Search

The adaptability of microscale devices allows microtechnologies to be used for a wide range of applications. Biology and medicine are among those fields that, in recent decades, have applied microtechnologies to achieve new and improved functionality. However, despite their ability to achieve assay sensitivities that rival or exceed conventional standards, silicon-based microelectromechanical systems remain underutilised for biological and biomedical applications. Although microelectromechanical resonators and actuators do not always exhibit optimal performance in liquid due to electrical double layer formation and high damping, these issues have been solved with some innovative fabrication processes or alternative experimental approaches. This paper focuses on several examples of silicon-based resonating devices with a brief look at their fundamental sensing elements and key fabrication steps, as well as current and potential biological/biomedical applications.

Background: In non-metastatic colorectal cancer (CRC), we evaluated prospectively the pertinence of longitudinal detection and quantification of circulating tumor DNA (ctDNA) as a prognostic marker of recurrence., Method: The presence of ctDNA was assessed from plasma collected before and after surgery for 184 patients classified as stage II or III and at each visit during 3-4 years of follow-up. The ctDNA analysis was performed by droplet-based digital polymerase chain reaction, targeting mutation and methylation markers, blindly from the clinical outcomes. Multivariate analyses were adjusted on age, gender, stage, and adjuvant chemotherapy., Results: Before surgery, 27.5% of patients were positive for ctDNA detection. The rate of recurrence was 32.7% and 11.6% in patients with or without detectable ctDNA respectively (P = 0.001). Time to recurrence (TTR) was significantly shorter in patients with detectable ctDNA before (adjusted hazard ratio [HR] = 3.58, 95% confidence interval [CI] 1.71-7.47) or immediately after surgery (adjusted HR = 3.22, 95% CI 1.32-7.89). The TTR was significantly shorter in patients with detectable ctDNA during the early postoperative follow-up (1-6 months) (adjusted HR = 5, 95% CI 1.9-12.9). Beyond this period, ctDNA remained a prognostic marker with a median anticipated diagnosis of recurrence of 13.1 weeks (interquartile range 28 weeks) when compared to imaging follow-up. The rate of ctDNA+ might be underestimated knowing that consensus pre-analytical conditions were not described at initiation of the study., Conclusion: This prospective study confirms the relevance of ctDNA as a recurrence risk factor in stage II and III CRC before surgery and as a marker of minimal residual disease after surgery that may predict recurrence several months before imaging techniques., Competing Interests: Conflict of interest statement The authors of this work have conflicts of interest to declare: Valerie Taly: Honoraria from Raindance Technologies and Boerhinger Ingehleim; cofounder Emulseo; board Emulseo. Pierre Laurent-Puig: honoraria and board: Amgen, Merck-Serono, Boehringer Ingelheim, Sanofi, Roche, Lilly. Julien Taieb: Honoraria from Merck, Amgen, Roche, Pierre Fabre, MSD, Sanofi, Lilly, Servier, Astra-Zeneca. AZ: consulting and/or advisory boards: Roche, Merck Serono, Amgen, Sanofi, and Lilly. Olivier Bouché: honoraria and board: Merck, Amgen, Roche, Bayer, Servier, Pierre Fabre, MSD. Delphine Le Corre: Bio-Rad Employee. All remaining authors have declared no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Trypanosome parasites are infecting mammals in Sub-Saharan Africa and are transmitted between hosts through bites of the tsetse fly. The transmission from the insect vector to the mammal host causes a number of metabolic and physiological changes. A fraction of the population continuously adapt to the immune system of the host, indicating heterogeneity at the population level. Yet, the cell to cell variability in populations is mostly unknown. We develop here an analytical method for quantitative measurements at the single cell level based on encapsulation and cultivation of single-cell Trypanosoma brucei in emulsion droplets. We first show that mammalian stage trypanosomes survive for several hours to days in droplets, with an influence of droplet size on both survival and growth. We unravel various growth patterns within a population and find that droplet cultivation of trypanosomes results in 10-fold higher cell densities of the highest dividing cell variants compared to standard cultivation techniques. Some variants reach final cell titers in droplets closer to what is observed in nature than standard culture, of practical interest for cell production. Droplet microfluidics is therefore a promising tool for trypanosome cultivation and analysis with further potential for high-throughput single cell trypanosome analysis., (© 2021. The Author(s).)

