FAM134B oligomerization drives endoplasmic reticulum membrane scission for ER-phagy.

Degradation of endoplasmic reticulum (ER) by selective autophagy (ER-phagy) is crucial for ER homeostasis. However, it remains unclear how ER scission is regulated for subsequent autophagosomal sequestration and lysosomal degradation. Here, we show that oligomerization of ER-phagy receptor FAM134B (also referred to as reticulophagy regulator 1 or RETREG1) through its reticulon-homology domain is required for membrane fragmentation in vitro and ER-phagy in vivo. Under ER-stress conditions, activated CAMK2B phosphorylates the reticulon-homology domain of FAM134B, which enhances FAM134B oligomerization and activity in membrane fragmentation to accommodate high demand for ER-phagy. Unexpectedly, FAM134B G216R, a variant derived from a type II hereditary sensory and autonomic neuropathy (HSAN) patient, exhibits gain-of-function defects, such as hyperactive self-association and membrane scission, which results in excessive ER-phagy and sensory neuron death. Therefore, this study reveals a mechanism of ER membrane fragmentation in ER-phagy, along with a signaling pathway in regulating ER turnover, and suggests a potential implication of excessive selective autophagy in human diseases.

Cyclic Multiple Primer Generation Rolling Circle Amplification Assisted Capillary Electrophoresis for Simultaneous and Ultrasensitive Detection of Multiple Pathogenic Bacteria.

Efficient, accurate, and economical detection of pathogenic bacteria is crucial in ensuring food safety and preventing foodborne illnesses. How to fulfill the highly sensitive and simultaneous detection of multiple trace pathogenic bacteria is a big challenge. In this work, capillary electrophoresis coupled with a cyclic multiple primer generation rolling circle amplification (cyclic MPG-RCA) was studied for highly sensitive and simultaneous detection of three kinds of pathogenic bacteria. The cyclic MPG-RCA was based on a carefully designed clover-shaped DNA probe, in which three "leaves" corresponded to three types of aimed pathogenic bacteria: Shigella dysenteriae (S. dysenteriae), Salmonella enterica subsp. enterica serovar Typhi (S. Typhi), and Vibrio parahaemolyticus (V. parahaemolyticus). Under the optimal experimental conditions, the limits of detection (S/N = 3) of this method for bacterial target DNA were 11.4 amol·L-1 (S. dysenteriae), 4.88 amol·L-1 (S. Typhi), and 14.9 amol·L-1 (V. parahaemolyticus), and the conversion concentrations for the target bacteria were 10 colony-forming units (CFU)·mL-1 (S. dysenteriae), 3 CFU·mL-1 (S. Typhi), and 12 CFU·mL-1 (V. parahaemolyticus). This method had been applied to the detection of tap water samples with good results, which proved that it could be used as an effective tool for trace pathogenic bacteria monitoring in foods, environments, and medicines.

Bibliometric and Visual Analysis of the Research Status and Knowledge Structure of Assisted Reproductive Therapy for Patients with Premature Ovarian Insufficiency.

