Ratiometric luminescent simultaneous sensing of aristolochic acids (I-IV) by a novel metal-organic framework and its nanowire.

Aristolochic acids (AAs), which are a group of nitrophenanthrene carboxylic acids formed by Aristolochia plant, have become an increasing serious threat to humans due to their nephrotoxicity and carcinogenicity. Fast and accurate approaches capable of simultaneous sensing of aristolochic acids (I-IV) are vital to avoid intake of such compounds. In this research, the novel ratiometric fluorescence zinc metal-organic framework and its nanowire have been prepared. The two different coordination modes (tetrahedral configuration and twisted triangular bipyramidal configuration) within zinc metal-organic framework lead to the significant double emissions. The ratiometric fluorescence approach based on nanowire provides a broader concentration range (3.00 × 10-7~1.00 × 10-4 M) and lower limit of detection (3.70 × 10-8 M) than that based on zinc metal-organic framework (1.00 × 10-6~1.00 × 10-4 M, 5.91 × 10-7 M). The RSDs of the results are in the range 1.4-3.5% (nanowire). The density functional theory calculations and UV-Vis absorption verify that the sensing mechanism is due to charge transfer and energy transfer. Excellent spiked recoveries for AAs(I-IV) in soil and water support that nanowire is competent to simultaneously detect these targets in real samples, and the proposed approach has potential as a fluorescence sensing platform for the simultaneous detection of AAs (I-IV) in complex systems.

Bright and Efficient Sensitized Near-Infrared Photoluminescence from an Organic Neodymium-Containing Composite Material System.

Intense organic neodymium (Nd3+) emission is obtained with near-infrared (NIR) emission equivalent in intensity to that of an organic semiconductor emitting material. The advantage of Nd3+ emission is its narrow line width and NIR emission, which is enhanced by ∼3000 times at low excitation power through an efficient sensitization effect from a composite organic sensitizer. This performance is optimized at high concentrations of Nd3+ ions, and the organic perfluorinated system provides the ion excitations with a quantum efficiency of ∼40%. The material system is applicable to thin films that are compatible with integrated optics applications.

Long non-coding RNA NORAD functions as a microRNA-204-5p sponge to repress the progression of Parkinson's disease in vitro by increasing the solute carrier family 5 member 3 expression.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders. Long non-coding RNAs have important regulatory values in various human diseases. Non-coding RNA Activated by DNA Damage (NORAD) was reported to regulate PD progression in vitro, but its functional mechanism is fully unknown. We used 1-methyl-4-phenylpyridinium (MPP+ ) to establish the cell-based PD model. NORAD, microRNA-204-5p (miR-204-5p), and solute carrier family 5 member 3 (SLC5A3) levels were quantified using the quantitative real-time polymerase chain reaction. Cell viability and apoptosis were determined by Cell Counting Kit-8 and flow cytometry, respectively. The protein levels were analyzed via western blot. Cytotoxicity was assessed by the released lactate dehydrogenase level in cell supernatant. Oxidative stress and inflammation were measured by the standard indicators. Dual-luciferase reporter and RNA immunoprecipitation assays were performed for intergenic combination. First, we found that NORAD was obviously reduced in MPP+ -treated neuroblastoma cells and lightened the MPP+ -induced cytotoxicity, oxidative stress, and inflammatory response. Then, NORAD was shown to be a miR-204-5p sponge and avoided the injury induced by MPP+ in neuroblastoma cells via targeting miR-204-5p. SLC5A3 was a miR-204-5p target and could be regulated by NORAD/miR-204-5p axis. SLC5A3 knockdown assuaged the anti-miR-204-5p-induced protection for neuroblastoma cells from MPP+ . Altogether, NORAD played a neuroprotective role against the progression of MPP+ -induced PD model in neuroblastoma cells relying on the miR-204-5p/SLC5A3 axis. This study afforded the clear elaboration on the PD pathomechanism concerning NORAD.

A new method using machine learning for automated image analysis applied to chip-based digital assays.

