Hand-Powered Microfluidics for Parallel Droplet Digital Loop-Mediated Isothermal Amplification Assays.

Droplet digital loop-mediated isothermal amplification (ddLAMP) is an important assay for pathogen detection due to its high accuracy, specificity, and ability to quantify nucleic acids. However, performing ddLAMP requires expensive instrumentation and the need for highly trained personnel with expertise in microfluidics. To make ddLAMP more accessible, a ddLAMP assay is developed, featuring significantly decreased operational difficulty and instrumentation requirements. The proposed assay consists of three simplified steps: (1) droplet generation step, in which a LAMP mixture can be emulsified just by manually pulling a syringe connected to a microfluidic device. In this step, for the first time, we verify that highly monodispersed droplets can be generated with unstable flow rates or pressures, allowing untrained personnel to operate the microfluidic device and perform ddLAMP assay; (2) heating step, in which the droplets are isothermally heated in a water bath, which can be found in most laboratories; and (3) result analysis step, in which the ddLAMP result can be determined using only a fluorescence microscopy and an open-source analyzing software. Throughout the process, no droplet microfluidic expertise or equipment is required. More importantly, the proposed system enables multiple samples to be processed simultaneously with a detection limit of 10 copies/µL. The test is simple and intuitive to operate in most laboratories for multi-sample detection, significantly enhancing the accessibility and detection throughput of the ddLAMP technique.

A Multifunctional Microfluidic Device for Blood Typing and Primary Screening of Blood Diseases.

In this work, we demonstrate a multifunctional, portable, and disposable microfluidic device for blood typing and primary screening of blood diseases. Preloaded antibodies (anti-A, anti-B, and anti-D) interact with injected whole blood cells to cause an agglutination reaction that blocks a microslit in the microfluidic channel to accumulate red blood cells and form a visible red line that can be easily read to determine the blood type. Moreover, the different blood density and agglutination properties of normal and subtype blood groups, as well as different blood diseases, including anemia and polycythemia vera, generate different lengths of blood agglutination within the channels, which allows us to successfully screen these various conditions in as little as 2 min. The required blood volume for each test is just 1 µL, which can be obtained by minimally invasive finger pricking. This novel method of observing agglutinated red blood cells to distinguish blood types and diseases is both feasible and affordable, suggesting its promise for use in areas with limited resources.

Simultaneous Determination of l-Phenylalanine, Phenylethylamine, and Phenylacetic Acid Using Three-Color Whole-Cell Biosensors within a Microchannel Device.

The neurotransmitter phenylethylamine (PEA) is highly susceptible to oxidation to produce phenylacetic acid (PA). The fact that PEA and PA are both metabolites of phenylalanine (Phe) in humans makes them important indicators in the diagnosis of phenylketonuria. In this work, three-color whole-cell biosensors were developed to simultaneously detect these analytes (Phe, PEA, and PA). The tyrosine-responsive promoter was used to control the production of green fluorescent protein signals in response to Phe levels. The FeaR regulon was first used to indicate the presence of PEA, whereas the Paa regulon was used for the detection of PA. The combination of three sensor strains together made it possible to semiquantify the three analytes according to unique color outputs without cross-interference. We sought to optimize various modular components (ribosomal binding sites and fluorescent proteins) to ensure the rapid generation of fluorescent signals. Finally, the biosensors were implemented within a microchannel device to reduce sample consumption in point-of-care assays.

Mechanism of CNP-mediated DG-PKC and IP4 signaling pathway in diabetic rats with gastric motility disorder.