Objective: A large proportion of patients undergoing assisted reproductive therapy (ART) suffer from premature ovarian insufficiency (POI). The knowledge structure, research hotspots, and research trends related to ART for patients with POI are still unclear and have not been systematically summarized. We aimed to analyze the research status of ART for patients with POI and deeply explore its knowledge structure and research trends. Our findings may provide treatment recommendations for clinicians and guidance for researchers in further research. Methods: The PubMed database for publications on ART for patients with POI was searched. The Bibliographic Item Co-occurrence Matrix Builder (BICOMB) obtained the Co-word matrix and co-occurrence matrix. The H-index method was used to extract high-frequency main Medical Subject Headings (MeSH) terms/subheadings. Then we used software such as graphical clustering toolkit (gCluto), Microsoft Excel, Ucinet and NetDraw to carry out the biclustering analysis, strategic diagram analysis and social network analysis of the major MeSH terms/subheadings. Results: The high-frequency major MeSH terms/subheadings were analyzed by biclustering, strategic diagram, and social network analyses. A total of 431 articles from 1983 to 2023 were retrieved. Analysis showed that a total of 176 journals published relevant papers, including FERTILITY AND STERILITY, ranking first. In addition, we extracted 20 high-frequency major MeSH terms/subheadings. We grouped them into five categories: cryopreservation of oocyte and ovarian tissue, oocyte donation, in vitro activation (IVA) of primordial follicles, overview of therapy for patients with POI, therapy of iatrogenic POI. Within these five categories, there were 4, 4, 3, 4, and 5 major MeSH terms/subheadings, respectively. The major MeSH terms/subheadings were evenly distributed, and no particular group had a particular central tendency. Conclusion: The therapy of Iatrogenic POI is in the core position of research and is becoming increasingly mature. Oocyte donation and IVA of primordial follicles are the trends of future research. This study is helpful to understand the current research status, knowledge structure, and research trends of ART for patients with POI, and provide reference for improving ART for patients with POI in the future. Our study may guide clinicians to apply more established research to treat patients, which may lead to better treatment outcomes for patients. At the same time, we also suggest that researchers can conduct research in the field of future research trends, which may lead to greater research results.

Quantitative proteomics reveals PPAR signaling pathway regulates the cardiomyocyte activity of neonatal mouse heart.

Cardiovascular diseases (CVDs) are among the most morbid and deadly types of diseases worldwide, while the existing therapeutic approaches all have their limitations. Mouse heart undergoes a very complex postnatal developmental process, including the 1-week window in which cardiomyocytes (CMs) maintain relatively high cell activity. The underlying mechanism provides an attractive direction for CVDs treatment. Herein, we collected ventricular tissues from mice of different ages from E18.5D to P8W and performed iTRAQ-based quantitative proteomics to characterize the atlas of cardiac development. A total of 3422 proteins were quantified at all selected time points, revealing critical proteomic changes related to cardiac developmental events such as the metabolic transition from glycolysis to beta-oxidation. A cluster of significantly dysregulated proteins containing proteins that have already been reported to be associated with cardiac regeneration (Erbb2, Agrin, and Hmgb) was identified. Meanwhile, the peroxisome proliferator-activated receptor (PPAR) signaling pathway (Cpt1α, Hmgcs2, Plin2, and Fabp4) was also found specifically enriched. We further revealed that bezafibrate, a pan-activator of PPAR signaling pathway markedly enhanced H9C2 cardiomyocyte activity via enhancing Cpt1α expression. This work provides new hint that activation of PPAR signaling pathway could potentially be a therapeutic strategy for the treatment of CVDs.

Quantitative Proteomics Reveals Down-Regulated Glycolysis/Gluconeogenesis in the Large-Duct Type Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a lethal hepatobiliary malignancy that arises from the epithelial cells of the intrahepatic bile ducts, accounting for approximately 10% of cholangiocarcinoma (CCA). According to the 2019 World Health Organization (WHO) classification of tumors of the digestive system, iCCA is divided into small-duct type (SD-type) and large-duct type (LD-type). However, it remains unknown which molecular events contribute to the disparity. To explore the proteomic characteristics of iCCA, we used an isobaric tag for relative and absolute quantitation (iTRAQ) based quantitative proteomics strategy to investigate stably dysregulated proteins in the SD-type and LD-type of iCCA tissues. Importantly, we found three glycolysis/gluconeogenesis-related enzymes, triosephosphate isomerize 1 (TPI1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and phosphoglycerate kinase 1 (PGK1), were significantly down-regulated in the LD-type iCCA, which were further confirmed by immunohistochemistry using tissue microarray. Moreover, we demonstrated that the knockdown of these three candidate proteins by siRNAs notably increased the ability of proliferation in two CCA cell lines (HuH28 and RBE), suggesting that effective down-regulation of the glycolysis/gluconeogenesis pathway might be an underlying novel mechanism contributing to the LD-type iCCA.