With this personal account we show how the Integrative Chemistry, when combining the sol-gel process and concentrated emulsions, allows to trigger inorganic, hybrid or living materials when dedicated toward heterogeneous catalysis applications. In here we focus on 3D-macrocellular monolithic foams bearing hierarchical porosities and applications thereof toward heterogeneous catalysis where both activities and mass transport are enhanced. We thereby first depict the general background of emulsions, focusing on concentrated ones, acting as soft templates for the design of solid (HIPE) foams, HIPE being the acronym for High Internal Phase Emulsions while encompassing both sol-gel and polymer chemistry. Secondly we extend this approach toward the design of inorganic cellular materials labeled Si(HIPE) and hybrid organic-inorganic foams, labeled Organo-Si(HIPE), where heterogeneous catalysis applications are addressed considering acidic, metallic, enzymatic and bacterial-based modified Si-HIPE. Along, we will show how the fluid hydrodynamic within the macrocellular foams is offering advanced "out of the box" heterogeneous catalytic capabilities., (© 2018 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One way for phytoplankton to survive orthophosphate depletion is to utilize dissolved organic phosphorus by expressing alkaline phosphatase. The actual methods to assay alkaline phosphate activity-either in bulk or as a presence/absence of enzyme activity-fail to provide information on individual living cells. In this context, we develop a new microfluidic method to compartmentalize cells in 0.5 nL water-in-oil droplets and measure alkaline phosphatase activity at the single-cell level. We use enzyme-labeled fluorescence (ELF), which is based on the hydrolysis of ELF-P substrate, to monitor in real time and at the single-cell level both qualitative and quantitative information on cell physiology (i.e., localization and number of active enzyme sites and alkaline phosphatase kinetics). We assay the alkaline phosphatase activity of Tetraselmis sp. as a function of the dissolved inorganic phosphorus concentration and show that the time scale of the kinetics spans 1 order of magnitude. The advantages of subnanoliter-scale compartmentalization in droplet-based microfluidics provide a precise characterization of a population with single-cell resolution. Our results highlight the key role of cell physiology to efficiently access dissolved organic phosphorus.

We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies.

We demonstrate an accurate and sensitive quantification of mutated KRAS oncogene in genomic DNA, using droplet-based microfluidics and digital PCR.

We describe a droplet microfluidics method to screen for multiple mutations of a same oncogene in a single experiment using passive droplet fusion. Genomic DNA from H1573 cell-line was screened for the presence of the six common mutations of the KRAS oncogene as well as wild-type sequences with a detection efficiency of 98 %. Furthermore, the mutant allelic fraction of the cell-line was also assessed correctly showing that the technique is quantitative.

An emerging therapeutic strategy for cancer is to induce selective lethality in a tumor by exploiting interactions between its driving mutations and specific drug targets. Here we use a multi-species approach to develop a resource of synthetic lethal interactions relevant to cancer therapy. First, we screen in yeast ∼169,000 potential interactions among orthologs of human tumor suppressor genes (TSG) and genes encoding drug targets across multiple genotoxic environments. Guided by the strongest signal, we evaluate thousands of TSG-drug combinations in HeLa cells, resulting in networks of conserved synthetic lethal interactions. Analysis of these networks reveals that interaction stability across environments and shared gene function increase the likelihood of observing an interaction in human cancer cells. Using these rules, we prioritize ∼10(5) human TSG-drug combinations for future follow-up. We validate interactions based on cell and/or patient survival, including topoisomerases with RAD17 and checkpoint kinases with BLM., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Chemical inhibitors of the checkpoint kinases have shown promise in the treatment of cancer, yet their clinical utility may be limited by a lack of molecular biomarkers to identify specific patients most likely to respond to therapy. To this end, we screened 112 known tumor suppressor genes for synthetic lethal interactions with inhibitors of the CHEK1 and CHEK2 checkpoint kinases. We identified eight interactions, including the Replication Factor C (RFC)-related protein RAD17. Clonogenic assays in RAD17 knockdown cell lines identified a substantial shift in sensitivity to checkpoint kinase inhibition (3.5-fold) as compared to RAD17 wild-type. Additional evidence for this interaction was found in a large-scale functional shRNA screen of over 100 genotyped cancer cell lines, in which CHEK1/2 mutant cell lines were unexpectedly sensitive to RAD17 knockdown. This interaction was widely conserved, as we found that RAD17 interacts strongly with checkpoint kinases in the budding yeast Saccharomyces cerevisiae. In the setting of RAD17 knockdown, CHEK1/2 inhibition was found to be synergistic with inhibition of WEE1, another pharmacologically relevant checkpoint kinase. Accumulation of the DNA damage marker γH2AX following chemical inhibition or transient knockdown of CHEK1, CHEK2 or WEE1 was magnified by knockdown of RAD17. Taken together, our data suggest that CHEK1 or WEE1 inhibitors are likely to have greater clinical efficacy in tumors with RAD17 loss-of-function.

