In Situ Track-Generated DNA Walker for AND-Gate Logic Imaging of Telomerase and Flap Endonuclease 1 Activities in Living Cells.

In situ monitoring of the actions of correlated enzymes in living cells is crucial for expanding our understanding of disease progression and evaluating drug efficacy. However, due to the diverse functions of different enzymes, currently available methods for comprehensive analysis of these events are limited. Here, we present an in situ track-generated DNA walker for AND-gate logic imaging of telomerase (TE) and flap endonuclease 1 (FEN1) activities in live cells. TE is in charge of generating the tracks for the walking strands by extending the TE primer on a gold nanoparticle, while FEN1 is responsible for recognizing the overlapping structure formed by the walking strands and the tracks and then cleaving the fluorescent reporter to produce signals. By utilizing the DNA walker, we successfully determined the expression levels and activities of TE and FEN1 in various cancer cell lines, offering promising prospects for screening inhibitors and investigating the biomolecular mechanisms of diseases.

Ultrafast and Highly Specific Detection of One-Base Mutated Cell-Free DNA at a Very Low Abundance.

Liquid biopsy as well as genotyping plays important roles in guiding the use of tumor-targeted drugs and monitoring the generation of drug resistance. However, current methods, such as next-generation sequencing (NGS) and pyrosequencing, require long analysis time and complicated steps. To achieve ultrafast and highly specific detection of cell-free DNA (cfDNA) from blood, we improved our recently developed FEN1-aided RPA (FARPA), which combined flap endonuclease 1 (FEN1)-catalyzed invasive reactions with recombinase polymerase amplification (RPA) by inactivating the RPA enzymes before invasive reactions, designing short RPA primers, and changing invasive reaction conditions. Using the L858R and T790M mutations as examples, FARPA was sensitive to detect 5 copies of targeted mutants, specific to sense the mutants with an abundance as low as 0.01% from blood, and ultrafast to get results within 40 min. The method was readily expended to genotyping, and 15 min was enough to report the allele species directly from oral swab samples by coupling quick DNA extraction reagents. Validation was carried out by detecting clinical samples, including 20 cfDNA from patients with non-small cell lung cancer (NSCLC) for liquid biopsy and 43 human genomic DNA (gDNA) purified from blood (33) or lysed from oral swabs (10) for genotyping, giving 100% agreement with NGS and pyrosequencing, respectively. Furthermore, a portable battery-driven device with dual-channel fluorescence detection was successfully constructed to facilitate point-of-care testing (POCT) of liquid biopsy and genotyping, providing doctors with a potential tool to achieve genotyping- or mutant-guided personalized medicine at emergency or source-limited regions.

A DNA Nano Firework for Imaging and Inhibitor Screening of Flap Endonuclease 1 in Living Cells.

In situ observation of changes in the activity of marker proteins in living cells is crucial for both biomarker-based disease diagnosis and drug screening. Flap endonuclease 1 (FEN1) has been recognized as a broad-spectrum cancer biomarker and therapeutic target. However, simple and reliable methods for in situ studying the FEN1 activity changes in living cells are limited. Here, we introduce a nano firework as a fluorescent sensor to sense and report FEN1 activity changes in living cells through FEN1 recognizing the substrates on the surface of the nano firework to release and restore the fluorescence of the prequenched fluorophores. We verified the high selectivity, anti-interference ability, stability, and quantitative performance of the nano firework in tubes and living cells, respectively. A series of controlled experiments have demonstrated that the nano firework could accurately report changes in FEN1 activity in different cells, enabling "sensors in, results out" in the manner of simple addition to the cell culture medium. Using an in silico molecular docking study and experiments, we also explored the ability of the nano firework for rapid screening of FEN1 inhibitors and found two new candidate compounds myricetrin and neoisoliquritin, which could be used as FEN1 inhibitors for further research. These performances of the nano firework suggest that it can be used in high-throughput screening applications, providing a promising tool for biomarker-based new drug discovery.

PM2.5 exposure and its interaction of oxidative balance score on ovarian cancer survival: A prospective cohort study.

