A Randomized, Controlled Trial of Continuous Heparin Infusion to Prevent Asymptomatic Catheter-Related Thrombosis at Discharge in Infants After Cardiac Surgery: The CHIP-CRT Trial.

OBJECTIVES: There are conflicting results in preventing catheter-related thrombosis (CRT). Continuing infusion of unfractionated heparin (UFH) was a potential option for CRT. This study was to determine the effect of continuous UFH infusion on asymptomatic CRT at discharge in infants after cardiac surgery. STUDY DESIGN: This study was a randomized, placebo-controlled, clinical trial at a single center. All infants with central venous catheters after cardiac surgery, below 3 months of age, were eligible. Stratified by CRT, infants were randomly assigned to the UFH group or the normal saline group. UFH was initiated at a speed of 10 to 15 units/kg/h for infants with CRT and 2 to 3 units/kg/h without CRT. The primary outcome was to determine the rate of CRT at discharge. The secondary outcomes included thrombosis 6 months after surgery, adverse events of UFH, and post-thrombotic symptoms. RESULTS: Due to slow recruitment during the COVID-19 pandemic, this trial was prematurely stopped. Only 35 infants were randomly assigned to the UFH or control groups. There was no statistically significant difference in CRT rate at discharge (P=0.429) and 6 months after surgery (P=1.000) between groups. All CRTs except one disappeared at discharge. No thrombosis or post-thrombotic symptom was reported at follow-up evaluation. There was no difference between groups in duration of thrombus (P=0.088), D dimer (P=0.412), catheter in situ days (P=0.281), and post-thrombotic syndrome (P=1.000), except for activated partial thromboplastin time (P=0.001). CONCLUSIONS: With the early stop of this trial and limited data, it is difficult to draw a definitive conclusion about the efficacy of UFH on CRT. Meanwhile, considering the data from 6 months follow-up, in this population, asymptomatic CRT might resolve with no intervention.

Design and Application of Portable Centrifuge Inspired by a Hand-Powered Spinning Top.

Traditional centrifuges, extensively employed in biology, chemistry, medicine, and other domains for tasks such as blood separation and pathogen extraction, have certain limitations. Their high cost, substantial size, and reliance on electricity restrict their range of application. Contemporary centrifuges, inspired by everyday items like paper trays and egg beaters, boast characteristics such as ease of operation, independence from electricity, and portability. These features offer unique advantages in specific situations, such as electricity shortages, inadequate infrastructure, and challenging medical conditions. Consequently, we designed a hand-powered portable centrifuge driven by pulling a rope. Our experiments revealed significant performance factors, including load capacity, rope length, and frequency of rope pulling. The results demonstrated that the revolutions per minute (RPM) of a hand-powered portable centrifuge were directly proportional to the length of the rope and the frequency of pulling, up to a certain limit, while inversely proportional to the load. When used for separating and washing polystyrene microspheres, the portable centrifuge's performance equaled that of traditional centrifuges. According to relevant calculations, this centrifuge could be capable of meeting the application of blood separation. Therefore, we believe this portable centrifuge will find meaningful applications in similar areas, particularly in resource-poor settings.

Research progress of electrode shapes in EWOD-based digital microfluidics.

Digital microfluidics (DMF) is an innovative technology used for precise manipulation of liquid droplets. This technology has garnered significant attention in both industrial applications and scientific research due to its unique advantages. Among the key components of DMF, the driving electrode plays a role in facilitating droplet generation, transportation, splitting, merging, and mixing. This comprehensive review aims to present an in-depth understanding of the working principle of DMF particularly focusing on the Electrowetting On Dielectric (EWOD) method. Furthermore, it examines the impact of driving electrodes with varying geometries on droplet manipulation. By analyzing and comparing their characteristics, this review offers valuable insights and a fresh perspective on the design and application of driving electrodes in DMF based on the EWOD approach. Lastly, an assessment of the development trend and potential applications of DMF concludes the review, providing an outlook for future prospects in the field.

Highly paralleled emulsion droplets for efficient isolation, amplification, and screening of cancer biomarker binding phages.