Purpose: KRAS mutations are predictive of nonresponse to anti-EGFR therapies in metastatic colorectal cancer (mCRC). However, only 50% of nonmutated patients benefit from them. KRAS-mutated subclonal populations nondetectable by conventional methods have been suggested as the cause of early progression. Molecular analysis technology with high sensitivity and precision is required to test this hypothesis., Experimental Design: From two cohorts of patients with mCRC, 136 KRAS, NRAS, and BRAF wild-type tumors with sufficient tumor material to perform highly sensitive picodroplet digital PCR (dPCR) and 41 KRAS-mutated tumors were selected. All these patients were treated by anti-EGFR therapy. dPCR was used for KRAS or BRAF mutation screening and compared with qPCR. Progression-free survival (PFS) and overall survival (OS) were analyzed according to the KRAS-mutated allele fraction., Results: In addition to the confirmation of the 41 patients with KRAS-mutated tumors, dPCR also identified KRAS mutations in 22 samples considered as KRAS wild-type by qPCR. The fraction of KRAS-mutated allele quantified by dPCR was inversely correlated with anti-EGFR therapy response rate (P < 0.001). In a Cox model, the fraction of KRAS-mutated allele was associated with worse PFS and OS. Patients with less than 1% of mutant KRAS allele have similar PFS and OS than those with wild-type KRAS tumors., Conclusions: This study suggests that patients with mCRC with KRAS-mutated subclones (at least those with a KRAS-mutated subclones fraction lower or equal to 1%) had a benefit from anti-EGFR therapies., (©2014 American Association for Cancer Research.)

Due to current challenges in the early detection, less than 40% of individuals diagnosed with hepatocellular carcinoma (HCC) are viable candidates for surgical intervention. Therefore, validating and launching of a novel precise diagnostic approach is essential for early diagnosis. Based on developing evidence using circulating tumor cells and their derivatives, circulating miRNAs, and extracellular vesicles (EVs), liquid biopsy may offer a reliable platform for the HCC's early diagnosis. Each liquid biopsy analyte may provide significant areas for diagnosis, prognostic assessment, and treatment monitoring of HCC patients depending on its kind, sensitivity, and specificity. The current review addresses potential clinical applications, current research, and future developments for liquid biopsy in HCC management. [ABSTRACT FROM AUTHOR]

Polymerase chain reaction based techniques have been widely used in laboratory settings. Several applications in oncology, virology or prenatal diagnosis require highly sensitive detection methods, which cannot be achieved with conventional techniques. Digital PCR (dPCR) was developed from the association of PCR and limiting dilution procedures. It is based on the compartmentalization of DNA molecules in small volumes. Controlling the size and the content of each compartment is crucial to obtain a high sensitivity with a single molecule resolution. Microfluidics offers promising tools to isolate DNA fragments such as microdroplets, microchambers or microwells with volumes ranging from few picoliters to nanoliters. The review provides an overview of recent developments of microfluidics dPCR platforms and how this technology can influence the management of cancer patients., (© 2015 médecine/sciences – Inserm.)

Background: Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample., Methods: We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR., Results: Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected., Conclusions: This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.

Background: Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA., Methods: Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues., Results: One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants., Conclusions: The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples., (© 2013 American Association for Clinical Chemistry.)

The efficient analysis and noninvasive detection of molecules such as DNA, mRNA, and miRNA for clinical diagnostics requires sensitive, high-throughput methods. By segregating individual sequences within separate compartments, digital procedures allow identification of very rare sequences. These procedures are based on the limiting dilution of biological samples in individual compartments such as droplets of a water-in-oil emulsion, and relies on the discrete counting of a given event, providing an absolute value and quantitative data. Coupled with microfluidic systems, digital procedures could become an essential diagnostic tool for the study of diseases as well as patient management., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment., (This journal is © The Royal Society of Chemistry 2011)

Background: Today, the prognostic tools available at the time of diagnosis in colorectal cancer (CRC) are limited. Better prognostic tools are a prerequisite for personalised treatment. This study aimed to investigate whether circulating tumour DNA (ctDNA) markers found in plasma before clinical diagnosis of CRC could contribute to the prediction of poor prognosis. Methods: This observational cohort study included patients diagnosed with CRC stage I-III within 24 months following participation in the Trøndelag Health Study (n = 85). Known methylated ctDNA biomarkers of CRC were analysed by PCR in plasma. Outcomes were overall survival (OS), recurrence-free survival (RFS) and poor prognosis (PP). Candidate clinical and methylated ctDNA predictors of the outcomes were identified by Cox regression analyses. Results: Methylated GRIA4 (HR 1.96 (1.06–3.63)), RARB (HR 9.48 (3.00–30.00)), SLC8A1 (HR 1.97 (1.03–3.77)), VIM (HR 2.95 (1.22–7.14)) and WNT5A (HR 5.83 (2.33–14.56)) were independent predictors of OS, methylated RARB (HR 9.67 (2.54–36.81)), SDC2 (HR 3.38 (1.07–10.66)), SLC8A1 (HR 2.93 (1.01–8.51)) and WNT5A (HR 6.95 (1.81–26.68)) were independent predictors of RFS and methylated RARB (HR 6.11 (1.69–22.18)), SDC2 (HR 2.79 (1.20–6.49)) and WNT5A (HR 5.57 (3.04–15.26)) were independent predictors of PP (p < 0.05). Conclusions: Prediagnostic ctDNA markers are promising contributors to predicting poor prognosis in CRC, potentially becoming one of the tools guiding more personalised treatment. [ABSTRACT FROM AUTHOR]