Quantitative proteomics reveals arsenic attenuates stem-loop binding protein stability via a chaperone complex containing heat shock proteins and ERp44.

Arsenic pollution impacts health of millions of people in the world. Inorganic arsenic is a carcinogenic agent in skin and lung cancers. The stem-loop binding protein (SLBP) binds to the stem-loop of the canonical histone mRNA and regulates its metabolism during cell cycle. Our previous work has shown arsenic induces ubiquitin-proteasome dependent degradation of SLBP and contributes to lung cancer. In this study, we established the first comprehensive SLBP interaction network by affinity purification-mass spectrometry (AP-MS) analysis, and further demonstrated arsenic enhanced the association between SLBP and a crucial chaperone complex containing heat shock proteins (HSPs) and ERp44. Strikingly, knockdown of these proteins markedly rescued the protein level of SLBP under arsenic exposure conditions, and abolished the increasing migration capacity of BEAS-2B cells induced by arsenic. Taken together, our study provides a potential new mechanism that a chaperone complex containing HSPs and ERp44 attenuates the stability of SLBP under both normal and arsenic exposure conditions, which could be essential for arsenic-induced high cell migration.

Global Phosphoproteomic Analysis Reveals Significant Metabolic Reprogramming in the Termination of Liver Regeneration in Mice.

Phosphorylation is crucial in regulating various biological processes. However, comprehensive phosphoproteomic profiling in the termination of liver regeneration (LR) is still missing. Here, we used Tandem Mass Tag (TMT) labeling coupled with phosphopeptide enrichment and two-dimensional (2D) liquid chromatography-mass spectrometry (LC-MS)/MS analysis to establish a global phosphoproteomic map in the liver of mice at day 5 after partial hepatectomy (PH). Altogether, 9731 phosphosites from 3443 proteins were identified and 7802 phosphosites from 2980 proteins were quantified. Motif analysis of the identified phosphosites revealed a diverse array of consensus sequences, suggesting that multiple kinase families including ERK/MAPK, PKA/PKC, CaMK-II, CKII, and CDK may be involved in the termination of LR. Functional clustering analysis of proteins with dysregulated phosphosites showed that they mainly participate in metabolic pathways, DNA replication, and tight junction. More importantly, the deletion of PP2Acα in the liver remarkably changes the overall phosphorylation profile, indicating its critical role in regulating the termination of LR. Finally, several differentially phosphorylated sites were validated by co-immunoprecipitation and Western blot. Taken together, our data unravel the first comprehensive phosphoproteomic map in the termination of LR in mice, which greatly expands our knowledge in the complicated regulation of this process and provides new directions for the treatment of liver cancer using liver resection.

IKK-Mediated Regulation of the COP9 Signalosome via Phosphorylation of CSN5.

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex, which controls protein degradation through deneddylation and inactivation of cullin-RING ubiquitin E3 ligases (CRLs). Recently, the CSN complex has been linked to the NF-κB signaling pathway due to its association with the IKK complex. However, how the CSN complex is regulated in this signaling pathway remains unclear. Here, we have carried out biochemical experiments and confirmed the interaction between the CSN and IKK complexes. In addition, we have determined that overexpression of IKKα or IKKß leads to enhanced phosphorylation of CSN5, the catalytic subunit for CSN deneddylase activity. Mutational analyses have revealed that phosphorylation at serine 201 and threonine 205 of CSN5 impairs CSN-mediated deneddylation activity in vitro. Interestingly, TNF-α treatment not only enhances the interaction between CSN and IKK but also induces an IKK-dependent phosphorylation of CSN5 at serine 201, linking CSN to TNF-α signaling through IKK. Moreover, TNF-α treatment affects the CSN interaction network globally, especially the associations of CSN with the proteasome complex, eukaryotic translation initiation factor complex, and CRL components. Collectively, our results provide new insights into IKK-mediated regulation of CSN associated with the NF-κB signaling pathway.