In the precedent research conducted by the same team, it concluded that the activities in C-type natriuretic peptide (CNP)/cyclic guanosine monophosphate (cGMP)/cyclic adenosine monophosphate (cAMP)/ß-type phospholipase C (PLCß) pathways of rat antral smooth muscle were changed due to diabetes, which was the key pathogenetic mechanism for diabetic gastric dysmotility. As the follow-on step, this study was designed to probe into the downstream signaling pathway of CNP/PLCß. The results showed that level of α-type protein kinase C (PKCα),cell membrane to cytoplasm ratio of PKCα, cell membrane to cytoplasmic ratio of ßI-type protein kinase C (PKCßI) and level of Phosphor-PKCα (P-PKCα) were significantly reduced in diabetes rat antral smooth muscle samples. The content of tetraphosphate inositol (IP4) in gastric antral smooth muscle of diabetic rats reduced, and the content of diacyl-glycerol (DG) was unchanged. CNP significantly decreased the content of IP4 and DG, this effect was more obvious in diabetic rats. Subsequent to the addition of protein kinase A (PKA) blocker N-[2- (p-Bromocin-namylamino)ethyl]-5 -isoquinolinesulfonamide dihydrochloride (H-89) before CNP treatment, the inhibitory effect of CNP was reduced; subsequent to the addition of protein kinase G (PKG) blocker KT5823 before CNP treatment, the inhibitory effect of CNP was also reduced. With the addition of the combination of H-89 and KT5823 before CNP treatment, the inhibition by CNP could be offset. These results were concluded that CNP inhibited the activity of PKC family in rat smooth muscle and reduced the levels of IP4 and DG through the PKG/PKA-PLCß pathways, causing inhibited muscular contractions, which may be a key pathogenetic factor for diabetic gastroparesis.

Correction to: Effects of AMPK on Apoptosis and Energy Metabolism of Gastric Smooth Muscle Cells in Rats with Diabetic Gastroparesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effects of AMPK on Apoptosis and Energy Metabolism of Gastric Smooth Muscle Cells in Rats with Diabetic Gastroparesis.

This study aimed to investigate the effect of AMPK on apoptosis and energy metabolism of gastric smooth muscle cells in diabetic rats and to explore the role of AMPK in the pathogenesis of diabetic gastroparesis (DGP). After establishment of a diabetic rat model, rats were divided into normal control (NC), 4-week (DM4W), 6-week (DM6W), and 8-week (DM8W) diabetic model groups. The gastric residual pigment ratio, intestinal transit rate, and intestinal propulsion rate in each group were detected to confirm the successful establishment of the DGP model. The spontaneous contraction in isolated gastric smooth muscle strips of the NC and DM8W groups was experimentally observed. The expression of phospho-AMPK, AMPK, phospho-LKB1, LKB1, phospho-TAK1, TAK1, and CaMMKß in rat gastric smooth muscle tissues was detected by western blot analysis; ADP, AMP, ATP contents, and the energy charge were detected using Elisa; and apoptosis of gastric smooth muscle cells was detected by flow cytometry. The rat gastric smooth muscle cells were cultured in vitro, and treated with an AMPK inhibitor and an agonist. At 24 and 48 h, the effects of AMPK on apoptosis and energy metabolism of gastric smooth muscle cells were observed. Reduced spontaneous contractions, AMPK activation, cell apoptosis, and energy metabolism disorders were observed in gastric smooth muscle tissues of a diabetic rat, and AMPK activation was associated with an increased ratio of ADP/ATP, AMP/ATP, LKB1 activity, and CaMMKß expression. From in vitro cell culture experiments, we found that AMPK activation of high-glucose conditions promoted cell apoptosis. Inhibition of AMPK had no obvious effect on apoptosis at the early stage with high glucose, but the inhibitory effect was significant at the late stage with high glucose. AMPK can regulate both mitochondrial metabolism and glycolysis pathways under high-glucose conditions. During the early stage with high glucose, AMPK was the main promotion factor of the mitochondrial metabolism pathway, but did not increase the ATP production, AMPK also promoted the glycolysis pathway. During the late stage with high glucose, AMPK was a major inhibitor of the mitochondrial pathway, and still played a role in promoting the glycolytic pathway, which acted as the main regulator. Apoptosis and energy metabolism disorders were present in gastric smooth muscle cells during the occurrence of DGP. Under high-glucose condition, AMPK was activated, which can promote apoptosis, change the energetic metabolism pathway of cells, inhibit mitochondrial energy metabolism, and promote glycolysis.

Effects of insulin-like growth factor-1 on endoplasmic reticulum stress and autophagy in rat gastric smooth muscle cells cultured at different glucose concentrations in vitro.