Directed evolution is a powerful technique for creating biomolecules such as proteins and nucleic acids with tailor-made properties for therapeutic and industrial applications by mimicking the natural evolution processes in the laboratory. Droplet microfluidics improved classical directed evolution by enabling time-consuming and laborious steps in this iterative process to be performed within monodispersed droplets in a highly controlled and automated manner. Droplet microfluidic chips can generate, manipulate, and sort individual droplets at kilohertz rates in a user-defined microchannel geometry, allowing new strategies for high-throughput screening and evolution of biomolecules. In this review, we discuss directed evolution studies in which droplet-based microfluidic systems were used to screen and improve the functional properties of biomolecules. We provide a systematic overview of basic on-chip fluidic operations, including reagent mixing by merging continuous fluid streams and droplet pairs, reagent addition by picoinjection, droplet generation, droplet incubation in delay lines, chambers and hydrodynamic traps, and droplet sorting techniques. Various microfluidic strategies for directed evolution using single and multiple emulsions and biomimetic materials (giant lipid vesicles, microgels, and microcapsules) are highlighted. Completely cell-free microfluidic-assisted in vitro compartmentalization methods that eliminate the need to clone DNA into cells after each round of mutagenesis are also presented. [ABSTRACT FROM AUTHOR]

The randomized FIRE-4.5 (AIO KRK0116) trial compared first-line therapy with FOLFOXIRI (folinic acid, fluorouracil, oxaliplatin, and irinotecan) plus either cetuximab or bevacizumab in B-Raf proto-oncogene, serine/threonine kinase (BRAF) V600E-mutant metastatic colorectal cancer (mCRC) patients. This study was accompanied by a prospective translational project analyzing cell-free circulating tumor DNA (ctDNA) in plasma to test whether ctDNA analysis may help to guide clinical treatment decision making. FIRE-4.5 included mCRC patients with BRAF V600E mutation detected by tissue-based analyses. Liquid biopsies (LBs) were collected at baseline (pre-treatment) and during therapy. Digital droplet PCR (ddPCR) technology was applied for determination of BRAF mutations and the in vitro diagnostics (IVD)-certified ONCOBEAM RAS procedure for analysis of RAS mutations. The BRAF V600E variants in ctDNA were analyzable in 66 patients at start of the therapy, at baseline. No BRAF V600E mutations were detected in 26% (17/66) of patients and was associated with a significantly longer progression-free survival (PFS: 13.2 vs 6.5 months; HR 0.47; P = 0.014) and overall survival (OS: 36.8 vs 13.2 months; HR 0.35; P = 0.02) as compared to ctDNA mutant patients. Patients with detectable BRAF mutations showed a clear superiority of FOLFOXIRI plus bevacizumab with regard to PFS (10.4 vs 5.7 months; HR 0.4; P = 0.009) and OS (16.6 vs 11.6 months; HR 0.5; P = 0.15), while this was not the case for BRAF wild-type patients. Follow-up LBs were obtained from 51 patients. Patients converting from BRAF V600E mutant to a BRAF V600 wild-type status (36%, N = 18) had a superior PFS (8.6 vs 2.3 months; P = 0.0002) and OS (17.4 vs 5.1 months; P < 0.0001) compared to patients with stable or increased mutational allele frequency (12%, N = 6). Those patients also achieved a significantly greater disease control rate (89% vs 20%; P = 0.008). In conclusion, LB evaluating ctDNA is informative and may help to guide treatment in patients with BRAF V600E-mutated mCRC., (© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Cell-free DNA (cfDNA), a burgeoning class of molecular biomarkers, has been extensively studied across a variety of biomedical fields. As a key component of liquid biopsy, cfDNA testing is gaining prominence in disease detection and management due to the convenience of sample collection and the abundant wealth of genetic information it provides. However, the broader clinical application of cfDNA is currently impeded by a lack of standardization in the preanalytical procedures for cfDNA analysis. A number of fundamental challenges, including the selection of appropriate preanalytical procedures, prevention of short cfDNA fragment loss, and the validation of various cfDNA measurement methods, remain unaddressed. These existing hurdles lead to difficulties in comparing results and ensuring repeatability, thereby undermining the reliability of cfDNA analysis in clinical settings. This review discusses the crucial preanalytical factors that influence cfDNA analysis outcomes, including sample collection, transportation, temporary storage, processing, extraction, quality control, and long-term storage. The review provides clarification on achievable consensus and offers an analysis of the current issues with the goal of standardizing preanalytical procedures for cfDNA analysis. [ABSTRACT FROM AUTHOR]