Chip-based digital assays such as the digital polymerase chain reaction (digital PCR), digital loop-mediated amplification (digital LAMP), digital enzyme-linked immunosorbent assay (digital ELISA) and digital proximity ligation assay (digital PLA) need high-throughput quantification of the captured fluorescence image data. However, traditional methods that are mainly based on image segmentation using either a fixed threshold or an automated hard threshold failed to extract valid signals over a broad range of image characteristics. In this study, we introduce a new method for automated image analysis to extract signals applied to chip-based digital assays. This approach precisely locates each micro-compartment based on the structure design of the chip, thereby eliminating the interference of non-signal noise in the image. Utilizing the principle that the human eyes can distinguish between the positive micro-compartments and the negative micro-compartments, we take the parameters of each micro-compartment together with its surrounding micro-compartments as the training dataset of the Random Forest classifier to classify the micro-compartments and extract valid signals, thus solving the problem caused by the differences among images. Furthermore, we adopted the iteration methodology that adds the output of a model's prediction to the input of the next model's training dataset, until the output of a model's prediction reaches the accuracy we expected, which improves the work efficiency during data training greatly. We demonstrate the method on the dPCR dataset and it performs well without any manual adjustment of settings. The results show that our proposed method can recognize the positive signals from the fluorescence images with an accuracy of 97.78%. With minor modification, bio-instrument companies or researchers can integrate this method into their digital assay devices' software conveniently.

High throughput single cell separation and identification using a self-priming isometric and Equant screw valve-based (SIES) microfluidic chip.

The emergence of various single cell separation and identification platforms has greatly promoted the development of single cell research. Among these platforms, microfluidic chip-based strategies occupy a significant position in single cell separation and identification. Here, we proposed a self-priming isometric and Equant screw valve-based microfluidic chip (SIES chip) for high throughput single cell isolation and identification. With several special designs, such as a peripheral water tank to balance negative pressure distribution in a marginal area of the chip, a screw valve to preserve the suction power during the step-by-step sample loading, and multistage branching "T" shape channels to separate cells evenly into the chambers, up to 2000 single cells can be well dispersed and analyzed at the same time using this chip. We applied this chip for the isolation and identification of single A549 cells targeting the activated leukocyte cell adhesion molecule (ALCAM) gene. The results showed that only a small proportion (approximately 5.1%) of A549 cells expressed ALCAM, which can potentially provide a reference for A549 cell reclassification. Besides being inexpensive, user-friendly and portable, our chip can be used in some resource-limited settings and may have a great potential in POC (Point-of-Care) applications.

Comparison of Endoscopic and Minimally Invasive Transforaminal Lumbar Interbody Fusion for Lumbar Degenerative Diseases: A Meta-analysis.

STUDY DESIGN: Systematic review and meta-analysis. OBJECTIVE: To compare the results of endoscopic transforaminal lumbar interbody fusion (Endo-TLIF) and minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) for patients with lumbar degenerative diseases. SUMMARY OF BACKGROUND DATA: The MIS-TLIF has been widely used in lumbar degenerative diseases and achieved favorable clinical effects. The main disadvantage is the limited working space and visualization, especially in the deeper operational field, for preparing fusion bed. In recent years, with the development of surgical techniques, optical technology, and special instruments, Endo-TLIF has gradually been applied. MATERIALS AND METHODS: A systematic review and meta-analysis of cohort studies between Endo-TLIF and MIS-TLIF in the lumbar degenerative diseases. The following outcome measures were extracted: visual analog scale (VAS), Oswestry Disability Index, fusion rate, disk height, segmental lordosis, operative time, length of hospital stay and complications. Data analysis was performed by RevMan 5.3. RESULTS: Eight studies comprising 687 patients were included in this meta-analysis. The pooled result revealed there was no significant differences in the VAS of leg, Oswestry Disability Index, fusion rate, disk height, segmental lordosis, and complication rate between the 2 groups ( P >0.05). However, the VAS of back in the Endo-TLIF group was significantly less than those in the MIS-TLIF group within 2 weeks after surgery [weighted mean difference (WMD)=-1.33 (-1.98, -0.68), P <0.0001] and at 3 months postoperatively [WMD=-0.72(-0.85, -0.59), P <0.00001]. The Endo-TLIF group also seemed to fewer VAS of back at the last follow-up (≥12 mo) [WMD=-0.12 (-0.25, -0.00), P =0.05]. Compared with the MIS-TLIF group, the Endo-TLIF group was associated with longer operation time [WMD=26.74 (2.14, 51.34), P =0.03], but shorter length of hospital stay [WMD=-1.98(-2.91, -1.05), P <0.0001]. CONCLUSIONS: Compared with minimally invasive TLIF, endoscopic TLIF achieved comparable improvement of symptoms and intervertebral fusion, longer operation time, and smaller surgical trauma. Endoscopic TLIF, which requires a demanding learning curve, maybe a feasible and effective technique for the patients with symptomatic lumbar degenerative diseases.

