The Development of a Sensitive Droplet Digital Polymerase Chain Reaction Test for Quantitative Detection of Goose Astrovirus.

(1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections.

A new method incorporating deep learning with shape priors for left ventricular segmentation in myocardial perfusion SPECT images.

Accurate segmentation of the left ventricle (LV) is crucial for evaluating myocardial perfusion SPECT (MPS) and assessing LV functions. In this study, a novel method combining deep learning with shape priors was developed and validated to extract the LV myocardium and automatically measure LV functional parameters. The method integrates a three-dimensional (3D) V-Net with a shape deformation module that incorporates shape priors generated by a dynamic programming (DP) algorithm to guide its output during training. A retrospective analysis was performed on an MPS dataset comprising 31 subjects without or with mild ischemia, 32 subjects with moderate ischemia, and 12 subjects with severe ischemia. Myocardial contours were manually annotated as the ground truth. A 5-fold stratified cross-validation was used to train and validate the models. The clinical performance was evaluated by measuring LV end-systolic volume (ESV), end-diastolic volume (EDV), left ventricular ejection fraction (LVEF), and scar burden from the extracted myocardial contours. There were excellent agreements between segmentation results by our proposed model and those from the ground truth, with a Dice similarity coefficient (DSC) of 0.9573 ± 0.0244, 0.9821 ± 0.0137, and 0.9903 ± 0.0041, as well as Hausdorff distances (HD) of 6.7529 ± 2.7334 mm, 7.2507 ± 3.1952 mm, and 7.6121 ± 3.0134 mm in extracting the LV endocardium, myocardium, and epicardium, respectively. Furthermore, the correlation coefficients between LVEF, ESV, EDV, stress scar burden, and rest scar burden measured from our model results and the ground truth were 0.92, 0.958, 0.952, 0.972, and 0.958, respectively. The proposed method achieved a high accuracy in extracting LV myocardial contours and assessing LV functions.

Automatic reorientation by deep learning to generate short-axis SPECT myocardial perfusion images.

BACKGROUND: Single photon emission computed tomography (SPECT) myocardial perfusion images (MPI) can be displayed both in traditional short-axis (SA) cardiac planes and polar maps for interpretation and quantification. It is essential to reorient the reconstructed transaxial SPECT MPI into standard SA slices. This study is aimed to develop a deep-learning-based approach for automatic reorientation of MPI. METHODS: A total of 254 patients were enrolled, including 226 stress SPECT MPIs and 247 rest SPECT MPIs. Fivefold cross-validation with 180 stress and 201 rest MPIs was used for training and internal validation; the remaining images were used for testing. The rigid transformation parameters (translation and rotation) from manual reorientation were annotated by an experienced nuclear cardiologist and used as the reference standard. A convolutional neural network (CNN) was designed to predict the transformation parameters. Then, the derived transform was applied to the grid generator and sampler in spatial transformer network (STN) to generate the reoriented image. A loss function containing mean absolute errors for translation and mean square errors for rotation was employed. A three-stage optimization strategy was adopted for model optimization: (1) optimize the translation parameters while fixing the rotation parameters; (2) optimize rotation parameters while fixing the translation parameters; (3) optimize both translation and rotation parameters together. RESULTS: In the test set, the Spearman determination coefficients of the translation distances and rotation angles between the model prediction and the reference standard were 0.993 in X axis, 0.992 in Y axis, 0.994 in Z axis, 0.987 along X axis, 0.990 along Y axis and 0.996 along Z axis, respectively. For the 46 stress MPIs in the test set, the Spearman determination coefficients were 0.858 in percentage of profusion defect (PPD) and 0.858 in summed stress score (SSS); for the 46 rest MPIs in the test set, the Spearman determination coefficients were 0.9 in PPD and 0.9 in summed rest score (SRS). CONCLUSIONS: Our deep learning-based LV reorientation method is able to accurately generate the SA images. Technical validations and subsequent evaluations of measured clinical parameters show that it has great promise for clinical use.

Stable and tunable single-mode lasers based on cholesteric liquid crystal microdroplets.