Digital PCR (dPCR) is a technique for absolute quantification of nucleic acid molecules. To develop a dPCR technique that enables more accurate nucleic acid detection and quantification, we established a novel dPCR apparatus known as centrifugal force real-time dPCR (crdPCR). This system is efficient than other systems with only 2.14% liquid loss by dispensing samples using centrifugal force. Moreover, we applied a technique for analyzing the real-time graph of the each micro-wells and distinguishing true/false positives using artificial intelligence to mitigate the rain, a persistent issue with dPCR. The limits of detection and quantification were 1.38 and 4.19 copies/μL, respectively, showing a two-fold higher sensitivity than that of other comparable devices. With the integration of this new technology, crdPCR will significantly contribute to research on next-generation PCR targeting absolute micro-analysis. [ABSTRACT FROM AUTHOR]

This study aims to experimentally evaluate the mechanical properties of concrete slabs reinforced with two types of fibers: metallic fibers (MF) and grids based on polypropylene fibers (PPG). The metallic fibers, sourced from machining waste of steel parts, are randomly distributed in the concrete, while the polypropylene fibers are arranged in grids. The investigation includes punching tests conducted on slabs measuring [25x50x7] cm³, as well as compression tests on cylinders with a diameter of Ø16 cm and a height of H32 cm. Mechanical resistance and tearing characteristics of the fibers were also assessed. The concrete's composition was determined using the experimental "Dreux-Gorisse" method. Five different metal fiber contents (MF) were studied (W=0.2%, W=0.4%, W=0.6%, W=0.8%, and W=1%), alongside a control concrete sample (BT) with no fibers (W=0%) having the same composition as the matrix. Two variants of grids based on small-mesh polypropylene fibers (PPG/SM) and large-mesh (PPG/LM) configurations were considered. The comparative analysis of the results highlights that, at a fiber content of W=0.8%, metallic fibers (MF) enhance both resistance and rigidity more effectively than polypropylene fibers in small meshes (PPG/SM) and large meshes (PPG/LM), thereby limiting the formation of puncturing cracks. [ABSTRACT FROM AUTHOR]

Introduction: Induced modification of plant gene expression is of both fundamental and applied importance. Cis-acting regulatory elements (CREs) are major determinants of the spatiotemporal strength of gene expression. Yet, there are few examples where induced genetic variation in predetermined CREs has been exploited to improve or investigate crop plants. Methods: The digital PCR based FIND-IT technology was applied to discover barley mutants with CRE variants in the promoter of the nutritional important barley grain phytase (PAPhy_a) gene. Results and discussion: Mutants with higher or lower gene expression and ultimately higher or lower mature grain phytase activity (MGPA), respectively, were discovered. Field trials and inositol phosphate profiling during germination showed that PAPhy_a does not influence agronomic performance under the trial conditions but it does shorten the lag time of phosphate mobilization during germination. Higher endogenous MGPA is an improvement of grain quality for feed use as it improves the phosphate bioavailability for monogastric animals. Moreover, as the targeted CRE motifs of the PAPhy_a promoter are shared with a range of seed expressed genes like key cereal and legume storage genes, the current results demonstrates a concept for modulating individual gene expression levels of a range of seed genes. [ABSTRACT FROM AUTHOR]

Thanks to recent and continuing technological innovations, modern microfluidic systems are increasingly offering researchers working across all fields of biotechnology exciting new possibilities (especially with respect to facilitating high throughput analysis, portability, and parallelization). The advantages offered by microfluidic devices—namely, the substantially lowered chemical and sample consumption they require, the increased energy and mass transfer they offer, and their comparatively small size—can potentially be leveraged in every sub-field of biotechnology. However, to date, most of the reported devices have been deployed in furtherance of healthcare, pharmaceutical, and/or industrial applications. In this review, we consider examples of microfluidic and miniaturized systems across biotechnology sub-fields. In this context, we point out the advantages of microfluidics for various applications and highlight the common features of devices and the potential for transferability to other application areas. This will provide incentives for increased collaboration between researchers from different disciplines in the field of biotechnology. [ABSTRACT FROM AUTHOR]