Recent evidence advises particles with a diameter of 2.5 µm or less (PM2.5) might be a prognostic factor for ovarian cancer (OC) survival. The oxidative balance score (OBS) incorporates diet-lifestyle factors to estimate individuals' anti-oxidant exposure status which may be relevant to cancer prognosis. We aimed to investigate the roles of PM2.5, and OBS and their interaction in OC prognosis. 663 patients with OC were enrolled in the current study. Satellite-derived annual average exposures to PM2.5 based on patients' residential locations. The OBS was calculated based on 16 different diet-lifestyle components derived using an acknowledged self-reported questionnaire. The Cox regression model was performed to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) for overall survival (OS). We also assessed the effect of modification between PM2.5 and OS by OBS via interaction terms. During a median follow-up of 37.57 (interquartile:35.27-40.17) months, 123 patients died. Compared to low-concentration PM2.5 exposure, high PM2.5 during 1 year before diagnosis was associated with worse OC survival (HR= 1.19, 95% CI = 1.01-1.42). We observed an improved OS with the highest compared with the lowest OBS (HR = 0.46, 95% CI = 0.27-0.79, P for trend < 0.05). Notably, we also found an additive interaction between low OBS and high exposure to PM2.5, with the corresponding associations of PM2.5 being more pronounced among participants with lower OBS (HR = 1.42, 95% CI = 1.09-1.86). PM2.5 may blunt OC survival, but high OBS represented an antioxidative performance that could alleviate the adverse association of PM2.5 and OS.

Association of long-term particulate matter exposure with all-cause mortality among patients with ovarian cancer: A prospective cohort.

BACKGROUND: Evidence of the association between particles with a diameter of 2.5 µm or less (PM2.5) in long term and ovarian cancer (OC) mortality is limited. METHODS: This prospective cohort study analyzed data collected between 2015 and 2020 from 610 newly diagnosed OC patients, aged 18-79 years. The residential average PM2.5 concentrations 10 years before the date of OC diagnosis were assessed by random forest models at a 1 km × 1 km resolution. Cox proportional hazard models fully adjusted for the covariates (including age at diagnosis, education, physical activity, kitchen ventilation, FIGO stage, and comorbidities) and distributed lag non-linear models were used to estimate the hazard ratios (HRs) and 95 % confidence intervals (CIs) of PM2.5 and all-cause mortality of OC. RESULTS: During a median follow-up of 37.6 months (interquartile: 24.8-50.5 months), 118 (19.34 %) deaths were confirmed among 610 OC patients. One-year PM2.5 exposure levels before OC diagnosis was significantly associated with an increase in all-cause mortality among OC patients (single-pollutant model: HR = 1.22, 95 % CI: 1.02-1.46; multi-pollutant models: HR = 1.38, 95 % CI: 1.10-1.72). Furthermore, during 1 to 10 years prior to diagnosis, the lag-specific effect of long-term PM2.5 exposure on the all-cause mortality of OC had a risk increase for lag 1-6 years, and the exposure-response relationship was linear. Of note, significant interactions between several immunological indicators as well as solid fuel use for cooking and ambient PM2.5 concentrations were observed. CONCLUSION: Higher ambient PM2.5 concentrations were associated with an increased risk of all-cause mortality among OC patients, and there was a lag effect in long-term PM2.5 exposure.

The association of macronutrient quality and its interactions with energy intake with survival among patients with ovarian cancer: results from a prospective cohort study.

BACKGROUND: Emerging evidence supports shifting the focus from the quantity of macronutrients to quality to obtain greater benefits for the prognosis of ovarian cancer (OC). Additionally, despite the high relevance between macronutrient quality and quantity, the interaction of these parameters on OC survival remains unknown. OBJECTIVE: A multidimensional macronutrient quality index (MQI) was applied to investigate the association between overall macronutrient quality and the survival of patients with OC. METHODS: A prospective cohort study was conducted with 701 females diagnosed with OC who were enrolled from 2015 to 2020. Dietary intake information was obtained from a validated food frequency questionnaire. The MQI was calculated based on 3 quality indices: carbohydrate quality index (CQI), fat quality index (FQI), and protein quality index (PQI). Cox proportional hazards regression was conducted to calculate HRs and 95% CIs. Furthermore, we evaluated whether energy intake status (total energy intake and energy balance) modified the association between MQI and OC survival. RESULTS: During a median follow-up period of 38 (interquartile: 35-40) mo, 130 deaths occurred. The prediagnosis high MQI scores were associated with substantially improved survival among females with OC (HRtertile 3 vs. tertile 1 = 0.50, 95% CI: 0.33, 0.77). For sub-indices of the MQI, higher CQI (HR = 0.60, 95% CI: 0.36, 0.99), higher FQI (HR = 0.55, 95% CI: 0.34, 0.87), and higher PQI (HR = 0.58, 95% CI: 0.35, 0.94) scores were all associated with better survival. Notably, significant interactions were observed for the MQI score with total energy intake and energy balance as well as the quantity and quality of carbohydrates on survival. CONCLUSIONS: Intake of high-quality macronutrients before diagnosis was associated with improved survival among females with OC, especially for those with energy imbalance.