A cytosolic chaperone complex controls folding and degradation of type III CD38.

Cluster of differentiation 38 (CD38) is the best-studied enzyme catalyzing the synthesis of the Ca2+ messenger cyclic ADP-ribose. It is a single-pass transmembrane protein, but possesses dual orientations. We have documented the natural existence of type III CD38 in cells and shown that it is regulated by a cytosolic activator, calcium- and integrin-binding 1 (CIB1). However, how type III CD38 can be folded correctly in the reductive cytosol has not been addressed. Using the yeast two-hybrid technique with CD38's catalytic domain (sCD38) as bait, here we identified a chaperone, Hsp70-interacting protein (Hip), that specifically interacts with both the type III CD38 and sCD38. Immunoprecipitation coupled with MS identified a chaperone complex associated specifically with sCD38. Pharmacological and siRNA-mediated knockdown of Hsp90 chaperones decreased the expression levels of both sCD38 and type III CD38, suggesting that these chaperones facilitate their folding. Moreover, knockdown of Hsc70 or DNAJA2 increased the levels of both CD38 types, consistent with the roles of these proteins in mediating CD38 degradation. Notably, Hip knockdown decreased type III CD38 substantially, but only marginally affected sCD38, indicating that Hip was selective for the former. More remarkably, DNAJA1 knockdown decreased sCD38 but increased type III CD38 levels. Mechanistically, we show that Hsc70 mediates lysosomal degradation of type III CD38, requiring the lysosomal receptor Lamp2A and the C19-motif in the C terminus of CD38. Our results indicate that folding and degradation of type III CD38 is effectively controlled in cells, providing further strong support of its physiological relevance.

The transferrin receptor CD71 regulates type II CD38, revealing tight topological compartmentalization of intracellular cyclic ADP-ribose production.

The CD38 molecule (CD38) catalyzes biogenesis of the calcium-mobilizing messenger cyclic ADP-ribose (cADPR). CD38 has dual membrane orientations, and type III CD38, with its catalytic domain facing the cytosol, has low abundance but is efficient in cyclizing cytosolic NAD to produce cADPR. The role of cell surface type II CD38 in cellular cADPR production is unknown. Here we modulated type II CD38 expression and assessed the effects of this modulation on cADPR levels. We developed a photoactivatable cross-linking probe based on a CD38 nanobody, and, combining it with MS analysis, we discovered that cell surface CD38 interacts with CD71. CD71 knockdown increased CD38 levels, and CD38 knockout reciprocally increased CD71, and both could be cocapped and coimmunoprecipitated. We constructed a chimera comprising the N-terminal segment of CD71 and a CD38 nanobody to mimic CD71's ligand property. Overexpression of this chimera induced a dramatically large decrease in CD38 via lysosomes. Remarkably, cellular cADPR levels did not decrease correspondingly. Bafilomycin-mediated blockade of lysosomal degradation greatly elevated active type II CD38 by trapping it in the lysosomes but also did not increase cADPR levels. Retention of type II CD38 in the endoplasmic reticulum (ER) by expressing an ER construct that prevented its transport to the cell surface likewise did not change cADPR levels. These results provide first and direct evidence that cADPR biogenesis occurs in the cytosol and is catalyzed mainly by type III CD38 and that type II CD38, compartmentalized in the ER or lysosomes or on the cell surface, contributes only minimally to cADPR biogenesis.

PP2Acα inhibits PFKFB2-induced glycolysis to promote termination of liver regeneration.