An optimization study is presented to enhance optical dielectrophoretic (ODEP) performance for effective manipulation of an oil-immersed droplet in the floating electrode optoelectronic tweezers (FEOET) device. This study focuses on understanding how the droplet's position and size, relative to light illumination, affect the maximum ODEP force. Numerical simulations identified the characteristic length (Lc) of the electric field as a pivotal factor, representing the location of peak field strength. Utilizing 3D finite element simulations, the ODEP force is calculated through the Maxwell stress tensor by integrating the electric field strength over the droplet's surface and then analyzed as a function of the droplet's position and size normalized to Lc. Our findings reveal that the optimal position is xopt= Lc+ r, (with r being the droplet radius), while the optimal droplet size is ropt = 5Lc, maximizing light-induced field perturbation around the droplet. Experimental validations involving the tracking of droplet dynamics corroborated these findings. Especially, a droplet sized at r = 5Lc demonstrated the greatest optical actuation by performing the longest travel distance of 13.5 mm with its highest moving speed of 6.15 mm/s, when it was initially positioned at x0= Lc+ r = 6Lc from the light's center. These results align well with our simulations, confirming the criticality of both the position (xopt) and size (ropt) for maximizing ODEP force. This study not only provides a deeper understanding of the position- and size-dependent parameters for effective droplet manipulation in FEOET systems, but also advances the development of low-cost, disposable, lab-on-a-chip (LOC) devices for multiplexed biological and biochemical analyses. [ABSTRACT FROM AUTHOR]

Introduction and Objective. Colorectal cancer (CRC), a malignant neoplasm of the gastrointestinal tract, affects the colon and rectum, its incidence is high, being the third most common neoplasm in men, with two million cases/year and survival <70%/5 years. The pathophysiology and progression of CRC are closely related to endoplasmic reticulum stress (ERE) and the unfolded or misfolded protein response (UPR). ERE can be triggered by various oxidative stress and inflammation factors with high UPR load followed by physicochemical and conformational interactions. The aim of the review is to present recent evidence on the relationships between endoplasmic reticulum stress, unfolded protein response and colorectal cancer. Review Methods. An expanded integrative review was carried out of scientific information from PubMed, LILACS and SciELO health databases. Articles containing key words were selected for abstract fast readings, followed by full text selections of works containing targeted subjects. From a total of 198 articles, 96 were selected (92% = 8 years) for inclusion in the review. Brief description of the state of knowledge. New developments in CRC research are presented within approaches to molecular pathophysiological pathways, a spectrum of therapeutic targets and suggestive diets with a view of intestinal microbiota and dysbiosis, considering progression stages and evidences correlating CRC to socio-environmental and innate or acquired genetic load. Putative CRC target compounds and drugs, such as Aspirin, Fucoidan, PERK inhibitor, antimicrobial and current natural antioxidants are briefly presented and discussed. Summary. Chaperone proteins may accumulate misfolded proteins in the endoplasmic reticulum, causing disruption of ERE proteostasis. While CRC progression is closely related to these signaling pathways, a better understanding is vital for new target-specific anticarcinogenic molecules. [ABSTRACT FROM AUTHOR]

There are many instances of hollow-structure morphogenesis in the development of tissues. Thus, the fabrication of hollow structures in a simple, high-throughput and homogeneous manner with proper natural biomaterial combination is valuable for developmental studies and tissue engineering, while it is a significant challenge in biofabrication field. We present a novel method for the fabrication of a hollow cell module using a coaxial co-flow capillary microfluidic device. Sacrificial gelatin laden with cells in the inner layer and GelMa in the outer layer are used via a coaxial co-flow capillary microfluidic device to produce homogenous micro-beads. The overall and core sizes of core–shell microbeads were well controlled. When using human vein vascular endothelial cells to demonstrate how cells line the inner surface of core–shell beads, as the core liquifies, a hollow cell ball with asymmetric features is fabricated. After release from the GelMa shell, individual cell balls are obtained and deformed cell balls can self-recover. This platform paves way for complex hollow tissue modeling in vitro , and further modulation of matrix stiffness, curvature and biochemical composition to mimic in vivo microenvironments. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei is the causative agent of African trypanosomiasis and is transmitted by the tsetse fly (Glossina spp.). All stages of this extracellular parasite possess a single flagellum that is attached to the cell body and confers a high degree of motility. While several stages are amenable to culture in vitro, longitudinal high-resolution imaging of free-swimming parasites has been challenging, mostly due to the rapid flagellar beating that constantly twists the cell body. Here, using microfabrication, we generated various microfluidic devices with traps of different geometrical properties. Investigation of trap topology allowed us to define the one most suitable for single T. brucei confinement within the field of view of an inverted microscope while allowing the parasite to remain motile. Chips populated with V-shaped traps allowed us to investigate various phenomena in cultured procyclic stage wild-type parasites, and to compare them with parasites whose motility was altered upon knockdown of a paraflagellar rod component. Among the properties that we investigated were trap invasion, parasite motility, and the visualization of organelles labelled with fluorescent dyes. We envisage that this tool we have named "Tryp-Chip" will be a useful tool for the scientific community, as it could allow high-throughput, high-temporal and high-spatial resolution imaging of free-swimming T. brucei parasites. [ABSTRACT FROM AUTHOR]