Polyphenols functionalized MOF encapsulated BPQDs for synergistic photothermal/photodynamic antibacterial properties and multifunctional food preservation.

Active antibacterial materials play an important role in solving food safety problems caused by pathogen contamination. In this study, a composite active antibacterial material with the synergistic antibacterial effectiveness of photothermal, photodynamic and the surface charge of polyphenols was developed, where the multi-porous polyphenol functionalized metal-organic frameworks (ZIF-8-TA) were used as the framework carrier, and black phosphorus quantum dots (BPQDs) were used as the photosensitive source. The resulted ZIF-8-TA/PBQDs possesses excellent photothermal conversion efficiency (27.92%), photodynamic performance and surface charge, and these factors ensure the outstanding broad-spectrum antibacterial performance (100%). Multifunctional characteristics and excellent biocompatibility endow the materials with vast potential for foodstuff packaging. The results showed that the composite antibacterial film produced by doping ZIF-8-TA/PBQDs into chitosan could effectively prolong the shelf life of foodstuff compared with commercial membrane. The successful implementation of this research provides a new idea for controlling microbial contamination and developing multifunctional antibacterial materials.

Fluorescence sensing of water in various organic solvents based on a novel cyclic polymer.

A sensor capable of sensing of water in various organic solvents ranging from water-soluble to water-miscible solvents is still a challenging task. In this research, a cyclic polymer fluorescence chemosensor (CPFC) has been developed for sensing of water by turn-on model in 9 organic solvents and turn-off model in DMA, where the broadest concentration range and the lowest detection limit was obtained for water in DMA (10 %-90 %) and dioxane (0.011 %), respectively. The sensing mechanism is explored by theory calculation and experimental investigation. The amphiphilic nature endows the polymer probe with great potential for measuring various contaminants from aqueous and nonaqueous mediums. Furthermore, the present search highlights the potential applications of cyclic polymer as fluorescence probes in the field of sensing.

Development and validation of a DNA damage repair-related gene-based prediction model for the prognosis of lung adenocarcinoma.

Background: Lung cancer is the leading cause of morbidity and mortality among all cancer types, with lung adenocarcinoma (LUAD) being the most prevalent subtype. DNA damage repair (DDR)-related genes are closely associated with cancer progression and treatment, with emerging evidence highlighting their correlation with tumor development. However, the relationship between LUAD prognosis and DDR-related genes remains unclear. Methods: RNA sequencing (RNA-seq) data and clinical information were obtained from The Cancer Genome Atlas (TCGA) database. The GSE31210 dataset, utilized for external validation, was retrieved from the Gene Expression Omnibus (GEO) database. Differentially expressed DDR genes were identified, and a DDR-related prognostic model was established and validated using Kaplan-Meier (KM) survival analysis, time-dependent receiver operating characteristic (ROC) curves, gene set enrichment analysis (GSEA), tumor mutational burden (TMB) analysis, and immune cell infiltration. A P value of less than 0.05 was considered statistically significant. Results: A total of 514 patients with LUAD from TCGA database were divided into distinct subtypes to characterize the diversity within the DDR pathway. DDR-activated and DDR-suppressed subgroups showed distinct clinical characteristics, molecular characteristics, and immune profiles. Nine genes were identified as hub DDR-related genes, including CASP14, DKK1, ECT2, FLNC, HMMR, IGFBP1, KRT6A, TYMS, and FCER2. By using the expression levels of these selected genes, the corresponding risk scores for each sample was predicted. In the training group, KM survival analysis revealed that the high-risk group exhibited significantly diminished overall survival (OS) [hazard ratio (HR) =3.341, P=1.38e-08]. The corresponding area under the curve (AUC) values for the 1-year follow-up periods was 0.767, respectively. Upon validation in the external cohort, patients with higher risk scores manifested significantly reduced OS (HR =2.372, P=1.87e-03). The AUC values of the ROC curves for the 1-year OS in the validation cohort was 0.87, respectively. Moreover, advanced DDR risk score was correlated with increased TMB scores, a heightened frequency of TP53 mutations, an increased abundance of cancer-testicular antigens (CTAs), and a lower tumor immune dysfunction and exclusion (TIDE) score in patients with LUAD (P<0.05). Conclusions: A nine-gene risk signature associated with DDR in LUAD was effectively developed, demonstrating its potential as a robust and reliable classification tool for clinical practice. This model exhibited the capability to accurately predict the prognosis and survival outcomes of LUAD patients.