Based on the linkage of genotype and phenotype, display technology has been widely used to generate specific ligands for profiling, imaging, diagnosis and therapy applications. However, due to the lack of effective monoclonal manipulation and affinity evaluation methods, traditional display technology has to undergo tedious steps of selection, clone isolation, amplification, sequencing, synthesis and characterization to obtain the binding sequences. To directly acquire high-affinity clones, we propose a double monoclonal display approach (dm-Display) for peptide screening based on highly paralleled monoclonal manipulation in emulsion droplets. dm-Display can monoclonally link the genotype, phenotype and affinity to realize integrated monoclonal separation, amplification, recognition and staining in one droplet so that discrete high-affinity clones can be quickly extracted. Monoclonal manipulations highly-parallelly occur in millions of droplets so that molecular screening of a highly diverse phage library is achieved. We have screened specific peptide ligands against CD71 and GPC1, proving the feasibility and generality of dm-Display. As a highly efficient ligand screening platform, dm-Display will promote the further development of molecular screening.

Inhibition of protein FAK enhances 5-FU chemosensitivity to gastric carcinoma via p53 signaling pathways.

The small molecule drug 5-fluorouracil (5-FU) is widely used in the treatment for gastric cancer (GC), however, it exerts poor efficacy and is associated with acquired and intrinsic resistance. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays a key role in adhesion, migration, and proliferation of gastric carcinoma cells, suggesting that this kinase may be a promising therapeutic target. Differentially expressed FAK in GC tissue was detected by RT-qPCR and TCGA database analysis. To investigate the biological functions of FAK, loss-of-function experiments were performed. CCK-8 assay, colony formation assay, flow cytometry, dual-luciferase reporter assays, and western blot assays were conducted to determine the underlying mechanisms of FAK in 5-FU chemosensitivity in GC. FAK is overexpressed in GC patients, and positively correlated with poor prognosis. The use of shRNA interference to target FAK decreased proliferation and increased apoptosis of GC cells in vitro. Importantly, FAK silencing enhanced the therapeutic efficacy of 5-FU, leading to reduced tumor growth in vivo. We further demonstrated that FAK silencing increased 5-FU-induced caspase-3 activity, and promoted p53 transcriptional activities. Clinical data also has shown that patients with higher levels of FAK had significantly shorter overall survival (OS) and time to first progression (FP) than those with lower levels of FAK. These findings indicate that FAK plays a critical role in 5-FU chemosensitivity in GC, and the use of FAK inhibitors as an adjunct to 5-FU might be an effective strategy for patients who undergo chemotherapy.

A universal aptasensing platform based on cryonase-assisted signal amplification and graphene oxide induced quenching of the fluorescence of labeled nucleic acid probes: application to the detection of theophylline and ATP.

This study describes a universal fluorometric method for sensitive detection of analytes by using aptamers. It is based on the use of graphene oxide (GO) and cryonase-assisted signal amplification. GO is a strong quencher of FAM-labeled nucleic acid probes, while cryonase digests all types of nucleic acid probes. This makes the platform widely applicable to analytes for which the corresponding aptamers are available. Theophylline and ATP were chosen as model analytes. In the absence of targets, dye-labeled aptamers are in a flexible single strand state and adsorb on the GO. As a result, the probes are non-fluorescent due to the efficient quenching of dyes by GO. Upon the addition of a specific target, the aptamer/target complex desorbed from the GO surface and the probe becomes fluorescent. The released complex will immediately become a substrate for cryonase digestion and subsequently releasing the target to bind to another aptamer to initiate the next round of cleavage. This cyclic reaction will repeat again and again until all the related-probes are consumed and all fluorophores light up, resulting in significant fluorescent signal amplification. The detection limits are 47 nM for theophylline and 22.5 nM for ATP. This is much better than that of known methods. The assay requires only mix-and-measure steps that can be accomplished rapidly. In our perception, the detection scheme holds great promise for the design enzyme-aided amplification mechanisms for use in bioanalytical methods. Graphical abstract A cryonase-assisted signal amplification (CASA) method has been developed by using graphene oxide (GO) conjugated with a fluorophore-labeled aptamer for fluorescence signal generation. It has a large scope because it may be applied to numerous analytes.

Selection and identification of transferrin receptor-specific peptides as recognition probes for cancer cells.

Since the transferrin receptor (CD71 or TFRC) is known to be highly expressed in numerous cancers, CD71 has become an attractive target in cancer research. Acquiring specific molecular probes for CD71, such as small molecular ligands, aptamers, peptides, or antibodies, is of great importance for cancer cell recognition and capture. In this work, we chose CD71 as the target for phage display, and after four rounds of positive selection and one round of negative selection, the specific phage library was enriched. After verification and sequence analysis, six peptides were identified to be able to bind to CD71 with high specificity. The specific recognition of the CD71-positive cells was confirmed by flow cytometry and confocal microscopy. Competition experiments demonstrated that peptide Y1 and transferrin (TF) were bound to distinct sites on CD71, indicating that peptide Y1 could replace TF as a potential probe for cell imaging and drug delivery, thus avoiding competition by endogenous TF and side effects. Graphical abstract Six peptides were successfully isolated using in vitro biopanning against CD71 with high specificity and affinity. Peptides Y1 and Y2 would be powerful tools in biosensors and biomedicine due to their unique properties.