The microfluidic droplet polymerase chain reaction (PCR), which enables simultaneous DNA amplification in numerous droplets, has led to the discovery of various applications that were previously deemed unattainable. Decades ago, it was demonstrated that the temperature holding periods at the denaturation and annealing stages in thermal cycles for PCR amplification could be essentially eliminated if a rapid change of temperature for an entire PCR mixture was achieved. Microfluidic devices facilitating the application of such fast thermocycling protocols have significantly reduced the time required for PCR. However, in microfluidic droplet PCR, ensuring successful amplification from single molecules within droplets has limited studies on accelerating assays through fast thermocycling. Our developed microfluidic cartridge, distinguished for its convenience in executing single-molecule droplet PCR with common laboratory equipment, features droplets positioned on a thin glass slide. We hypothesized that applying fast thermocycling to this cartridge would achieve single-molecule droplet PCR amplification. Indeed, the application of this fast protocol demonstrated successful amplification in just 22 min for 30 cycles (40 s/cycle). This breakthrough is noteworthy for its potential to expedite microfluidic droplet PCR assays, ensuring efficient single-molecule amplification within a remarkably short timeframe. [ABSTRACT FROM AUTHOR]

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. In this study, we report an integrated microfluidic chip designed for the immunodetection of prostate‐specific antigens (PSAs). The microfluidic chip is based on the three‐dimensional structure of quartz capillaries. The outlet channel extends to 1.8 cm, effectively facilitating the generation of uniform droplets ranging in size from 3 to 50 μm. Furthermore, we successfully immobilized the captured antibodies onto the surface of magnetic beads using an activator, and we constructed an immunosandwich complex by employing biotinylated antibodies. A key feature of this microfluidic chip is its integration of microfluidic droplet technology advantages, such as high‐throughput parallelism, enzymatic signal amplification, and small droplet size. This integration results in an exceptionally sensitive PSA detection capability, with the detection limit reduced to 7.00 ± 0.62 pg/mL. [ABSTRACT FROM AUTHOR]

Background/Aim: Circulating tumor DNA (ctDNA), which is shed from cancer cells into the bloodstream, offers a potential minimally invasive approach for cancer diagnosis and monitoring. This research aimed to assess the preoperative ctDNA levels in ovarian tumors patients' plasma and establish correlations with clinicopathological parameters and patient prognosis. Patients and Methods: Tumor DNA was extracted from ovarian tumor tissue from 41 patients. Targeted sequencing using a panel of 127 genes recurrently mutated in cancer was performed to identify candidate somatic mutations in the tumor DNA. SAGAsafe digital PCR (dPCR) assays targeting the candidate mutations were used to measure ctDNA levels in patient plasma samples, obtained prior to surgery, to evaluate ctDNA levels in terms of mutant copy number/ml and variant allele frequency. Results: Somatic mutations were found in 24 tumor samples, 17 of which were from ovarian cancer patients. The most frequently mutated gene was TP53. Preoperative plasma ctDNA levels were detected in 14 of the 24 patients. With higher stage, plasma ctDNA mutant concentration increased (p for trend <0.001). The overall survival of cancer patients with more than 10 ctDNA mutant copies/ml in plasma was significantly worse (p=0.008). Conclusion: Pre-operative ctDNA measurement in ovarian cancer patients' plasma holds promise as a predictive biomarker for tumor staging and prognosis. [ABSTRACT FROM AUTHOR]