The effect of early tracheal extubation combined with physical training on pulmonary rehabilitation of patients after lung transplantation: a randomized controlled trial.

Background: This study aimed to explore the effect of early extubation combined with physical training on pulmonary rehabilitation of patients after lung transplantation. Methods: This is an open parallel randomized controlled trial. A total of 96 lung transplant patients admitted to Wuxi People's Hospital (July 2018 to June 2019) were included. Inclusion criteria: (I) aged 18-75; (II) lung transplantation; (III) communicate normally; (IV) voluntary participation. According to the random number method, they were divided into the control group (routine nursing intervention) and the observation group (early extubation combined with a physical training program). The indwelling tracheal intubation time, discharge time, intensive care unit (ICU) stay time, lung function, 6 Minutes Walk Distance (6MWD), Modified Barthel Index (MBI) and satisfaction rate were recorded and analyzed. Results: The observation group's first-time postoperative ambulation (t=2.10, P=0.039), indwelling tracheal intubation time (Z=2.864, P=0.004), and discharge time (t=3.111, P<0.001) were shorter than the control group, while the difference of ICU stay time was not statistically significant (Z=-1.658, P=0.097). Before treatment, there was no significant difference in the lung function, 6MWD, and MBI of the two groups (P>0.05). After treatment, the Forced Expiratory Volume In 1 s (FEV1)% (t=-2.707, P<0.001), forced vital capacity (FVC)% (t=-3.716, P<0.001), FEV1/FVC (t=-3.539, P<0.001), 6MWD (t=-5.567, P<0.001), and MBI indexes (t=-4.073, P<0.001) were better than in observation group. The satisfaction rate of the observation group was better than the control group (P<0.05). Conclusions: For lung transplant recipients, early extubation combined with a physical training program is scientific, safe, and feasible. This approach is helpful to promote the postoperative recovery of lung transplant patients, reduce the length of hospitalization, help patients improve their lung function and ability to engage in activities of daily living, and increase the satisfaction rate of postoperative recovery. Results show that the combination of early extubation and a physical training program is worthy of clinical promotion for lung transplant recipients. Trial Registration: Chinese Clinical Trial Registry ChiCTR2100051954.

Enhancement of inhibition rate of antibiotic against bacteria by molecularly imprinted nanoparticles targeting alarmone nucleotides as antibiotic adjuvants.

Antibiotic tolerance and resistance in bacteria have caused a great threat to humankind. Bacteria can rapidly accumulate alarmone nucleotides (guanosine tetra- and pentaphosphate, usually denoted as (p)ppGpp) to repair damaged DNA under adverse conditions. The inhibition synthetase enzyme activity of (p)ppGpp, indirectly preventing synthesis, or promoting degradation, has been reported; however, transferring these strategies to practical applications is still a challenging task due to the lack of highly effective molecules for these purposes. Here, an approach based on molecularly imprinted polymer nanoparticles (MIP-NPs) as antibiotic adjuvants was proposed, where MIP-NPs with specific recognition sites were used to capture alarmone nucleotides released by bacteria during stringent response activation. Enhanced inhibition rates of 40-80% were achieved in the presence of the MIP-NPs. The dose of antibiotic could be greatly reduced by utilizing the MIP-NPs as adjuvants for a similar deactivation effectiveness. Good biocompatibility (no obvious hemolysis or cytotoxic effects) and apparent antimicrobial efficiency for resisting wound infection in vivo support the fact that well-designed MIP-NPs have a bright future in dealing with the growing threat of antibiotic tolerance and resistance.