The mechanisms underlying the initiation and proliferation of liver regeneration (LR) has been extensively studied using the partial hepatectomy (PHx) model, while little is known about the termination of LR. PP2Acα (protein phosphatase 2 A catalytic subunit α isoform) is the catalytic subunit of protein phosphatase 2 A (PP2A), accounting for most of intracellular serine/threonine phosphatase activity. We have previously observed that termination of LR delayed in PP2Acα liver-specific knockout (LKO) mice after PHx. In our study, we used phospho explorer antibody array analysis to screen the potential phosphorylation targets of PP2Acα, and PP2Acα had a great influence on the hepatic phosphoproteomic signaling in the termination of LR after PHx. We then tested the phosphorylation changes and metabolic function of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB2), an isoform of the key glycolytic enzyme PFKFB, which was significantly regulated by PP2Acα knockout. PP2Acα knockout enhanced glycolysis in vivo and in vitro, while adenoviral-mediated RNAi of PFKFB2 reversed the extension of postoperative liver regeneration in KO mice along with the downregulation of glycolysis. Therefore, we demonstrated that PP2Acα liver-specific knockout regulated the hepatocytes glycolysis via activating PFKFB2, thus enhancing liver regeneration during the termination stage.

Atom-Ratio-Conducted Tailoring of PdAu Bimetallic Nanocrystals with Distinctive Shapes and Dimensions for Boosting the ORR Performance.

Systematically manipulating the shape, dimension, and surface structure of PdAu nanocrystals is an active subject because it offers a powerful means to regulate and investigate their structure-activity relationship. Meanwhile, it is still urgent to reduce the use of two-dimensional precious-metal-based nanomaterials. This work demonstrates that PdAu nanocrystals with a variety of shapes/dimensions, including 1D anisotropic nanowires, 2D porous nanosheets, and 3D penetrative nanoflowers, can be systematically synthesized by simply adjusting the atomic ratio or the reaction time in the same protocol. The resultant PdAu nanocrystals with distinctive shapes, but the same building blocks triumphantly avoid the effects of facet and surface properties; this represents an ideal platform for directly comparing the oxygen reduction reaction (ORR) activity. 2D porous PdAu nanosheets demonstrate superior ORR performance (Eonset = 1.040 V, E1/2 = 0.932 V) compared with other-dimension-based samples and commercial Pd black; this is attributed to the abundant surface atoms and omni-directional mass-transfer channels. This work not only paves the way for systematically measuring a series of distinctive PdAu nanocrystals as non-Pt electrocatalysts, but also sheds light on the study of structures/dimensions in tuning the catalytic properties of bimetallic nanocrystals.

Significant Down-Regulation of Urea Cycle Generates Clinically Relevant Proteomic Signature in Hepatocellular Carcinoma Patients with Macrovascular Invasion.

Vascular invasion is considered as the critical risk factor of hepatocellular carcinoma (HCC). To reveal the molecular mechanisms underlying macrovascular invasion (MaVI) in HCC, we performed an iTRAQ based proteomic study to identify notably dysregulated proteins from eight HCC patients with differential vascular invasion and further confirmed them in the other 53 HCC patients. Forty-seven proteins were found significantly down-regulated in HCC with MaVI. More importantly, 30 of them were not changed in HCC without MaVI. Gene ontology analysis of these 47 proteins shows the top three enriched biological processes are urea cycle, gluconeogenesis, and arginine biosynthetic process. We validated nine remarkably dysregulated candidates in HCC patients with MaVI by Western blot including eight down-regulated proteins (CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin A6, and CES1) and one up-regulated protein (CKAP4). Furthermore, dysregulation of CPS1, ASL, and ARG1, key enzymes involved in urea cycle, together with Annexin A6 and CES1, major proteins in regulating cholesterol homeostasis and fatty acid ester metabolism, was verified using immunohistochemical staining. The significant down-regulation of urea cycle generates clinically relevant proteomic signature in HCC patients with macrovascular invasion, which may provide possible insights into the molecular mechanisms of metastasis and new therapeutic targets of HCC.

Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.