The Neo RAS phenomenon is defined as the conversion of tumor RAS status from mutant-type (MT) to wild-type (WT) after systemic chemotherapy in metastatic colorectal cancer (mCRC). Cetuximab, an anti-epidermal growth factor receptor (EGFR) antibody, is effective in patients with RAS WT mCRC but ineffective in those with RAS MT mCRC; however, its outcome in patients with Neo RAS WT mCRC is unclear. Herein, we report two cases of Neo RAS WT mCRC that responded clinically to anti-EGFR treatment. The first was a 40-year-old man with synchronous peritoneal metastatic rectosigmoid cancer. The first RAS testing on tumor tissue revealed a KRAS G12C mutation, which was converted to RAS WT after two lines of chemotherapy, as assessed by liquid biopsy. After initiating irinotecan plus cetuximab treatment, a computed tomography (CT) scan revealed that malignant ascites had resolved. The treatment was discontinued after 4 months because of disease progression. The second was a 68-year-old male patient with synchronous liver metastasis from sigmoid colon cancer. The KRAS G12D mutation, initially detected in tumor tissue, was not detected by liquid biopsy after six lines of chemotherapy. Cetuximab monotherapy was initiated, and the liver metastases shrank significantly. The patient continued cetuximab monotherapy for 8 months without disease progression. Our cases demonstrate the efficacy of anti-EGFR therapy for Neo RAS WT mCRC and highlight the importance of capturing the gene mutation profile throughout the clinical course for optimal treatment selection. [ABSTRACT FROM AUTHOR]

Despite numerous efforts, the therapeutic advancement for neuroblastoma and other cancer treatments is still ongoing due to multiple challenges, such as the increasing prevalence of cancers and therapy resistance development in tumors. To overcome such obstacles, drug combinations are one of the promising applications. However, identifying and implementing effective drug combinations are critical for achieving favorable treatment outcomes. Given the enormous possibilities of combinations, a rational approach is required to predict the impact of drug combinations. Thus, CRISPR-Cas-based and other approaches, such as high-throughput pharmacological and genetic screening approaches, have been used to identify possible drug combinations. In particular, the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful tool that enables us to efficiently identify possible drug combinations that can improve treatment outcomes by reducing the total search space. In this review, we discuss the rational approaches to identifying, examining, and predicting drug combinations and their impact. [ABSTRACT FROM AUTHOR]

Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2–3 times larger than conventional messenger RNA. However, quality assessment of long RNA transcripts is challenging using standard techniques. Here, we utilized a multiplex droplet digital polymerase chain reaction (ddPCR) assay to assess the quality of saRNA produced from an in vitro transcription reaction and the replication kinetics in human cell lines. Using the one-step reverse transcription ddPCR, we show that an in vitro transcription generates 50–60% full-length saRNA transcripts. However, we note that the two-step reverse transcription ddPCR assay results in a 20% decrease from results obtained using the one-step and confirmed using capillary gel electrophoresis. Additionally, we provided three formulas that differ in the level of stringency and assumptions made to calculate the fraction of intact saRNA. Using ddPCR, we also showed that subgenomic transcripts of saRNA were 19-to-108-fold higher than genomic transcripts at different hours post-transfection of mammalian cells in copies. Therefore, we demonstrate that multiplex ddPCR is well suited for quality assessment of long RNA and replication kinetics of saRNA based on absolute quantification. [ABSTRACT FROM AUTHOR]

Pancreatic cancer is one of the most challenging cancers due to its high mortality rates. Considering the late diagnosis and the limited survival benefit with current treatment options, it becomes imperative to optimize early detection, prognosis and prediction of treatment response. To address these challenges, significant research efforts have been undertaken in recent years to develop liquid-biopsy-based biomarkers for pancreatic cancer. In particular, an increasing number of studies point to cell-free DNA (cfDNA) methylation analysis as a promising non-invasive approach for the discovery and validation of epigenetic biomarkers with diagnostic or prognostic potential. In this review we provide an update on recent advancements in the field of cfDNA methylation analysis in pancreatic cancer. We discuss the relevance of DNA methylation in the context of pancreatic cancer, recent cfDNA methylation research, its clinical utility, and future directions for integrating cfDNA methylation analysis into routine clinical practice. [ABSTRACT FROM AUTHOR]

Abstract—Digital PCR (dPCR) is a nucleic acid quantification method that is widely used in genetic analysis. One of the most significant advantages of dPCR over other methods is the possibility of absolute quantitative determination of genetic material without construction of calibration curves, which allows one to detect even single molecules of nucleic acids, and, hence, provides early diagnosis of diseases. One specific characteristic of dPCR is the detection of the analyzed biological object in each microreaction, followed by the presentation of the analysis results in a binary system, thereby giving the method its name. The key aspects of developing the dPCR method, i.e., from the first devices based on microfluidic chip technology to modern systems capable of measuring a target at a concentration of up to 1 in 100 000 copies are shown in the current work. We analyzed the data on the detection of various pathogens using dPCR, as well as summarizing various study results demonstrating the innovativeness of this method. Both the possibilities of multiplex dPCR analysis and its potential in clinical practice are presented. This review also addresses the issue of the role of dPCR in the development of noninvasive methods for analysis of oncological diseases. Possible ways of developing dPCR technology were emphasized, including its use as a "point-of-care" system. [ABSTRACT FROM AUTHOR]