Purine Intake and All-Cause Mortality in Ovarian Cancer: Results from a Prospective Cohort Study.

BACKGROUND: Current biological evidence suggests that purine involvement in purine metabolism may contribute to the development and progression of ovarian cancer (OC), but the epidemiological association is currently unknown. METHODS: A total of 703 newly diagnosed patients with OC aged 18-79 years were included in this prospective cohort study. Utilizing a verified food-frequency questionnaire, the participants' dietary consumption was gathered. Using medical records and ongoing follow-up, the deaths up until 31 March 2021 were determined. To assess the hazard ratios (HRs) and 95% confidence intervals (CIs) of purine intake with OC mortality, Cox proportional-hazard models were utilized. RESULTS: During the median follow-up of 31 months (interquartile: 20-47 months), 130 deaths occurred. We observed an improved survival for the highest tercile of total purine intake compared with the lowest tercile (HR = 0.39, 95% CI = 0.19-0.80; p trend < 0.05), and this protective association was mainly attributed to xanthine intake (HR = 0.52, 95% CI = 0.29-0.94, p trend < 0.05). Additionally, we observed a curving relationship in which OC mortality decreased with total purine intake, and the magnitude of the decrease was negatively correlated with intake (p non-linear < 0.05). Significant inverse associations were also observed in subgroup analyses and sensitivity analyses according to demographic and clinical characteristics. Moreover, we observed that xanthine intake and hypoxanthine intake had a multiplicative interaction with ER and PR expression (p < 0.05), respectively. CONCLUSION: A high total purine and xanthine intake was linked to a lower risk of OC mortality. Further clarification of these findings is warranted.

Nutrients-Rich Food Index Scores and the Overall Survival of Ovarian Cancer Patients: Results from the Ovarian Cancer Follow-Up Study, a Prospective Cohort Study.

Background: The nutrients-rich food (NRF) index provides a score of diet quality. Although high diet quality is associated with survival of ovarian cancer (OC), the associations between NRF index scores and OC survival remain unevaluated. Methods: The prospective cohort study enrolled 703 women with newly diagnosed epithelial OC to assess the correlations between NRF index scores and overall survival (OS) in OC patients. Dietary consumption was evaluated through a food frequency questionnaire and diet quality was calculated based on NRF index scores, including three limited nutrients and six (NRF6.3), nine (NRF9.3), or eleven (NRF11.3) benefit nutrients. All-cause deaths were ascertained through medical records combined with active follow-up. Immunohistochemistry (IHC) analyses were conducted to evaluate the expression of IHC indicators (including Estrogen Receptor, Progesterone Receptor, p53, Vimentin, and Wilms' tumor 1), which were identified by two independent pathologists. The Cox proportional hazards regression models were applied for estimating the hazard ratios (HRs) and 95% confidence intervals (CIs). Moreover, we performed the penalized cubic splines model to assess the curvilinear associations of NRF index scores with OC survival. Results: During the median follow-up of 37.17 (interquartile: 24.73-50.17) months, 130 deaths were documented. Compared to the lowest tertiles, the highest tertile of index scores [NRF9.3 (HR = 0.63, 95% CI = 0.41-0.95), NRF6.3 (HR = 0.59, 95% CI = 0.39-0.89), and NRF11.3 (HR = 0.57, 95% CI = 0.38-0.87)] were correlated to better OS, showing an obvious linear trend (all p trend < 0.05). Interestingly, the curvilinear association between the NRF6.3 index score and OC survival was also observed (p non-linear < 0.05). Subgroup analyses, stratified by clinical, demographic, and IHC features, showed similar risk associations as the unstratified results. Furthermore, there were significant multiplicative interactions between NRF index scores and Progestogen Receptors as well as Wilms' tumor 1 expressions (all p interaction < 0.05). Conclusions: Higher NRF index scores were associated with an improved OS in OC patients.