Deficiency of NEAT1 prevented MPP+-induced inflammatory response, oxidative stress and apoptosis in dopaminergic SK-N-SH neuroblastoma cells via miR-1277-5p/ARHGAP26 axis.

Noncoding RNAs including long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have been documented to play prominent role in neurodegenerative diseases including Parkinson's disease (PD). This study intended to investigate the role of lncRNA nuclear enriched assembly transcript 1 (NEAT1) in MPP+-induced PD model in dopaminergic neuronblastoma SK-N-SH cells, as well as its mechanism through sponging miRNA (miR)-1277-5p. Real-time PCR and western blotting revealed that NEAT1 and ARHGAP26 were upregulated, and miR-1277-5p was downregulated in MPP+-treated SK-N-SH cells in a certain of concentration- and time- dependent manner. MPP+ induced apoptosis in SK-N-SH cells, as evidenced by decreased cell viability and Bcl-2 expression, and elevated apoptosis rate and levels of Bax and cleaved caspase-3, which were examined by MTT assay, flow cytometry and western blotting. Moreover, commercial assay kits indicated that inflammatory response and oxidative stress were provoked in response to MPP+, due to promoted contents of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, malondialdehyde, and lactate dehydrogenase, accompanied with suppressed superoxide dismutase and glutathione peroxidase levels. Notably, MPP+-induced apoptosis, inflammatory response and oxidative stress in SK-N-SH cells were mitigated by NEAT1 knockdown and/or miR-1277-5p overexpression. Moreover, silencing of miR-1277-5p could abrogate the suppression of NEAT1 deficiency on MPP+-induced cell injury. Similarly, upregulating miR-1277-5p-elicited neuroprotection in MPP+-induced SK-N-SH cells was reversed by ARHGAP26 restoration. Dual-luciferase reporter assay demonstrated a direct interaction between miR-1277-5p and NEAT1 or ARHGAP26. Collectively, NEAT1 upregulation might contribute to MPP+-induced neuron injury via NEAT1-miR-1277-5p-ARHGAP26 competing endogenous RNAs (ceRNAs) pathway.

Knockdown of SNHG14 Alleviates MPP+-Induced Injury in the Cell Model of Parkinson's Disease by Targeting the miR-214-3p/KLF4 Axis.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease. Long non-coding RNA (lncRNA) small nucleolar RNA host gene 14 (SNHG14) has been demonstrated as an important regulator in PD pathology. However, the functional mechanisms played by SNHG14 in PD remain largely unclear. METHODS: We used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+) to establish PD mouse and cell models. The levels of SNHG14, miR-214-3p, and Krüppel-like factor 4 (KLF4) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot analysis. Cell viability and apoptosis were determined using the Cell Counting-8 Kit (CCK-8) assay and flow cytometry, respectively. The levels of inflammatory cytokines were evaluated by ELISA. The relationships among SNHG14, miR-214-3p, and KLF4 were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. RESULTS: Our data indicated that SNHG14 was upregulated and miR-214-3p was downregulated in PD models. SNHG14 knockdown ameliorated MPP+-stimulated damage in SK-N-SH cells, as evidenced by the enhancement in cell viability and the suppression in cell apoptosis and pro-inflammatory cytokine production. Mechanistically, SNHG14 directly targeted miR-214-3p via binding to miR-214-3p, and SNHG14 knockdown protected SK-N-SH cell from MPP+-stimulated cytotoxicity by upregulating miR-214-3p. KLF4 was a direct target of miR-214-3p, and SNHG14 regulated KLF4 expression by acting as a miR-214-3p sponge. Furthermore, miR-214-3p overexpression alleviated MPP+-stimulated damage in SK-N-SH cells by downregulating KLF4. CONCLUSION: Our current study first demonstrated the protective effect of SNHG14 knockdown on MPP+-stimulated cytotoxicity in SK-N-SH cells at least partially by targeting the miR-214-3p/KLF4 axis, illuminating a promising target for PD intervention and treatment.