Advance in phage display technology for bioanalysis.

Phage display technology has emerged as a powerful tool for target gene expression and target-specific ligand selection. It is widely used to screen peptides, proteins and antibodies with the advantages of simplicity, high efficiency and low cost. A variety of targets, including ions, small molecules, inorganic materials, natural and biological polymers, nanostructures, cells, bacteria, and even tissues, have been demonstrated to generate specific binding ligands by phage display. Phages and target-specific ligands screened by phage display have been widely used as affinity reagents in therapeutics, diagnostics and biosensors. In this review, comparisons of different types of phage display systems are first presented. Particularly, microfluidic-based phage display, which enables screening with high throughput, high efficiency and integration, is highlighted. More importantly, we emphasize the advances in biosensors based on phages or phage-derived probes, including nonlytic phages, lytic phages, peptides or proteins screened by phage display, phage assemblies and phage-nanomaterial complexes. However, more efficient and higher throughput phage display methods are still needed to meet an explosion in demand for bioanalysis. Furthermore, screening of cyclic peptides and functional peptides will be the hotspot in bioanalysis.

Evolution of DNA aptamers for malignant brain tumor gliosarcoma cell recognition and clinical tissue imaging.

Gliosarcoma, a variant of glioblastoma multiforme (GBM), is a highly invasive malignant tumor. Unfortunately, this disease still marked by poor prognosis regardless of modern treatments. It is of great significance to discover specific molecular probes targeting gliosarcoma for early cancer diagnosis and therapy. Herein, we have selected a group of DNA aptamers with high affinity and selectivity against gliosarcoma cells K308 using cell-SELEX. All the dissociation constants of these aptamers against gliosarcoma cells were in the nanomolar range and aptamer WQY-9 has the highest affinity and good selectivity among them. Furthermore, truncated aptamer sequence, WQY-9-B, shows similar recognition ability to aptamer WQY-9. In addition, WQY-9-B was found to be able to bind selectively and internalize into cytoplasm of target cancer cell at 37 °C. More importantly, compared to a random sequence, aptamer WQY-9-B showed excellent recognition rate (73.3%) for tissue sections of clinical gliosarcoma samples. These data suggests that aptamer WQY-9-B has excellent potential as an effective molecular probe for gliosarcoma diagnosis.

A signal-on split aptasensor for highly sensitive and specific detection of tumor cells based on FRET.

We present here a signal-on fluorescence biosensor for highly sensitive and specific detection of tumor cells with a split aptamer based on fluorescence resonance energy transfer (FRET). This sensor holds considerable potential for simple, rapid, sensitive and specific tumor cell detection in early clinical diagnosis.

Cell-SELEX based selection and optimization of DNA aptamers for specific recognition of human cholangiocarcinoma QBC-939 cells.

Cholangiocarcinoma (CCA) is a very aggressive biliary tract malignancy with no efficient early diagnosis and therapeutics available, so there is a call for effective molecular probes. Herein, we performed cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) to obtain aptamers for the specific recognition of human cholangiocarcinoma QBC-939 cells. By coordinating sequence homology analysis and secondary structure analysis, we successfully obtained two aptamers with dissociation constants (Kd) in the low nanomolar range. A 23 nt truncated sequence was identified after further analysis on the secondary structure. More importantly, because hepatocellular carcinoma SMMC-7721 cells were employed as the control in the counter selection, the obtained aptamers demonstrated excellent specificity to the target cells, and no binding to several other hepatocellular carcinoma cell lines was observed. Moreover, the aptamers were initially found to recognize membrane proteins, giving them great potential in the field of biomarker discovery. These newly generated aptamers may play a key role in the early diagnosis and clinical treatment of CCA.

Discrimination of hemoglobins with subtle differences using an aptamer based sensing array.

Discrimination of hemoglobins with subtle differences was achieved using an aptamer based sensing array. Linear discriminant analysis (LDA) showed that the sensing array can discriminate human hemoglobins from hemoglobins of different species.

In vivo evaluation of renal function using diffusion weighted imaging and diffusion tensor imaging in type 2 diabetics with normoalbuminuria versus microalbuminuria.

