Chronotoxici-Plate Containing Droplet-Engineered Rhythmic Liver Organoids for Drug Toxicity Evaluation.

The circadian clock coordinates the daily rhythmicity of biological processes, and its dysregulation is associated with various human diseases. Despite the direct targeting of rhythmic genes by many prevalent and World Health Organization (WHO) essential drugs, traditional approaches can't satisfy the need of explore multi-timepoint drug administration strategies across a wide range of drugs. Here, droplet-engineered primary liver organoids (DPLOs) are generated with rhythmic characteristics in 4 days, and developed Chronotoxici-plate as an in vitro high-throughput automated rhythmic tool for chronotherapy assessment within 7 days. Cryptochrome 1 (Cry1) is identified as a rhythmic marker in DPLOs, providing insights for rapid assessment of organoid rhythmicity. Using oxaliplatin as a representative drug, time-dependent variations are demonstrated in toxicity on the Chronotoxici-plate, highlighting the importance of considering time-dependent effects. Additionally, the role of chronobiology is underscored in primary organoid modeling. This study may provide tools for both precision chronotherapy and chronotoxicity in drug development by optimizing administration timing.

Protocol to encapsulate cerebral organoids with alginate hydrogel shell to induce volumetric compression.

In vitro organoids, including cerebral organoids, are usually developed without mechanical compression, which may contribute to a delay in maturation. Here, we present a protocol for encapsulating cerebral organoids with a thin shell of low-concentration alginate hydrogel. We describe steps for organoid generation, microfluidic chip culture, Matrigel coating, expansion culture, and alginate encapsulation. We then detail procedures for maturation culture and organoid characterization. The moderate compressive stimulation that the shell provides promotes cell proliferation and neuronal maturation. For complete details on the use and execution of this protocol, please refer to Tang et al.1.

Pleiotropic immunoregulation by growth-blocking peptide in Ostrinia furnacalis.

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth-blocking peptide (GBP) is a member of the stress-responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity-related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens.

Multi-Person Pose Regression with Distribution-Aware Single-Stage Models.

Understanding human posture is a challenging topic, which encompasses several tasks, e.g., pose estimation, body mesh recovery and pose tracking. In this paper, we propose a novel Distribution-Aware Single-stage (DAS) model for the pose-related tasks. The proposed DAS model estimates human position and localizes joints simultaneously, which requires only a single pass. Meanwhile, we utilize normalizing flow to enable DAS to learn the true distribution of joint locations, rather than making simple Gaussian or Laplacian assumptions. This provides a pivotal prior and greatly boosts the accuracy of regression-based methods, thus making DAS achieve comparable performance to the volumetric-based methods. We also introduce a recursively update strategy to progressively approach the regression target, reducing the difficulty of regression and improving the regression performance. We further adapt DAS to multi-person mesh recovery and pose tracking tasks and achieve considerable performance on both tasks. Comprehensive experiments on CMU Panoptic and MuPoTS-3D demonstrate the superior efficiency of DAS, specifically 1.5 times speedup over previous best method, and its state-of-the-art accuracy for multi-person pose estimation. Extensive experiments on 3DPW and PoseTrack2018 indicate the effectiveness and efficiency of DAS for human body mesh recovery and pose tracking, respectively, which prove the generality of our proposed DAS model.

Volumetric compression by heterogeneous scaffold embedding promotes cerebral organoid maturation and does not impede growth.

Although biochemical regulation has been extensively studied in organoid modeling protocols, the role of mechanoregulation in directing stem cell fate and organoid development has been relatively unexplored. To accurately replicate the dynamic organoid development observed in nature, in this study, we present a method of heterogeneous embedding using an alginate-shell-Matrigel-core system. This approach allows for cell-Matrigel remodeling by the inner layer and provides short-term moderate-normal compression through the soft alginate outer layer. Our results show that the time-limited confinement contributes to increased expression of neuronal markers such as neurofilament (NF) and microtubule-associated protein 2 (MAP2). Compared with non-alginate embedding and alginate compression groups, volume growth remains unimpeded. Our findings demonstrate the temporary mechanical regulation of cerebral organoid growth, which exhibits a regular growth profile with enhanced maturation. These results highlight the importance and potential practical applications of mechanoregulation in the establishment of brain organoids. A record of this paper's transparent peer review process is included in the supplemental information.

Isopsoralen promotes osteogenic differentiation of human jawbone marrow mesenchymal cells through Notch signaling pathway.