A dual-functional MOF for high proton conduction and sensitive detection of ascorbic acid.

A new metal-organic framework (MOF), [Eu2(HBDPP)2(H2O)2(DMF)2](H2O)2 (H4BDPP = 3,5-bis(3,5-dicarboxylphenyl) pyridine; DMF = N,N-dimethylformamide) (Eu-MOF), has been successfully synthesized under solvothermal conditions. A 1D chain was formed by the adjacent Eu2(COO)24+ dinuclear cluster and HBDPP3-, and further connected by HBDPP3- to form an infinitely extended 3D structure. In order to further improve the proton conductivity of the Eu-MOF, imidazole was encapsulated in its pores to form a composite material named Im@Eu-MOF. AC impedance analysis shows that the highest proton conductivity of the Im@Eu-MOF reaches up to 4.53 × 10-4 S cm-1 at 348 K and 98% RH, which is about 10 times higher than that of the Eu-MOF. In addition, the Eu-MOF can be considered an excellent luminescence-based sensor with a high sensitivity and low detection limit (0.1 µM) for the detection of trace amounts of ascorbic acid.

The endogenous hydrogen sulfide producing enzyme cystathionine-beta synthase contributes to visceral hypersensitivity in a rat model of irritable bowel syndrome.

BACKGROUND: The pathogenesis of visceral hypersensitivity, a characteristic pathophysiological feature of irritable bowel syndrome (IBS), remains elusive. Recent studies suggest a role for hydrogen sulfide (H2S) in pain signaling but this has not been well studied in visceral models of hyperalgesia. We therefore determined the role for the endogenous H2S producing enzyme cystathionine-beta-synthetase (CBS) in a validated rat model of IBS-like chronic visceral hyperalgesia (CVH). CVH was induced by colonic injection of 0.5% acetic acid (AA) in 10-day-old rats and experiments were performed at 8-10 weeks of age. Dorsal root ganglion (DRG) neurons innervating the colon were labeled by injection of DiI (1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate) into the colon wall. RESULTS: In rat DRG, CBS-immunoreactivity was observed in approximately 85% of predominantly small- and medium-sized neurons. Colon specific DRG neurons revealed by retrograde labeling DiI were all CBS-positive. CBS-positive colon neurons co-expressed TRPV1 or P2X3 receptors. Western blotting analysis showed that CBS expression was significantly increased in colon DRGs 8 weeks after neonatal AA-treatment. Furthermore, the CBS inhibitor hydroxylamine markedly attenuated the abdominal withdrawal reflex scores in response to colorectal distention in rats with CVH. By contrast, the H2S donor NaHS significantly enhanced the frequency of action potentials of colon specific DRG neurons evoked by 2 times rheobase electrical stimulation. CONCLUSION: Our results suggest that upregulation of CBS expression in colonic DRG neurons and H2S signaling may play an important role in developing CVH, thus identifying a specific neurobiological target for the treatment of CVH in functional bowel syndromes.

A highly integrated real-time digital PCR device for accurate DNA quantitative analysis.