Patients with obesity have a high prevalence of non-alcoholic fatty liver disease (NAFLD) and, in parallel, increased susceptibility to fibrosis/cirrhosis/hepatocellular carcinoma (HCC). Herein, we report that a high-fat diet (HFD) can augment glycolysis and then accelerate NAFLD-fibrosis progression by downregulating the expression of geranylgeranyl diphosphate synthase (GGPPS), which is a critical enzyme in the mevalonate pathway. Long-term HFD overloading decreases GGPPS expression in mice, which shifts the fuel preference from fatty acids towards glucose. Liver-specific Ggpps deficiency drives the Warburg effect by impairing mitochondrial function, and then induces hepatic inflammation, thus exacerbating fibrosis. Ggpps deficiency also enhances the hyperfarnesylation of liver kinase B1, and promotes metabolic reprogramming by regulating 5'-AMP-activated protein kinase activity. Clinical data further imply that GGPPS expression can predict the stage of NAFLD and recurrence of NAFLD-associated HCC. We conclude that the level of GGPPS is a susceptibility factor for NAFLD-fibrosis progression, and requires more stringent surveillance to ensure early prediction and precision of treatment of NAFLD-related HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Polycomb group RING finger proteins 3/5 activate transcription via an interaction with the pluripotency factor Tex10 in embryonic stem cells.

Polycomb group (PcG) proteins are epigenetic transcriptional repressors that orchestrate numerous developmental processes and have been implicated in the maintenance of embryonic stem (ES) cell state. More recent evidence suggests that a subset of PcG proteins engages in transcriptional activation in some cellular contexts, but how this property is exerted remains largely unknown. Here, we generated ES cells with single or combined disruption of polycomb group RING finger protein 3 (Pcgf3) and Pcgf5 with the CRISPR-Cas9 technique. We report that although these mutant cells maintained their self-renewal and colony-forming capacity, they displayed severe defects in mesoderm differentiation in vitro and in vivo Using RNA-seq to analyze transcriptional profiles of ES cells with single or combined Pcgf3/5 deficiencies, we found that in contrast to the canonical role of the related polycomb repressive complex 1 (PRC1) in gene repression, Pcgf3/5 mainly function as transcriptional activators driving expression of many genes involved in mesoderm differentiation. Proteomic approaches and promoter occupancy analyses helped to establish an extended Pcgf3/5 interactome and identified several novel Pcgf3/5 interactors. These included testis-expressed 10 (Tex10), which may directly contribute to transcriptional activation via the transcriptional co-activator p300. Furthermore, Pcgf3/5 deletion in ES cells substantially reduced the occupancy of Tex10 and p300 at target genes. Finally, we demonstrated that Pcgf3/5 are essential for regulating global levels of the histone modifier H2AK119ub1 in ES cells. Our findings establish Pcgf3/5 as transcriptional activators that interact with Tex10 and p300 in ES cells and point to redundant activity of Pcgf3/5 in pluripotency maintenance.

Enzyme-Free Nucleic Acid Amplification Assay Using a Cellphone-Based Well Plate Fluorescence Reader.

Nucleic acids, DNA and RNA, provide important fingerprint information for various pathogens and have significant diagnostic value; however, improved approaches are urgently needed to enable rapid detection of nucleic acids in simple point-of-care formats with high sensitivity and specificity. Here, we present a system that utilizes a series of toehold-triggered hybridization/displacement reactions that are designed to convert a given amount of RNA molecules (i.e., the analyte) into an amplified amount of signaling molecules without any washing steps or thermocycling. Fluorescent probes for signal generation were designed to consume products of the catalytic reaction in order to push the equilibrium and enhance the assay fold amplification for improved sensitivity and reaction speed. The system of toehold-assisted reactions is also modeled to better understand its performance and capabilities, and we empirically demonstrate the success of this approach with two analytes of diagnostic importance, i.e., influenza viral RNA and a micro RNA (miR-31). We also show that the amplified signal permits using a compact and cost-effective smartphone-based fluorescence reader, an important requirement toward a nucleic-acid-based point-of-care diagnostic system.

The constitutive activation of Egr-1/C/EBPa mediates the development of type 2 diabetes mellitus by enhancing hepatic gluconeogenesis.