The purpose of the study was to observe changes in endoplasmic reticulum stress (ERS)- and autophagy-related proteins in gastric smooth muscle tissues of diabetic rats with gastroparesis, investigate the effect of insulin-like growth factor 1 (IGF-1) on ERS and autophagy in rat gastric smooth muscle cells cultured under different glucose concentrations, and explore the influence of IGF-1 on development of diabetic gastroparesis (DGP). After establishing a rat model of DGP, rats were divided into normal control (NC) and 6-week diabetic model (DM6W) groups. Expression of ERS-related and autophagy-related proteins was detected by western blot analysis and immunofluorescence assay in rat gastric smooth muscle tissue and in vitro-cultured rat gastric smooth muscle cells exposed to different glucose concentrations and treatment with IGF-1 for 24 or 48 h. Changes in glucose-regulated-protein-78 (GRP78), growth arrest and DNA damage-inducible gene 153 (CHOP), and microtubule-associated protein 1A/1B light chain 3B (LC3) expression levels were detected by western blot analysis, and GRP78 and LC3 expression were examined by confocal laser-scanning microscopy. In vivo expression levels of GRP78, CHOP, and LC3 were significantly higher in the DM6W group compared with the NC group (p < 0.001). Twenty-four hours after cells were cultured at different glucose concentrations in vitro, expression of GRP78, CHOP, and LC3II/I was significantly higher in the high glucose-treated group compared with the normal glucose group (p < 0.05). After IGF-1 intervention, CHOP and GRP78 expression were significantly higher in the normal glucose + IGF-1 group compared with the normal glucose group (p < 0.01), while no significant difference was found between high glucose and high glucose + IGF-1 groups. LC3II/I expression was significantly lower in the normal glucose + IGF-1 group compared with the normal glucose group, and was significantly lower in the high glucose and high glucose + IGF-1 groups (p < 0.05). After 48 h of culture, CHOP expression was significantly higher and LC3II/I expression was significantly lower in the high glucose group compared with the normal glucose group (p < 0.05), but no significant change in GRP78 expression was observed between these two groups. After IGF-1 intervention, there was no difference in CHOP or GRP78 expression between normal glucose + IGF-1 and normal glucose groups. However, CHOP and GRP78 expression were significantly lower in the high glucose + IGF-1 group compared with the high glucose group (p < 0.05). There was no significant difference in LC3II/I expression between normal glucose + IGF-1 and normal glucose groups, or high glucose + IGF-1 and high glucose groups. Results of confocal laser-scanning microscopy showed significantly lower expression of LC3II/I in the high glucose + IGF-1 group compared with the high glucose group (p < 0.05). ERS and autophagy were involved in the occurrence of DGP. IGF-1 exerted an inhibitory effect on ERS in rat gastric smooth muscle cells cultured under high glucose conditions, and this inhibitory effect increased with time. IGF-1 inhibited the level of autophagy in rat gastric smooth muscle cells cultured under high glucose conditions at early stages, which may be achieved through inhibition of ERS.

The role of CNP-mediated PKG/PKA-PLCß pathway in diabetes-induced gastric motility disorder.

Our previous work demonstrated that the C-type natriuretic peptide (CNP)/cyclic guanosine monophosphate (cGMP)/cyclic adenosine monophosphate (cAMP) pathway in gastric antrum smooth muscle of rats with diabetes was upregulated and played an important role in the development of diabetic gastric dysmotility. Our goal for this study was to explore the downstream signaling pathways of CNP. We found that the expressions of protein kinase G (PKG) and protein kinase A (PKA) in gastric smooth muscle tissue of rats with diabetes were significantly upregulated. The expressions of ß-type phospholipase C 3(PLCß3) and ß-type phospholipase C 1(PLCß1) protein were reduced, whereas Phosphor-PLCß3Ser1105 (P-PLCß3Ser1105) was increased. The inhibitory effect of CNP on gastric antral smooth muscle in diabetic rats was significantly greater than in the normal group. The content of trisphosphate inositol (IP3) in the gastric antral smooth muscle of rats with diabetes was significantly lower than that of the normal group. After blocking PKA with N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89, a blockage PKA), the effect of CNP on the production of IP3 was decreased, while blocking PKG with KT5823 (a blockage PKG) simultaneously, and CNP can no longer reduce the IP3 production. CNP promoted the phosphorylation of PLCß3Ser1105, thereby inhibiting the activity of PLCß3 in gastric smooth muscle tissue of rats with diabetes; this effect can be abolished by blocking PKA and PKG. These results suggested that CNP can decrease IP3 level in gastric smooth muscle cells and thus inhibit gastric smooth muscle contraction through PKG/PKA-PLCß pathway, which may play an important role in the development of diabetic gastroparesis.