BACKGROUND: The aim of this study was to investigate the effect of isopsoralen on osteogenic differentiation of human jawbone marrow mesenchymal cells and its possible mechanism. METHOD: The cytotoxicity and proliferation of cells were measured by a cell counting kit 8. Alkaline phosphatase activity analysis was then used to determine the optimal concentration of isopsoralen to promote the differentiation. Western blot, qRT-PCR and Alizarin Red S staining were used to evaluate the role of Notch signaling pathway in isopsoralen-induced osteogenic differentiation. This study also investigated the anti-osteoporotic effects of ISO using in vivo osteoporosis models. RESULTS: Our results showed that 1 × 10-6 mol / L isopsoralen can effectively promote the proliferation and osteogenic differentiation of cells. Moreover, we found that activation of notch signaling pathway inhibited isopsoralen-induced osteogenesis and inhibition of Notch signal promoted the differentiation of osteoblasts induced by isopsoralen. In vivo experiments revealed that ISO significantly inhibited OVX-induced bone mineral density loss and restored the impaired bone structural properties in osteoporosis model mice. CONCLUSION: Our findings demonstrated that isopsoralen induced osteogenic differentiation by inhibiting Notch signaling and it might be a potential therapeutic agent for treating or preventing osteoporosis.

Predicting Overall Survival for Patients with Malignant Mesothelioma Following Radiotherapy via Interpretable Machine Learning.

PURPOSE/OBJECTIVES: Malignant pleural mesothelioma (MPM) is a rare but aggressive cancer arising from the cells of the thoracic pleura with a poor prognosis. We aimed to develop a model, via interpretable machine learning (ML) methods, predicting overall survival for MPM following radiotherapy based on dosimetric metrics as well as patient characteristics. MATERIALS/METHODS: Sixty MPM (37 right, 23 left) patients treated on a Tomotherapy unit between 2013 and 2018 were retrospectively analyzed. All patients received 45 Gy (25 fractions). The multivariable Cox regression (Cox PH) model and Survival Support Vector Machine (sSVM) were applied to build predictive models of overall survival (OS) based on clinical, dosimetric, and combined variables. RESULTS: Significant differences in dosimetric endpoints for critical structures, i.e., the lung, heart, liver, kidney, and stomach, were observed according to target laterality. The OS was found to be insignificantly different (p = 0.18) between MPM patients who tested left- and right-sided, with 1-year OS of 77.3% and 75.0%, respectively. With Cox PH regression, considering dosimetric variables for right-sided patients alone, an increase in PTV_Min, Total_Lung_PTV_Mean, Contra_Lung_Volume, Contra_Lung_V20, Esophagus_Mean, and Heart_Volume had a greater hazard to all-cause death, while an increase in Total_Lung_PTV_V20, Contra_Lung_V5, and Esophagus_Max had a lower hazard to all-cause death. Considering clinical variables alone, males and increases in N stage had greater hazard to all-cause death; considering both clinical and dosimetric variables, increases in N stage, PTV_Mean, PTV_Min, and esophagus_Mean had greater hazard to all-cause death, while increases in T stage and Heart_V30 had lower hazard to all-cause-death. In terms of C-index, the Cox PH model and sSVM performed similarly and fairly well when considering clinical and dosimetric variables independently or jointly. CONCLUSIONS: Clinical and dosimetric variables may predict the overall survival of mesothelioma patients, which could guide personalized treatment planning towards a better treatment response. The identified predictors and their impact on survival offered additional value for translational application in clinical practice.

Adaptation of the Tumor Antigen Presentation Machinery to Ionizing Radiation.

Ionizing radiation (IR) can reprogram proteasome structure and function in cells and tissues. In this article, we show that IR can promote immunoproteasome synthesis with important implications for Ag processing and presentation and tumor immunity. Irradiation of a murine fibrosarcoma (FSA) induced dose-dependent de novo biosynthesis of the immunoproteasome subunits LMP7, LMP2, and Mecl-1, in concert with other changes in the Ag-presentation machinery (APM) essential for CD8+ T cell-mediated immunity, including enhanced expression of MHC class I (MHC-I), ß2-microglobulin, transporters associated with Ag processing molecules, and their key transcriptional activator NOD-like receptor family CARD domain containing 5. In contrast, in another less immunogenic, murine fibrosarcoma (NFSA), LMP7 transcripts and expression of components of the immunoproteasome and the APM were muted after IR, which affected MHC-I expression and CD8+ T lymphocyte infiltration into NFSA tumors in vivo. Introduction of LMP7 into NFSA largely corrected these deficiencies, enhancing MHC-I expression and in vivo tumor immunogenicity. The immune adaptation in response to IR mirrored many aspects of the response to IFN-γ in coordinating the transcriptional MHC-I program, albeit with notable differences. Further investigations showed divergent upstream pathways in that, unlike IFN-γ, IR failed to activate STAT-1 in either FSA or NFSA cells while heavily relying on NF-κB activation. The IR-induced shift toward immunoproteasome production within a tumor indicates that proteasomal reprogramming is part of an integrated and dynamic tumor-host response that is specific to the stressor and the tumor and therefore is of clinical relevance for radiation oncology.

Low sodium intake ameliorates hypertension and left ventricular hypertrophy in mice with primary aldosteronism.