Association between pre-diagnostic dietary antioxidant vitamin consumption and ovarian cancer survival: a prospective cohort study.

Background: Epidemiological evidence regarding the relationship between dietary antioxidant vitamin intake and ovarian cancer (OC) survival is not clear. Herein, we aimed to first evaluate this topic in a prospective cohort study in China. Methods: The present study included participants from the Ovarian Cancer Follow-Up Study, which was a hospital-based prospective cohort study including OC patients who were aged 18 to 79 years during 2015-2020. The information on the intake of antioxidant vitamins, consisting of vitamin A, retinol, α-carotene, ß-carotene, vitamin C, and vitamin E, and other diet information was obtained through a 111-item food frequency questionnaire. Deaths were recorded until March 31, 2021. Hazard ratios (HRs) and 95% confidence intervals (CIs) for overall survival were evaluated using Cox proportional hazards models. Results: There were 130 (18.49%) deaths among 703 OC patients during a median 37.19 months follow-up. In the multivariable-adjusted model, the highest tertile of dietary vitamin C (HR = 0.43, 95% CI = 0.25-0.75, P for trend <0.05) and ß-carotene intake (HR = 0.52, 95% CI = 0.31-0.87, P for trend <0.05) was inversely associated with the overall survival of OC when compared with the lowest tertile group. Retinol, vitamin A, vitamin E, and α-carotene consumption showed no association with OC survival. Of note is that the multiplicative interaction was identified between vitamin C intake and residual lesions in OC survival (P for interaction <0.05). Conclusion: Our findings indicate that pre-diagnostic higher vitamin C and ß-carotene intake was associated with improved OC survival.

Molecular profiles of single circulating tumor cells from early breast cancer patients with different lymph node statuses.

BACKGROUND: Characterization of early breast cancer circulating tumor cells (CTCs) may provide valuable information on tumor metastasis. METHODS: We used immunomagnetic nanospheres to capture CTCs from the peripheral blood of eight early breast cancer patients and then performed single-cell RNA sequencing using our proposed bead-dd-seq method. RESULTS: CTCs displayed obvious tumor cell characteristics, such as the activation of oxidative stress, proliferation, and promotion of metastasis. CTCs were clustered into two subtypes significantly correlated with the lymph node metastasis status of patients. CTCs in subtype 1 showed a strong metastatic ability because these CTCs have the phenotype of partial epithelial-mesenchymal transition and enriched transcripts, indicating breast cancer responsiveness and proliferation. Furthermore, DNA damage repair pathways were significantly upregulated in subtype 1. We performed in vitro and in vivo investigations, and found that cellular oxidative stress and further DNA damage existed in CTCs. The activated DNA damage repair pathway in CTCs favors resistance to cisplatin. A checkpoint kinase 1 inhibitor sensitized CTCs to cisplatin in mouse models of breast cancer metastasis. CONCLUSION: The present study dissects the molecular characteristics of CTCs from early-stage breast cancer, providing novel insight into the understanding of CTC behavior in breast cancer metastasis.

Phytosterol intake and overall survival in newly diagnosed ovarian cancer patients: An ambispective cohort study.

Background: Phytosterol is a bioactive compound existing in all plant foods, which might have anticancer properties. The aim of this study was to first assess the impact of the pre-diagnosis phytosterol intake on overall survival (OS) of patients with ovarian cancer (OC). Materials and methods: This ambispective cohort study recruited 703 newly diagnosed OC patients to investigate the aforementioned associations. Dietary intake was assessed using a validated 111-item food frequency questionnaire. Deaths were ascertained until March 31, 2021, through active follow-up and medical records. Cox proportional hazards regression models were applied to calculate the hazard ratios (HRs) and 95% confidence intervals (CIs). Results: During the median follow-up of 37.17 months, 130 deaths occurred. The median age at diagnosis of 703 OC patients was 53.00 (interquartile: 48.00-60.00) years. Of these, almost half patients (48.08%) were diagnosed in advanced International Federation of Gynecology and Obstetrics (FIGO) stage (III-IV). Additionally, more than half patients were serous carcinoma (68.14%), poorly differentiated (85.21%), and no residual lesions (78.66%). Patients consumed the highest tertile of dietary campesterol (HR = 0.54, 95% CI = 0.31-0.94, P trend < 0.05), stigmasterol (HR = 0.60, 95% CI = 0.37-0.98), and ß-sitosterol (HR = 0.63, 95% CI = 0.40-0.99) were significantly associated with better OS compared with those with the lowest tertile of intake. The curvilinear associations were observed between total phytosterols and ß-sitosterol intake and OC survival (P non-linear < 0.05). Significant associations were generally consistent across different subgroups stratified by demographical, clinical, and immunohistochemical characteristics. Moreover, there were significant interactions between phytosterol intake and age at diagnosis, body mass index, as well as expressions of Wilms' tumor-1 and Progestogen Receptor (all P interaction < 0.05). Conclusion: Pre-diagnosis higher campesterol, stigmasterol, and ß-sitosterol intake were associated with better survival among OC patients.

