AUTHOR]