Although many studies on cholesteric liquid crystal (CLC) microdroplet single-mode lasers are available, it has been shown that the stability and tunability of such microdroplets are difficult to achieve simultaneously. In this paper, a new, to the best of our knowledge, method is proposed for the mass and rapid preparation of stable and tunable monodisperse CLC microdroplet single-mode lasers. This is based on the formation of polymer networks on the surface of the microdroplet via interfacial polymerization and a disruption of the orderliness of the polymer networks by increasing the temperature during polymerization, which results in a single pitch inside the microdroplets. This approach enables CLC microdroplet single-mode lasers to achieve improved environmental robustness, while maintaining the same temperature tunability as the unpolymerized sample. Our method has promising future applications in integrated optics, flexible devices, and sensors.

LncRNA FAF attenuates hypoxia/ischaemia-induced pyroptosis via the miR-185-5p/PAK2 axis in cardiomyocytes.

Pyroptosis is associated with various cardiovascular diseases. Increasing evidence suggests that long noncoding RNAs (lncRNAs) have been implicated in gene regulation, but how lncRNAs participate in the regulation of pyroptosis in the heart remains largely unknown. In this study, we aimed to explore the antipyroptotic effects of lncRNA FGF9-associated factor (FAF) in acute myocardial infarction (AMI). The expression patterns of lncRNA FAF, miR-185-5p and P21 activated kinase 2 (PAK2) were detected in hypoxia/ischaemia-induced cardiomyocytes. Hoechst 33342/PI staining, lactate dehydrogenase (LDH) release assay, immunofluorescence and Western blotting were conducted to assay cell pyroptosis. The interaction between lncRNA FAF, miR-185-5p and PAK2 was verified by bioinformatics analysis, small RNA sequencing luciferase reporter assay and qRT-PCR. The expression of LncRNA FAF was downregulated in hypoxic cardiomyocytes and myocardial tissues. Overexpression of lncRNA FAF could attenuate cardiomyocyte pyroptosis, improve cell viability and reduce infarct size during the procession of AMI. Moreover, lncRNA FAF was confirmed as a sponge of miR-185-5p and promoted PAK2 expression in cardiomyocytes. Collectively, our findings reveal a novel lncRNA FAF/miR-185-5p/PAK2 axis as a crucial regulator in cardiomyocyte pyroptosis, which might be a potential therapeutic target of AMI.

MNK2-eIF4E axis promotes cardiac repair in the infarcted mouse heart by activating cyclin D1.

Adult mammals have limited potential for cardiac regeneration after injury. In contrast, neonatal mouse heart, up to 7 days post birth, can completely regenerate after injury. Therefore, identifying the key factors promoting the proliferation of endogenous cardiomyocytes (CMs) is a critical step in the development of cardiac regeneration therapies. In our previous study, we predicted that mitogen-activated protein kinase (MAPK) interacting serine/threonine-protein kinase 2 (MNK2) has the potential of promoting regeneration by using phosphoproteomics and iGPS algorithm. Here, we aimed to clarify the role of MNK2 in cardiac regeneration and explore the underlying mechanism. In vitro, MNK2 overexpression promoted, and MNK2 knockdown suppressed cardiomyocyte proliferation. In vivo, inhibition of MNK2 in CMs impaired myocardial regeneration in neonatal mice. In adult myocardial infarcted mice, MNK2 overexpression in CMs in the infarct border zone activated cardiomyocyte proliferation and improved cardiac repair. In CMs, MNK2 binded to eIF4E and regulated its phosphorylation level. Knockdown of eukaryotic translation initiation factor (eIF4E) impaired the proliferation-promoting effect of MNK2 in CMs. MNK2-eIF4E axis stimulated CMs proliferation by activating cyclin D1. Our study demonstrated that MNK2 kinase played a critical role in cardiac regeneration. Over-expression of MNK2 promoted cardiomyocyte proliferation in vitro and in vivo, at least partly, by activating the eIF4E-cyclin D1 axis. This investigation identified a novel target for heart regenerative therapy.

P16ink4a overexpression ameliorates cardiac remodeling of mouse following myocardial infarction via CDK4/pRb pathway.