Galectin-3 enhances trastuzumab resistance by regulating cancer malignancy and stemness in HER2-positive breast cancer cells.

PURPOSE: The aim of this study was to explore the role of galectin-3 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells and the potential mechanism. METHODS: Kaplan-Meier (KM)-plot and The Cancer Genome Atlas (TCGA) databases were used to study the role of galectin-3 in the prognosis of HER2-positive breast cancer. The effects of galectin-3 on cell proliferation, migration, invasion, and colony formation ability in HER2-positive breast cancer cells were examined. The relationship between galectin-3 and important components in the HER2 pathways, including HER2, epidermal growth factor receptor (EGFR), protein kinase B (AKT), and phosphatase and tensin homolog (PTEN), was further studied. Lentivirus and CRISPR/Cas9 were used to construct stable cell lines. Cell counting kit-8 (CCK-8) and apoptosis assays were used to study the relationship between galectin-3 and trastuzumab. The effect of galectin-3 on cell stemness was studied by mammosphere formation assay. The effects of galectin-3 on stemness biomarkers and the Notch1 pathway were examined. Tumorigenic models were used to evaluate the effects of galectin-3 on tumorigenesis and the therapeutic effect of trastuzumab in vivo. RESULTS: HER2-positive breast cancer patients with a high expression level of LGALS3 (the gene encoding galectin-3) messenger RNA (mRNA) showed a poor prognosis. Galectin-3 promoted cancer malignancy through phosphoinositide 3-kinase (PI3K)/AKT signaling pathway activation and upregulated stemness by activating the Notch1 signaling pathway in HER2-positive breast cancer cells. These two factors contributed to the enhancement of trastuzumab resistance in cells. Knockout of LGALS3 had a synergistic therapeutic effect with trastuzumab both in vitro and in vivo. CONCLUSIONS: Galectin-3 may represent a prognostic predictor and therapeutic target for HER2-positive breast cancer.

Lipid membrane anchoring and highly specific fluorescence detection of cancer-derived exosomes based on postfunctionalized zirconium-metal-organic frameworks.

Cancer-derived exosomes carry a variety of important biomarkers specific to the formation, invasion and metastasis of tumor tissue. Dynamic monitoring of exosomes originated from cancer cells has clinical significance. Here we proposed a novel method to employ zirconium-metal-organic frameworks (Zr-MOFs) for extracting and identifying exosomes from blood. At first UiO-66 was magnetically modified as the adsorbent to anchor exosomes by forming Zr-O-P bonds. Then UiO-66-NH2 modified with anti-EpCAM was used to construct the fluorescent probe to recognize the extracted EpCAM-positive exosomes by forming a "MOF-exosome-MOF" structure. The proposed fluorescence detection method was evaluated by quantifying MCF-7 cell-derived exosomes at the concentration as low as 16.72 particles/µl. This method was successfully applied to analyze exosomes in the plasma samples from healthy donors and breast cancer patients, demonstrating that our method might have a great potential in assisting the early diagnosis and in dynamically monitoring the efficacy of cancer treatment. We believe that the method could be extended to the detection of other biomarkers in exosomes derived from cancer cell.

Flap Endonuclease 1-Assisted DNA Walkers for Sensitively and Specifically Sensing ctDNAs.