This work aims to estimate the value of diffusion weighted imaging (DWI) and diffusion tensor imaging (DTI) in detecting early-stage kidney injury in type 2 diabetic patients with normoalbuminuria (NAU) versus microalbuminuria (MAU) prospectively. A total of 30 T2DM patients with normal kidney function were recruited and assigned to the NAU group (n = 14) or MAU group (n = 16) according to 8 h overnight urinary albuminuria excretion rate (AER) results. A contemporary cohort of health check-up recipients were included as controls (n = 12). DWI and DTI scans were performed on bilateral kidney using SE single-shot EPI, and apparent diffusion coefficient (ADC) and fractional anisotropy (FA) of the renal parenchyma was determined from ADC and FA maps of the three groups. ADC and FA values were compared among the three groups. According to DWI with a b value of 400 s/mm(2), the MAU and NAU groups showed significantly lowered mean ADC values compared with the healthy controls (P < 0.01). The mean ADC in the MAU group [(2.22 ± 0.07) × 10(-3)mm(2)/s] was slightly lower than that of the NAU group [(2.31 ± 0.22) × 10(-3)mm(2)/s], but this difference was not statistically significant (P > 0.05). The FA value in the MAU group was higher than that in the control group (0.45 ± 0.07 vs. 0.39 ± 0.03, P = 0.004) but did not differ from that in the NAU group (0.42 ± 0.03) (P > 0.05). ADC and FA values may be more sensitive than urine AER in reflecting early-stage kidney injury and, hence, may facilitate earlier detection and quantitative evaluation of kidney injury in T2DM patients. Combined evaluation of ADC and FA values may provide a better quantitative approach for identifying diabetic nephropathy at early disease stages.

Single-walled carbon nanotubes (SWCNTs)-assisted cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) for improving screening efficiency.

Separating the specific from the nonspecific bound single-strand DNA (ssDNA) is the most important step to improve the efficiency of selection procedure. However, most cell-SELEX protocols (where SELEX = systematic evolution of ligands by exponential enrichment) use simply washing only, which leads to incomplete separation. It is well-established that ssDNAs can be adsorbed on single-walled carbon nanotubes (SWCNTs). Based on this, herein, we developed a modified cell-SELEX approach termed "SWCNTs-assisted cell-SELEX". In our approach, SWCNTs are applied in the separation step, during which the unbound or the nonspecific ssDNAs are adsorbed onto SWCNTs, while the bound ssDNAs still remain on the cell surface, because of the stronger interaction between ssDNA and target. The cells can then be centrifuged to enrich the specifically binding aptamers. As a proof of concept, two nasopharyngeal carcinoma (NPC) cell lines-CNE2 cell and HONE cell-were used as the target cell and the negative cell, respectively. The result show that it takes only 6 cycles to enrich the aptamer pool through the SWCNTs-assisted cell-SELEX, which is much shorter than 15 cycles in the conventional cell-SELEX, thus improving the screening efficiency. Moreover, the achieved aptamers show high specificity and affinity with CNE2 cells, which are highly attractive for clinical diagnosis and biomedicine applications.

Proximity-dependent protein detection based on enzyme-assisted fluorescence signal amplification.

In this paper, we develop a sensitive fluorescence method for protein detection based on proximity extension and enzyme-assisted signal amplification. In this novel method, pairs of proximity probes are designed, and the recognition elements are integrated into the proximity probes. Then proteins are detected by transforming aptamer or antibody-protein binding signals into DNA detection based on proximity effect. In addition, nick sites are introduced into the proximity probes to amplify the detectable signal. As proof of concept, detection of human α-thrombin and human IgG are demonstrated in this study. The aptamers and antibodies are coupled in the proximity probes as recognition elements for human α-thrombin and human IgG respectively. In the presence of target protein, aptamer or antibody-protein binding signals are transformed into detectable signals by the proximity effect, and can be further amplified by enzyme-assisted strand displacement. The above mentioned strategies consequently bring the limit of detection (LOD) to as low as 1 pM for human α-thrombin and 6 pM for human IgG. Furthermore, this method might be extended to sensitive detection of other proteins by changing recognition elements of proximity probes.

Abnormal DNA methylation in CD4+ T cells from people with latent autoimmune diabetes in adults.