BACKGROUND: P16ink4a can accumulate in senescent cells and can be induced by different oncogenic stimulations. These functions make p16ink4a a biomarker of senescence and cancer. However, the exact role of p16ink4a remains unclear in cardiovascular disease. This study was aimed to investigate the role of p16ink4a in cardiac remodeling after myocardial infarction (MI). METHODS: In vivo, gain and loss of function experiments using p16ink4a overexpression and knockdown adenovirus were induced to determine the effect of p16ink4a on cardiac structure and function after MI. The in vitro effects of p16ink4a were evaluated by overexpression and knockdown adenovirus of p16ink4a on isolated neonatal mouse cardiac myocytes (NMCMs) and neonatal mouse cardiac fibroblasts (NMCFs). RESULTS: Expression level of p16ink4a was increased after MI and enriched in the infarction area. In vivo, overexpression of p16ink4a protected, while knockdown of p16ink4a worsened cardiac function. In vitro, p16ink4a did not influence the hypertrophy of NMCMs. Overexpression of p16ink4a inhibited the proliferation and migration of NMCFs and reduced the level of collagen I and α-SMA. Consistently, knockdown of p16ink4a in vitro displayed the opposite effects. Further mechanism studies revealed that p16ink4a affected the expression level of cyclin-dependent kinase 4 (CDK4) and phosphorylation of retinoblastoma (pRb), which could be a potential pathway in regulating cardiac remodeling after MI. CONCLUSION: Overexpression of 16ink4a in cardiac fibroblasts can ameliorate cardiac dysfunction and attenuate pathological cardiac remodeling in mice after MI by regulating the p16ink4a/CDK4/pRb pathway.

Tunable multi-mode laser based on robust cholesteric liquid crystal microdroplet.

To date, various studies have been dedicated to the development of cholesteric liquid crystal (CLC) microdroplet omnidirectional lasers. In this work, a stable and tunable multi-mode laser emission is achieved by designing a dye-doping CLC microdroplet. In such a structure, the polymer network only exists on the surface, maintaining stability while providing tunability, and due to the uneven distribution of the pitch, it leads to multi-mode laser emission. A large number of microdroplets are produced quickly via a new method based on ultrasonic separation. During the reaction, we introduce interfacial polymerization where monomers and photoinitiator are respectively distributed inside and outside the microdroplets through mutual diffusion, which enables one to make the polymer network exist on the surface instead of the interior. The obtained microdroplet-based multi-mode laser is shown to possess stability and tunability, demonstrating a great potential for flexible devices and 3D displays.

MicroRNA ssc-miR-124a exhibits antiviral activity against porcine reproductive and respiratory syndrome virus via suppression of host genes CD163.

Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease in the swine industry, which causes severe economic losses to current swine production worldwide. There are no effective antiviral strategies for preventing this disease. Previous studies showed that microRNAs (miRNAs) play important role in virus-host interactions. In this study, we demonstrated that the expression level of ssc-miR-124a was significantly downregulated during both high and low pathogenic PRRSV infection. Overexpression of ssc-miR-124a markedly inhibits PRRSV replication in PAMs. Luciferase reporter experiments and RISC immunoprecipitation assay were used to identify the ssc-miR-124a could directly target the 3'UTR of pig CD163 mRNA in a sequence-specific manner and that CD163 mRNA and protein levels were reduced in PAMs overexpressing ssc-miR-124a. These data not only provide new insights into virus-host interactions during PRRSV infection, but also suggest potential new antiviral strategies against PRRSV infection in the future.

Comparison of left ventricular mechanical dyssynchrony parameters between exercise and adenosine triphosphate stress tests using gated single-photon emission computed tomography myocardial perfusion imaging.

BACKGROUND: Left ventricular mechanical dyssynchrony (LVMD) can be induced after stress test. However, no studies have compared the influence of different stress­inducing methods on LVMD parameters. AIMS: The aim of the study was to determine whether there is a difference between exercise and adenosine triphosphate (ATP) stress tests in terms of changes in LVMD parameters assessed using gated single­photon emission computed tomography myocardial perfusion imaging (GSPECT MPI). METHODS: A total of190 patients who underwent 99mTc ­sestamibi GSPECT MPI were consecutively enrolled. Treadmill exercise and ATP stress tests were performed in 95 patients each. Normal myocardial perfusion was defined as the summed stress score (SSS) ≤3 and summed rest score (SRS) ≤3, myocardial ischemia as SSS >3 and SRS ≤3, and myocardial infarction as SSS >3 and SRS >3. Parameters of LVMD, including phase standard deviation (PSD), phase bandwidth (PBW), skewness, and kurtosis were compared. Subtraction was made between values during stress and rest phases to acquire ∆PSD, ∆PBW, ∆skewness, and ∆kurtosis Results: There were no differences in LVMD parameters between the exercise and ATP groups. The same results were obtained in the normal perfusion, ischemia, and infarction subgroups. Furthermore, no differences were observed in ∆PSD (median [interquartile range, IQR], 0.25 [-2.3 to 3.1] vs 0.42 (-1.7 to 3.1]; P = 0.73), ∆PBW (median [IQR], 1 [-7 to 11] vs 1 [-6 to 11]; P = 0.95), ∆skewness (mean [SD], -0.06 [0.63] vs 0 [0.81]; P = 0.53), and ∆kurtosis (median [IQR], -0.47 [-4.2 to 4.3] vs -0.42 [-4.8 to 5.2]; P = 0.73) between the exercise and ATP stress­inducing methods. CONCLUSIONS: There are no differences between the exercise and ATP stress tests in terms of changes in LVMD parameters. Thus, the 2 methods can be used alternatively.