[Association of the IL-18 gene polymorphism with susceptibility to colorectal cancer].

OBJECTIVE: To investigate single nucleotide polymorphisms(SNPs) and haplotypes of interleukin-18(IL-18) gene associated with the susceptibility to colorectal cancer(CRC). METHODS: Two SNPs of IL-18 gene promoter -137G/C and -607C/A in 170 patients with CRC and 160 healthy controls matched by age and sex in a Chinese population were analyzed using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) strategy. Frequency of haplotypes and linkage disequilibrium of IL-18 gene in different groups were analyzed by SHEsis programs. RESULTS: The distributions of IL-18 gene -607C/A polymorphism did not differ between CRC patients and healthy controls, but IL-18 gene -137G/C polymorphism was significantly different(P<0.05). The relative risk of C allele for CRC was 1.814 times of the G allele (OR=1.814,95% CI:1.246-2.642). Consistent with the results of the genotyping analyses, IL-18 -137G/C and -607C/A polymorphisms showed strong linkage disequilibrium(|D'|=0.945), frequency of the -137C/-607A haplotype in patients with CRC was significantly higher than that in healthy controls(P<0.05). The -137C/-607A haplotype was associated with a significantly increased risk of CRC(OR=1.637, 95% CI:1.100-2.437). CONCLUSIONS: IL-18 gene -137G/C polymorphism and -137C/-607A haplotype are associated with CRC. -137C allele may be an important genetic susceptibility gene for CRC.

DCE-MRI pixel-by-pixel quantitative curve pattern analysis and its application to osteosarcoma.

PURPOSE: To present a novel curve pattern analysis (CPA) method to characterize and quantify signal curves from the dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) data without any prerequisites such as arterial input function (AIF) or T(1) measurement. MATERIALS AND METHODS: CPA parameters represent characteristics of the scaled DCE signal curve. Simulations were performed to investigate the dependence of CPA parameters on T(1), TR, and flip angle. In vivo studies were performed on five pediatric patients with osteosarcoma. Parametric maps were generated using the CPA method and a pharmacokinetic model-based method for comparison. RESULTS: Simulations show that CPA parameters varied less than 2% when T(1) changed from 300 msec to 1500 msec, and less than 10% when the flip angle changed from 30 degrees to 40 degrees. Various curve patterns can be qualitatively identified and recognized from CPA parameter maps. Simulation and in vivo studies showed that the CPA parameter had a strong correlation with k(ep), with correlation coefficients of 0.9983 in the simulation and 0.95 in the in vivo studies. CONCLUSION: A novel CPA method is presented. Simulations and in vivo studies showed that the CPA method provides a feasible alternative to quantifying DCE-MRI studies with possibly higher repeatability by minimizing variations potentially induced by AIF and T(1) estimations and model dependence.

Dynamic contrast-enhanced magnetic resonance imaging parameters independent of baseline T10 values.

A baseline T(10) value is typically needed for dynamic contrast-enhanced (DCE-) MRI studies. However, an assumed baseline T(10) has to be used when T(10) measurements for patients are not available. In this work, we systematically investigate the dependence on T(10) of the commonly used DCE-MRI parameters (K(trans), k(ep), v(e) and IAUC) as well as several newly defined parameters [the normalized ratios (NRs) of k(ep), K(trans) and v(e), which are measures of relative changes in these parameters between two time points] for a spoiled gradient-echo pulse sequence using simulations and in vivo studies. Effects of various factors on the T(10) dependence, such as the true T(10) value, flip angle and the potential changes in T(10) due to treatment, were also assessed using simulations. We found that DCE-MRI parameters k(ep) and the NR of k(ep) are largely independent of T(10), especially when larger flip angles are used (e.g., 30-40 degrees). Their estimations therefore do not require any knowledge of T(10). The NRs of K(trans), v(e) and IAUC also exhibit independence to T(10), but only when T(10) remains constant between pre- and posttreatment. The estimation of parameters themselves (K(trans), v(e) and IAUC) is highly dependent on the T(10) value.

Evaluation of motion effects on parallel MR imaging with precalibration.