Misclassification of positive partitions in microfluidic digital polymerase chain reaction (dPCR) can cause the false positives and false negatives, which significantly alter the resulting estimate of target DNA molecules. To address this issue, establishing real-time fluorescence interrogation of each partition in microfluidic arrays is an effective way in which false positive and false negative partitions can be eliminated. However, currently available devices for real-time fluorescence interrogation are either not competent for microfluidic digital array, or they are bulky, expensive and entail peripheral equipment due to low integration. Therefore, in this study, a Raspberry Pi based, low-cost and highly integrated device is presented to achieve real-time fluorescence detection for microfluidic digital array, termed real-time dPCR device. In the device, uniform thermocycler, streamlined real-time fluorescence imaging setup, and compact data processing system are all integrated to undergo on-chip dPCR amplification, real-time fluorescence detection, and data analysis. Using this real-time dPCR device, the accuracy of DNA absolute quantification by dPCR is improved, since the misclassification of positive partitions is efficiently reduced based on the characteristic real-time fluorescence curves of positive partitions in a self-priming microfluidic chip. Compared with end-point dPCR on our device and commercialized QuantStudio™ 3D dPCR system, the real-time dPCR on our device exhibits a higher accuracy for DNA quantification. In addition, this real-time dPCR device is much smaller and cheaper than the commercialized Digital PCR system, but not sacrificing the capability of error correction for absolute quantitation analysis. Conclusively, this highly integrated real-time dPCR device is very beneficial for DNA quantitative analysis where the determination accuracy is pivotal.

Proximity ligation assays for precise quantification of femtomolar proteins in single cells using self-priming microfluidic dPCR chip.

The quantification of low concentration proteins can facilitate the discovery of some significant biomarkers, and provide us a more profound understanding of cell heterogeneity when applied to single cell analysis. However, most state-of- art single cell protein detection platforms are bulky, expensive and complicated. Here we report a simple and low cost microfluidic dPCR (digital polymerase chain reaction) chip-based proximity ligation assay (PLA) for the quantification of low concentration proteins. First, standard hCSTB (human cystatin B) protein was used to optimize the related experimental conditions. Comparing to ordinary PLA tests, the results showed that our method achieved femtomolar limit of detection (LOD) with a linear dynamic range over three to four orders of magnitude. Then human CD147 protein, a reported biomarker for hepatoma carcinoma, was detected in single HepG2 and L02 cells. The results showed that there were wide disparities in single cell CD147 abundance for both of the two cell lines. And the average CD147 protein content in single HepG2 cells displayed 2-fold increase in comparison to that in single L02 cells. Comparing to the research findings obtained at bulk level, our method can provide more useful information for diagnosis and targeted therapy of tumors.

Smartphone-based mobile digital PCR device for DNA quantitative analysis with high accuracy.

Digital polymerase chain reaction (dPCR) circumventing the external calibration and potentially providing absolute quantification of nucleic acids has become an increasingly popular manifestation of PCR in biological researches. However, currently reported or commercial dPCR devices are not suitable for applications in laboratories or zones with limited infrastructures, due to low function integration, cost-inefficiency, or weak mobility. Herein, in order to enable accurate DNA quantitative analysis in such situations, we have developed a smartphone-based mobile dPCR device integrated with thermal cycling control, on-chip dPCR, data acquisition, and result analysis. All the function units are automatically controlled using a customized Android software. The device is approximately 90 mm × 90 mm × 100 mm in size and about 500 g in weight, only costing about 320 dollars except the smartphone. Coupled with the self-priming dPCR chip previously developed by our lab, the device is able to accurately quantify down to 10 copies of the human 18 S ribosomal DNA fragment inserted in a plasmid. Comparing to the commercial QuantStudio™ 3D dPCR platform, our device achieves a comparable analytical accuracy. Besides, our device is capable of detecting single molecule of cancer biomarker gene CD147 in a low number of HepG2 cells. Therefore, our dPCR device as a low-cost, potable, and robust tool for highly accurate DNA quantitative analysis has a great potential in Point-of-care (POC) applications.

[Effects of sodium tanshinone II A sulfonate on proliferation of fibroblasts in scar and the mRNA and protein expressions of transforming growth factor beta 1 and alpha smooth muscle actin].