Left ventricular systolic and diastolic dyssynchrony to improve cardiac resynchronization therapy response in heart failure patients with dilated cardiomyopathy.

BACKGROUND: The systolic and diastolic dyssynchrony is physiologically related, but measure different left ventricular mechanisms. Left ventricular systolic mechanical dyssynchrony (systolic LVMD) has shown significant clinical values in improving cardiac resynchronization therapy (CRT) response in the heart failure patients with dilated cardiomyopathy (DCM). Our recent study demonstrated that LV diastolic dyssynchrony (diastolic LVMD) parameters have important prognostic values for DCM patients. However, there are a limited number of studies about the clinical value of diastolic LVMD for CRT. This study aims to explore the predictive values of both systolic LVMD and diastolic LVMD for CRT in DCM patients. METHODS: Eighty-four consecutive CRT patients with both DCM and complete left bundle branch block (CLBBB) who received gated resting SPECT MPI at baseline were included in the present study. The phase analysis technique was applied on resting gated short-axis SPECT MPI images to measure systolic LVMD and diastolic LVMD, characterized by phase standard deviation (PSD) and phase histogram bandwidth (PBW). CRT response was defined as ≥ 5% improvement of LVEF at 6-month follow-up. Variables with P < 0.10 in the univariate analysis were included in the multivariate cox analysis. RESULTS: During the follow-up period, 59.5% (50 of 84) patients were CRT responders. The univariate cox regression analysis showed that at baseline QRS duration, non-sustained ventricular tachycardia (NS-VT), systolic PSD, systolic PBW, diastolic PSD, diastolic PBW, scar burden and LV lead in the scarred myocardium were statistically significantly associated with CRT response. The multivariate cox regression analysis showed that QRS duration, NS-VT, systolic PSD, systolic PBW, diastolic PSD, and diastolic PBW were independent predictive factors for CRT response. Furthermore, the rate of CRT response was 94.4% (17 of 18) in patients whose LV lead was in the segments with both the first three late contraction and the first three late relaxation; by contrast, the rate of CRT response was only 6.7% (1 of 15, P < 0.000) in patients whose LV lead was in the segments with neither the first three late contraction nor the first three late relaxation. CONCLUSION: Both systolic LVMD and diastolic LVMD from gated SPECT MPI have important predictive values for CRT response in DCM patients. Pacing at LV segments with both late contraction and late relaxation has potential to increase the CRT response.

A strip test for the optical determination of influenza virus H3 subtype using gold nanoparticle coated polystyrene latex microspheres.

A strip test is described for the optical determination of influenza virus H3 subtype. It utilizes gold nanoparticle (AuNP) coated polystyrene latex microspheres (PS) as the label and a sandwich format. The AuNP and PS particles were linked using monoclonal antibodies against influenza virus as the bridge. Under the optimal conditions, the visual detection limit of the AuNP-PS-based strip test was as low as 1/16 hemagglutination unit (HAU). It was 64 times higher than that of 10 nm (4 HAU) AuNP-based strip tests. Quantitative analysis showed that the detection limit of the AuNP-PS-based strip is 0.016 HAU. The AuNP-PS-based strip test showed no cross-reactivity to the other subtypes (H1, H5, H7, or H9) of influenza viruses. Graphical abstract .

Prognostic value of left-ventricular systolic and diastolic dyssynchrony measured from gated SPECT MPI in patients with dilated cardiomyopathy.