The sequential secretion of insulin and glucagon delicately maintains glucose homeostasis by inhibiting or enhancing hepatic gluconeogenesis during postprandial or fasting states, respectively. Increased glucagon/insulin ratio is believed to be a major cause of the hyperglycemia seen in type 2 diabetes. Herein, we reveal that the early growth response gene-1 (Egr-1) can be transiently activated by glucagon in hepatocytes, which mediates glucagon-regulated gluconeogenesis by increasing the expression of gluconeogenesis genes. Blockage of Egr-1 function in the liver of mice led to lower fasting blood glucose, better pyruvate tolerance, and higher hepatic glycogen content. The mechanism analysis demonstrated that Egr-1 can directly bind to the promoter of C/EBPa and regulate the expression of gluconeogenesis genes in the later phase of glucagon stimulation. The transient increase of Egr-1 by glucagon kept the glucose homeostasis after fasting for longer periods of time, whereas constitutive Egr-1 elevation found in the liver of db/db mice and high serum glucagon level overactivated the C/EBPa/gluconeogenesis pathway and resulted in hyperglycemia. Blockage of Egr-1 activation in prediabetic db/db mice was able to delay the progression of diabetes. Our results suggest that dysregulation of Egr-1/C/EBPa on glucagon stimulation may provide an alternative mechanistic explanation for type 2 diabetes.

Quantification of multiple microRNAs by microchip electrophoresis assisted by strand displacement amplification.

MicroRNAs (miRNAs) are increasingly recognized as potential biomarkers for the early diagnosis of cancer. However, the concurrent detection of multiple miRNAs in biological samples presents a significant challenge due to their high homogeneity and low abundance. This study introduced a novel approach combining strand displacement amplification (SDA) with microchip electrophoresis (MCE) for the simultaneous quantitation of trace levels of three miRNAs associated with cancer: miRNA-21, miRNA-145, and miRNA-221. Specifically designed probes were utilized to selectively capture the target miRNAs, thereby initiating the SDA process in a single solution without cross-interference. Under optimized conditions, the SDA-MCE method achieved the limit of detection (LOD) as low as 0.02 fM (S/N = 3) and the limit of quantitation (LOQ) as low as 0.1 fM across a broad linear range spanning from 0.1 fM to 1 pM. The SDA reaction was completed in approximately 1.5 h, and all target products were separated within 135 s through MCE. Application of this method for the simultaneous detection of these three miRNAs in human lung cancer cell samples yielded satisfactory results. Featuring high sensitivity, rapid analysis, minimal reagent consumption, and straightforward operation, the proposed MCE-SDA strategy holds considerable promise for multi-miRNAs detection applications.

Isothermal strand displacement polymerase reaction (ISDPR)-assisted microchip electrophoresis for highly sensitive detection of cancer associated microRNAs.

More and more studies have found that microRNAs (miRNAs) are markers of cancer, and detection of miRNAs is beneficial for early diagnosis and prognosis of cancer. In this paper, the isothermal strand displacement polymerase reaction (ISDPR), which is an enzyme-assisted nucleic acid amplification method, was studied to combine with microchip electrophoresis (MCE) for a simultaneously detection of two cancer related miRNAs named microRNA-21 (miR-21) and microRNA-221 (miR-221). In the ISDPR amplification, two different DNA hairpins (HPs) were specifically designed, so that miR-21 and miR-221 could respectively bind to HPs and started ISDPR amplification to generate two different products which were ultimately detected by MCE. The optimal conditions of ISDPR were carefully investigated, and the limits of detection (LOD) of miR-21 and miR-221 were as low as 0.35 fM and 0.25 fM (S/N = 3) respectively under these conditions. The human lung tumor cells and serum samples were analyzed by this ISDPR-MCE method and satisfactory results were obtained, which means that this method is of high sensitivity, high efficiency, low reagent consumption and simple operation in miRNAs detection.