Several parallel imaging techniques such as SMASH, SENSE, k-space inherited parallel acquisition (KIPA) and others use reference (calibration) scans to find the parameters required for image reconstruction. Reference data is used to estimate coil sensitivity profiles for image domain techniques such as SENSE or reconstruction coefficients for k-space domain methods such as SMASH and KIPA. Any motion between the reference and accelerated imaging scans can make the reconstruction coefficients determined from the reference scan data suboptimal, resulting in an artifactual reconstruction. This work aims at comparing the effects of motion on the performance of three parallel imaging methods: SENSE, variable-density SENSE and KIPA, which all require one or more reference scans for calibration.

k-space inherited parallel acquisition (KIPA): application on dynamic magnetic resonance imaging thermometry.

In this study, a novel method for dynamic parallel image acquisition and reconstruction is presented. In this method, called k-space inherited parallel acquisition (KIPA), localized reconstruction coefficients are used to achieve higher reduction factors, and lower noise and artifact levels compared to that of generalized autocalibrating partially parallel acquisition (GRAPPA) reconstruction. In KIPA, the full k-space for the first frame and the partial k-space for later frames are required to reconstruct a whole series of images. Reconstruction coefficients calculated for different segments of k-space from the first frame data set are used to estimate missing k-space lines in corresponding k-space segments of other frames. The local determination of KIPA reconstruction coefficients is essential to adjusting them according to the local signal-to-noise ratio characteristics of k-space data. The proposed algorithm is applicable to dynamic imaging with arbitrary k-space sampling trajectories. Simulations of magnetic resonance thermometry using the KIPA method with a reduction factor of 6 and using dynamic imaging studies of human subjects with reduction factors of 4 and 6 have been performed to prove the feasibility of our method and to show apparent improvement in image quality in comparison with GRAPPA for dynamic imaging.

Improved accuracy and consistency in T1 measurement of flowing blood by using inversion recovery GE-EPI.

Problems associated with techniques currently used to measure the T1 of flowing blood are evaluated and a method to improve the consistency and repeatability of measurements is presented. Similar to some currently used techniques, the pulse sequence employs a nonselective adiabatic inversion pulse followed by a series of ECG-gated gradient echo EPI (echo planar imaging) images to obtain images where the blood (fluid) signal exhibits a T1-dependent inversion recovery signal from which the spin lattice relaxation constant (T1) of the flowing fluid can be measured. The new method combines curve fitting with a measure of the curve null point to acquire more accurate and consistent T1 values. Simulation and experimental results show that this combined fitting-nulling method is more stable and consistent in measuring the T1 of flowing fluid. The feasibility of temperature measurement of a flowing fluid based on the temperature dependence of the T1 of water protons is shown in this paper. ECG gating is used to reduce the effects of cyclic intensity changes for measurement of T1 in pulsatile flowing blood.

Clinical observation on acupuncture at Neimadian for analgesia postoperation of extremities / 中国针灸

<p><b>OBJECTIVE</b>To observe therapeutic effect of acupuncture at Neimadian on pain after operation of four limbs.</p><p><b>METHODS</b>Sixty-two patients were randomly divided into an observation group and a control group, 31 cases in each group. The observation group were treated with electroacupuncture at Neimadian for 30 min, and the control group with oral administration of tramadoli hydrochloridum. Changes of pain within 24 hours were observed.</p><p><b>RESULTS</b>The analgesic effect in the observation group was better than that in the control group (P < 0.05).</p><p><b>CONCLUSION</b>Analgesic effect of acupuncture at Neimadian on pain after operation of four limbs is superior to that of oral administration of tramadoli.</p>

Clinical observation on analgesic effect of electroacupuncture at Neimadian after operation of extremities / 中国针灸

<p><b>OBJECTIVE</b>To observe effectiveness and safety of electroacupuncture at Neimadian for analgesia in the extremities after orthopedic operation.</p><p><b>METHODS</b>Two hundred cases enrolled were divided into two groups. The test group of 100 cases were treated with electroacupuncture at Neimadian and oral administration of placebo, and the control group of 100 cases with oral administration of tramadoli hydrochloride.</p><p><b>RESULTS</b>The mean score for pain signs at all the time points before and after analgesic treatment in the test group had more decreases as compared with the control group (P < 0.001); and in the good rate after treatment, the test group was higher than the control group (P < 0.001, P < 0.05), and for safety, the test group was higher than the control group (P < 0.001).</p><p><b>CONCLUSION</b>The analgesic effect and safety of electroacupuncture at Neimadian are superior to the routine analgesic after operation of the extremities.</p>