Aberrant DNA methylation in T cells has been linked to pathogenesis of autoimmune diseases. To investigate genomic and gene-specific DNA methylation levels in CD4(+) T cells from patients with latent autoimmune diabetes in adults (LADA), and to investigate changes in the expression of genes that regulate methylation as well as the autoimmune-related gene FOXP3 in these patients. Global CD4(+) T cell DNA methylation was measured in 15 LADA patients and 11 healthy controls using a methylation quantification kit. mRNA levels of DNA methytransferases (DNMTs), methyl-DNA binding domain proteins (MBDs) and FOXP3 were measured by real time PCR. Methylation of a FOXP3 regulatory element region was determined by bisulphite genomic sequencing. Genomic DNA methylation in CD4(+) T cells from LADA patients was significantly increased compared to controls. DNMT3b mRNA levels were higher in CD4(+) T cells from LADA patients than in controls. DNMT3b expression positively correlated with global DNA methylation in LADA CD4(+) T cells. FOXP3 expression was decreased, and the FOXP3 promoter region was hypermethylated in CD4(+) T cells from LADA patients compared with controls. DNA methylation levels are altered in CD4(+) T cells from LADA patients, which may contribute to disease onset and progression by affecting the expression of autoimmune-related genes.

Gene expression changes in patients with fulminant type 1 diabetes.

BACKGROUND: Fulminant type 1 diabetes (F1D) is a complex disease. Microarray analysis was used to identify gene expression changes and obtain understanding of the underlying mechanisms. METHODS: Microarray analysis was performed on peripheral blood mononuclear cells from six F1D patients and six matched healthy subjects. Real-time polymerase chain reaction was used to verify the differentially expressed genes. NK cell activity was detected by methyl thiazoleterazolium assay. RESULTS: Microarray analysis identified 759 genes differing in expression between F1D patients and controls at a false discovery rate of 0.05. Expression of TLR9, ELF4 and IL1RAP were verified and consistent with changes in microarray results. NK cell activity was decreased in F1D. With use of a knowledge base, differentially expressed genes could be placed within different pathways with predicted functions including interleukin-1, and tumor necrosis factor-α signaling. CONCLUSIONS: These results identify several genes indicating possible mechanisms in F1D. NK cell dysfunction resulting from changes in expression of TLR9, ELF4 and IL1RAP, and pathways of interleukin-1 and tumor necrosis factor-α signaling might be involved in F1D through inducing ß-cell dysfunction.

Amplified electrochemical DNA sensor using peroxidase-like DNAzyme.

A novel electrochemical DNA sensor was developed here by using peroxidase-like G-quadruplex-based DNAzyme as a biocatalytic label. A hairpin structure including the G-quadruplex-based DNAzyme in a caged configuration and the target DNA probe were immobilized on Au-electrode surface. Upon hybridization with the target, the hairpin structure was opened, and the G-quadruplex-based DNAzyme was generated on the electrode surface, triggering the electrochemical oxidization of hydroquinone by H(2)O(2), which provide a quantitative measure for the detection of the target DNA. The DNA target was analyzed with a detection limit of 0.6 nM. This method is simple and easy to design without direct conjugation of redox-active element.

[Killer cell immunoglobin-like receptor and its ligand gene polymorphisms in Hunan Han patients with type 1 diabetes].

OBJECTIVE: To investigate the association of Killer cell immunoglobin-like receptor (KIR) gene and KIRs'ligand (HLA-C) gene polymorphisms with type 1 diabetes (T1DM). METHODS: Using polymerase chain reaction-sequence specific primer (PCR-SSP) to detect KIR and HLA-C genotype in 180 T1DM patients and 199 healthy controls from Hunan Han population. RESULTS: (1) The frequencies of KIR 2DL1 (98.9% vs 92.0%, OR = 7.78, P = 0.002), 3DL1 (94.3% vs 86.4%, OR = 2.67, P = 0.009) and 2DS4 (83.9% vs 70.9%, OR = 2.14, P = 0.003) were significantly higher in T1DM patients than those in the controls. (2) There were no differences in the frequencies of HLA-C1 and HLA-C2 between the patients and the controls, but the frequency of HLAC1+/C2+ (3.9% vs 9.6%, OR = 0.38, P = 0.03) was significantly lower in the T1DM patients. (3) The combination KIR2DL1-/HLA-C2-(0.6% vs 6.0%, OR = 0.087, P = 0.003) and KIR 2DS1-/HLA-C2-(53.3% vs 64.8%, OR = 0.62, P = 0.023) was significantly lower in the T1DM patients. CONCLUSION: The KIR gene polymorphism and KIR/HLA-C gene compatibility are associated with T1DM.