BACKGROUND: Left-ventricular systolic dyssynchrony (LVSD) has been an important prognostic factor in the patients with dilated cardiomyopathy (DCM). However, the association between the LV diastolic dyssynchrony (LVDD) and clinical outcome is not well established. This study aims to evaluate the prognostic values of both systolic and diastolic dyssynchrony in patients with DCM. METHODS: Fifty-two patients with DCM were enrolled and divided into two groups according to cardiac deaths from the follow-up data. The phase-analysis technique was applied on resting gated short-axis SPECT MPI images to measure LV systolic and diastolic dyssynchrony, including phase standard deviation (PSD), phase histogram bandwidth (PBW), and phase entropy (PE). Variables with P < 0.10 in the univariate analysis were included in the multivariate cox analysis. RESULTS: During the follow-up period (2.9 ± 1.7 years), 18 (34.6%) cardiac deaths were observed. Compared with survivors, patients with cardiac death had lower LVEF (P = 0.011), and more severe LV systolic and diastolic dyssynchrony. The univariate cox regression analysis showed that hypertension, NT-proBNP, LVEF, systolic PSD, systolic PE, and diastolic PBW were statistically significantly associated with cardiac death. The multivariate cox regression analysis showed that systolic PE and diastolic PE were independent predictive factors for cardiac death. Furthermore, the receiver operating characteristic (ROC) analysis, when applied into the combination of systolic PE and diastolic PE for predicting cardiac death, had an area under curve (AUC) of 0.766, a sensitivity of 0.765, and a specificity of 0.722. CONCLUSIONS: Both the LVSD and LVDD parameters from SPECT MPI have important prognostic values for DCM patients. Both systolic PE and diastolic PE are independent prognostic factors for cardiac death.

Myocardial stunning-induced left ventricular dyssynchrony on gated single-photon emission computed tomography myocardial perfusion imaging.

OBJECTIVES: Myocardial stunning provides additional nonperfusion markers of coronary artery disease (CAD), especially for severe multivessel CAD. The purpose of this study is to assess the influence of myocardial stunning to the changes of left ventricular mechanical dyssynchrony (LVMD) parameters between stress and rest gated single-photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI). PATIENTS AND METHODS: A total of 113 consecutive patients (88 males and 25 females) who had undergone both stress and rest Tc-sestamibi gated SPECT MPI were retrospectively enrolled. Suspected or known patients with CAD were included if they had exercise stress MPI and moderate to severe myocardial ischemia. Segmental scores were summed for the three main coronary arteries according to standard myocardial perfusion territories, and then regional perfusion, wall motion, and wall thickening scores were measured. Myocardial stunning was defined as both ischemia and wall dysfunction within the same coronary artery territory. Patients were divided into the stunning group (n=58) and nonstunning group (n=55). RESULTS: There was no significant difference of LVMD parameters between stress and rest in the nonstunning group. In the stunning group, phase SD and phase histogram bandwidth of contraction were significantly larger during stress than during rest (15.05±10.70 vs. 13.23±9.01 and 46.07±34.29 vs. 41.02±32.16, P<0.05). Phase SD and phase histogram bandwidth of relaxation were also significantly larger during stress than during rest (21.21±13.91 vs. 17.46±10.52 and 59.03±37.82 vs. 52.38±36.89, P<0.05). CONCLUSION: Both systolic and diastolic LVMD parameters deteriorate with myocardial stunning. This kind of change may have incremental values to diagnose CAD.

Effect of pre-miRNA-1658 gene polymorphism on chicken growth and carcass traits.

OBJECTIVE: Polymorphisms occurring in the precursor region of microRNAs (miRNAs) affect the target gene and alter the biogenesis of miRNAs, resulting in phenotypic variation. The purpose of the study was to investigate the genetic effects of rs16681031 (C>G) mutation in the precursor region of gga-miR-1658 on the economic traits of the Gushi-Anka chicken F2 resource population. METHODS: To explore the effect of miR-1658 polymorphisms on chicken economic traits, the SNP was genotyped by MassArray matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The association between the SNP and chicken body size, growth and carcass traits was determined by linear mixed models. RESULTS: The SNP was not only significantly associated with body weight at the age of 6, 8, 10, 12 weeks, respectively, but also with the breadth of the chicken chest, body slanting length and pelvic breadth at 4 weeks, chest depth at 8 weeks of age, and body slanting length at 12 weeks (p<0.05), respectively. CONCLUSION: Our data serve as a useful resource for further analysis of miRNA function, and represent a molecular genetic basis for poultry breeding.