OBJECTIVE: To study the effects of sodium tanshinone II A sulfonate (STS) on proliferation of fibroblasts (Fbs) in human hypertrophic scar (HS), the mRNA and protein expressions of transforming growth factor beta 1 (TGF-ß1) and alpha smooth muscle actin (α-SMA), and to investigate the scar inhibition mechanism of STS. METHODS: Fbs were isolated from HS tissues that were removed from eight patients after burn injury, and they were cultured in vitro. Cells from the 3rd to the 6th passages were used in the experiment. Fbs were divided into control group and experimental group according to the random number table, and cells in the experimental group was divided into 0.050, 0.075, 0.100, 0.125, 0.150, 0.200 mg/mL STS subgroups. Cells in each subgroup were cultured with the corresponding concentration of STS, and cells in control group were cultured in equal volume of serum-free medium. After being cultured for 24 and 48 h, cell morphology was observed with inverted phase contrast microscope; cell proliferation was determined with MTT method and the proliferation inhibition rate (IR) was calculated. After being cultured for 48 h, the protein levels of TGF-ß1 and α-SMA were determined with Western blotting; the mRNA expressions of TGF-ß1 and α-SMA were determined with RT-PCR (no 0.200 mg/mL STS subgroup was set for these two indicators). Data were processed with factorial analysis of variance; differences between groups were processed with LSD test or Games-Howell test for unequal variances. RESULTS: (1) Fbs grew well in control group, but reduction in adherence and disorderly arranged Fbs were observed in experimental group. The cells in experimental group became smaller and round, with increasing intracellular particles and necrosis. A large amount of necrotic debris of cells was observed in 0.200 mg/mL STS subgroup. (2) The absorbance value of Fbs in each experimental subgroup was significantly lower than that in control group (with P values all below 0.01). Along with the increase in the concentration of STS and extension of culture time, the IR value increased, showing a certain degree of time-concentration dependence. After being cultured with STS for 24 and 48 h, IR values of cells in the experimental subgroups were respectively 23.58%, 32.11%, 37.56%, 57.98%, 79.53%, 96.69% and 34.72%, 38.48%, 47.62%, 64.40%, 89.70%, 98.01%. (3) Except for the 0.050 mg/mL STS subgroup, the protein levels of TGF-ß1 and α-SMA in the other subgroups were significantly lower than those in control group (with F values respectively 57.674, 47.795, P values all below 0.001). The protein levels of TGF-ß1 and α-SMA reached the nadir in 0.150 mg/mL STS subgroup, respectively 0.34 ± 0.06, 0.33 ± 0.07. The relative expression amounts of TGF-ß1 and α-SMA mRNA in the experimental subgroups were obviously decreased compared with those in control group (with F values respectively 68.548, 47.522, P values all below 0.001), which was most significant in 0.150 mg/mL STS subgroup, with TGF-ß1 mRNA and α-SMA mRNA respectively 0.39 ± 0.07 and 0.42 ± 0.08. CONCLUSIONS: STS can inhibit the proliferation of Fbs, reduce the protein and mRNA expressions of TGF-ß1 and α-SMA, which may be beneficial to ameliorate the formation and contracture of HS, and it is assumed as a potential drug for treating scars.

Effects of trichlorfon on maternal estrous cycle, oocyte maturation, and near-term fetal developmental outcome in mice.

Trichlorfon (TCF) is a widely used broad-spectrum agricultural organophosphate (OP) pesticide. Few studies have evaluated the effects of TCF on reproductive toxicity after low-level exposure, especially after long-term exposure. This study assessed the direct effects of TCF on estrous cycle, oocyte maturation in female mice, and developmental outcome in near-term fetuses after 30 consecutive days of maternal exposure to 2, 10, or 50 mg/kg body weight/d TCF via drinking water. Both male and female fetuses in the 50 mg/kg/d TCF-treated group had significantly reduced body weights; but this did not occur in the 2 mg/kg/d and 10 mg/kg/d TCF-treated groups. No difference in oocyte maturation, including the percentages of germinal vesicle breakdown (GVBD) and first polar body (PB1) extrusion, or in estrous cycle was found between the control and TCF-treated groups. No increased incidence of fetal external malformations was observed in the TCF-treated groups. Significant decreases in maternal liver weights occurred in the 10 and 50 mg/kg/d TCF-treated groups in a dose-dependent manner. No significant changes were found in the weight of organs such as the ovaries, thymus, kidneys, spleen, lungs, heart or brain. The lack of effects of 2 mg/kg/d and 10 mg/kg/d TCF on any in vivo reproductive and developmental endpoints examined suggest that no TCF reproductive toxicity occurs at exposures less than 10 mg/kg/d.