DNA walkers have shown superior performance in biosensing due to their programmability to design molecular walking behaviors with specific responses to different biological targets. However, it is still challenging to make DNA walkers capable of distinguishing DNA targets with single-base differences, so that DNA walkers that can be used for circulating tumor DNA sensing are rarely reported. Herein, a flap endonuclease 1 (FEN 1)-assisted DNA walker has been proposed to achieve mutant biosensing. The target DNA is captured on a gold nanoparticle (AuNP) as a walking strand to walk by hybridizing to the track strands on the surface of the AuNP. FEN 1 is employed to report the walking events by cleaving the track strands that must form a three-base overlapping structure recognized by FEN 1 after hybridizing with the captured target DNA. Owing to the high specificity of FEN 1 for structure recognition, the one-base mutant DNA target can be discriminated from wild-type DNA. By constructing a sensitivity-enhanced DNA walker system, as low as 1 fM DNA targets and 0.1% mutation abundance can be sensed, and the theoretical detection limits for detecting the DNA target and mutation abundance achieve 0.22 fM and 0.01%, respectively. The results of epidermal growth factor receptor (EGFR) L858R mutation detection on cell-free DNA samples from 15 patients with nonsmall cell lung cancer were completely consistent with that of next-generation sequencing, indicating that our DNA walker has potential for liquid biopsy.

Circulating tumour cells at baseline and late phase of treatment provide prognostic value in breast cancer.

To determine the prognostic value of the timing of circulating breast tumour cell measurement during treatment, peripheral blood from 164 patients with breast disease was collected. Circulating tumour cells (CTCs) were enriched by using immunomagnetic nanospheres (IMNs) and were identified by using immunofluorescent staining. The CTC shows nuclear-positive, EpCAM-positive, CK19-positive, and CD45-negative. Patients with CTC positivity (> 19/7.5 mL blood) had shorter progression-free survival (PFS) and overall survival (OS) than those with negative results (≤ 19/7.5 mL blood) at baseline. Surgery caused an increase in the number and prevalence of CTCs, and the effect disappeared on day 14 after surgery. During adjuvant chemotherapy, CTCs detected before therapy was only correlated with PFS; however, CTCs at the end of adjuvant chemotherapy were correlated with both PFS and OS. The PFS and OS of the CTC-positive group were significantly shorter than those of the CTC-negative group at the end-point follow-up visit. The prognostic value of CTCs at different measurement time points was demonstrated during breast cancer treatment. Surgery and chemotherapy affected the prevalence of CTCs, leading to different prognostic relevance of CTCs at different treatment stages. CTCs detected at baseline or in the late phase of treatment are preferable for prognosis.

Multiplex Visualized Closed-Tube PCR with Hamming Distance 2 Code for 15 HPV Subtype Typing.

Cervical cancer is the fourth leading cause of death in women, especially in developing countries. Specific and economic methodologies for HPV typing are crucial in cancer diagnosis and further disease control. However, routine assays based on real-time polymerase chain reaction (qPCR) or DNA-chip hybridization are either incapable of offering detailed subtype information or involve tedious open-tube operations with the risk of cross-contamination from PCR amplicons. Herein, we proposed a multiplex visualized closed-tube PCR (Multi-Vision) for HPV typing. Using gold nanoparticle probes (AuNPs) as a color change indicator combined with a Hamming distance 2 coding scheme, 13 high-risk HPVs and two subtypes associated with high-incidence benign lesions were successfully typed by performing six closed-tube PCRs. The assay demonstrates high specificity with no cross-reaction among different subtypes under several artificial sample concentrations (from 100 to 103 copies per reaction) and enables highly sensitive detection of as low as 0.5 copies/µL. Further, 105 clinical samples were successfully analyzed using our method with a high concordance rate of 99.05% (104/105) compared to a HPV typing kit. The inconsistent sample was confirmed by sequencing to be consistent with the typing results determined by our method, indicating that Multi-Vision could be a useful tool for HPV detection, especially in resource-limited regions.

Multiplex detection of blood-borne pathogens on a self-driven microfluidic chip using loop-mediated isothermal amplification.

Detection of blood-borne pathogens such as hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV) is essential to ensure the safety of blood transfusion. However, traditional PCR-based pathogen nucleic acid detection methods require relatively high experimental facilities and are difficult to apply in areas with limited resources. In this study, a self-driven microfluidic chip was designed to carry out multiplex detection of HBV, HCV and HIV by using loop-mediated isothermal amplification (LAMP). Benefitting from the air permeability of the polydimethylsiloxane material, the chip could accomplish sample loading within 12 min driven by the pressure difference between the reaction chambers and vacuum chambers in the chip without using pumps or any injection devices. Multiplex detection is achieved by presetting LAMP primers specific to different targets in different reaction chambers. Calcein was used as an indicator to indicate the positive amplification reaction, and the result can be recorded by a smartphone camera. After 50 min of isothermal amplification at 63 °C, 2 copies/µL of HBV, HCV and HIV target nucleic acids could be detected. The results of HBV detection of 20 clinical plasma samples by using the chip are consistent with that of the qPCR-based kit, indicating that the LAMP-based self-driven chip has the clinical application potential for blood-borne pathogen detection, especially in resource-limited areas.

Sensitive quantitation of ESR1 mutations in cell-free DNA from breast cancer patients using base-specific invasive reaction assisted qPCR.

Acquired estrogen receptor 1 (ESR1) mutation is being promoted as a key mechanism of resistance to endocrine therapies in breast cancers. It is significative to monitor ESR1 mutations in real time, which provide an opportunity to alter therapy as these mutations emerge. Previous assays based on next-generation sequencing (NGS) and digital PCR (dPCR) usually due to high costs and complicated workflows hampered their clinical adoption in general medical institutions. Here, we proposed a new strategy using base-specific invasive reaction assisted qPCR measure for ESR1 mutations in cfDNA. Two pivotal steps involved in this strategy are target-specific signal generation and the quantification without adding any internal reference or making standard calibration curves. The strategy enabled a high specificity of 0.1% (better than traditional NGS-based method) and a minimum sensitivity of 0.1 copies µL-1. As validation, with the strategy, cfDNA from endocrine therapy-resistant breast cancers and untreated ones were successfully analyzed (20% mutation rate (2/10) with mutation abundance of 0.54-1.65% vs. 0% mutation rate (0/5)). By virtue of cost-effective, highly flexible and precise, the strategy could be readily implemented in general laboratory, showing promising application perspectives in analysis of other types of mutations.

Integrative analyses of scRNA-seq and scATAC-seq reveal CXCL14 as a key regulator of lymph node metastasis in breast cancer.

The potentially different genetics and epigenetics in the primary tumors and metastases affect the efficacy of treatment in breast cancer patients. Nevertheless, the cellular and molecular mechanisms of breast cancer lymph node metastasis still remain elusive. Here, we employed single-cell RNA sequencing to acquire the transcriptomic profiles of individual cells from primary tumors, negative lymph nodes (NLs) and positive lymph nodes (PLs). We also performed a single-cell assay for transposase-accessible chromatin (ATAC) sequencing (scATAC-seq) of the positive and NL samples to get the chromatin accessibility profile. We identified a novel cell subpopulation with an abnormally high expression level of CXCL14 in the PL of breast cancer patients. Cell trajectory analysis also revealed that CXCL14 was increased expressed in the late pseudo-time. Moreover, based on a tissue microarray of 55 patients and the Oncomine database, we validated that CXCL14 expression was significantly higher in breast cancer patients with lymph node metastasis. Furthermore, scATAC-seq identified several transcription factors that may be potential regulation factors for the lymph node metastasis of breast cancer. Thus, our findings will improve our current understanding of the mechanism for lymph node metastasis, and they are potentially valuable in providing novel prognosis markers for the lymphatic metastasis of breast cancer.

Multiplex-invasive reaction-assisted qPCR for quantitatively detecting the abundance of EGFR exon 19 deletions in cfDNA.

Exon 19 deletions (19-Del) on the epidermal growth factor receptor (EGFR) gene are vital biomarkers for guiding tyrosine kinase inhibitor (TKI) treatment and the diagnosis of non-small cell lung cancer (NSCLC). However, it is difficult for conventional qPCR to quantitatively detect all 19-Del targets of EGFR, especially for cfDNA samples. Herein, a multiplex invasive reaction-assisted qPCR was proposed by employing a multiplex invasive reaction to distinguish 19-Del DNA targets from wild DNA targets and report them with different fluorescence signals in each PCR cycle. As all 19-Del targets have the same amplification efficiency and very similar invasive reaction efficiencies, the 19-Del abundance in a sample could be quantified by using the difference between the Ct values (ΔCt) of the deletion targets and the wild targets without the requirement of a standard calibration curve. Combining the high sensitivity of PCR and the high specificity of the invasive reaction, this method can detect 10 copies of the deletion targets and lower than 0.1% deletion abundance. The results were 100% consistent with ARMS-PCR for the 38 tumor tissues tested and were in good agreement with next-generation sequencing for quantifying the abundance of EGFR 19-Del in 15 cfDNA samples, showing the great potential of the method for liquid biopsies.