The goal of this paper is to elucidate the effects of sodium restriction on hypertension and left ventricular (LV) hypertrophy in a mouse model with primary aldosteronism (PA). Mice with genetic deletion of TWIK-related acid-sensitive K (TASK)-1 and TASK-3 channels (TASK-/-) were used as the animal model of PA. Parameters of the LV were assessed using echocardiography and histomorphology analysis. Untargeted metabolomics analysis was conducted to reveal the mechanisms underlying the hypertrophic changes in the TASK-/- mice. The TASK-/- adult male mice exhibited the hallmarks of PA, including hypertension, hyperaldosteronism, hypernatremia, hypokalemia, and mild acid-base balance disorders. Two weeks of low sodium intake significantly reduced the 24-h average systolic and diastolic BP in TASK-/- but not TASK+/+ mice. In addition, TASK-/- mice showed increasing LV hypertrophy with age, and 2 weeks of the low-sodium diet significantly reversed the increased BP and LV wall thickness in adult TASK-/- mice. Furthermore, a low-sodium diet beginning at 4 weeks of age protected TASK-/- mice from LV hypertrophy at 8-12 weeks of age. Untargeted metabolomics demonstrated that the disturbances in heart metabolism in the TASK-/- mice (e.g., Glutathione metabolism; biosynthesis of unsaturated fatty acids; amino sugar and nucleotide sugar metabolism; pantothenate and CoA biosynthesis; D-glutamine and D-glutamate metabolism), some of which were reversed after sodium restriction, might be involved in the development of LV hypertrophy. In conclusion, adult male TASK-/- mice exhibit spontaneous hypertension and LV hypertrophy, which are ameliorated by a low-sodium intake.

Ultrathin Hydrogel Films toward Breathable Skin-Integrated Electronics.

On-skin electronics that offer revolutionary capabilities in personalized diagnosis, therapeutics, and human-machine interfaces require seamless integration between the skin and electronics. A common question remains whether an ideal interface can be introduced to directly bridge thin-film electronics with the soft skin, allowing the skin to breathe freely and the skin-integrated electronics to function stably. Here, an ever-thinnest hydrogel is reported that is compliant to the glyphic lines and subtle minutiae on the skin without forming air gaps, produced by a facile cold-lamination method. The hydrogels exhibit high water-vapor permeability, allowing nearly unimpeded transepidermal water loss and free breathing of the skin underneath. Hydrogel-interfaced flexible (opto)electronics without causing skin irritation or accelerated device performance deterioration are demonstrated. The long-term applicability is recorded for over one week. With combined features of extreme mechanical compliance, high permeability, and biocompatibility, the ultrathin hydrogel interface promotes the general applicability of skin-integrated electronics.

Bead-jet printing enabled sparse mesenchymal stem cell patterning augments skeletal muscle and hair follicle regeneration.

Transplantation of mesenchymal stem cells (MSCs) holds promise to repair severe traumatic injuries. However, current transplantation practices limit the potential of this technique, either by losing the viable MSCs or reducing the performance of resident MSCs. Herein, we design a "bead-jet" printer, specialized for high-throughput intra-operative formulation and printing of MSCs-laden Matrigel beads. We show that high-density encapsulation of MSCs in Matrigel beads is able to augment MSC function, increasing MSC proliferation, migration, and extracellular vesicle production, compared with low-density bead or high-density bulk encapsulation of the equivalent number of MSCs. We find that the high-density MSCs-laden beads in sparse patterns demonstrate significantly improved therapeutic performance, by regenerating skeletal muscles approaching native-like cell density with reduced fibrosis, and regenerating skin with hair follicle growth and increased dermis thickness. MSC proliferation within 1-week post-transplantation and differentiation at 3 - 4 weeks post-transplantation are suggested to contribute therapy augmentation. We expect this "bead-jet" printing system to strengthen the potential of MSC transplantation.

CNS Organoid Surpasses Cell-Laden Microgel Assembly to Promote Spinal Cord Injury Repair.

The choice of therapeutic agents remains an unsolved issue in the repair of spinal cord injury. In this work, various agents and configurations were investigated and compared for their performance in promoting nerve regeneration, including bead assembly and bulk gel of collagen and Matrigel, under acellular and cell-laden conditions, and cerebral organoid (CO) as the in vitro preorganized agent. First, in Matrigel-based agents and the CO transplantations, the recipient animal gained more axon regeneration and the higher Basso, Beattie, and Bresnahan (BBB) scoring than the grafted collagen gels. Second, new nerves more uniformly infiltrated into the transplants in bead form assembly than the molded chunks. Third, the materials loaded the neural progenitor cells (NPCs) or the CO implantation groups received more regenerated nerve fibers than their acellular counterparts, suggesting the necessity to transplant exogenous cells for large trauma (e.g., a 5 mm long spinal cord transect). In addition, the activated microglial cells might benefit from neural regeneration after receiving CO transplantation in the recipient animals. The organoid augmentation may suggest that in vitro maturation of a microtissue complex is necessary before transplantation and proposes organoids as the premium therapeutic agents for nerve regeneration.

Spatiotemporal pattern of reference crop evapotranspiration and its response to meteorological factors in Northwest China over years 2000-2019.

Agricultural irrigation water in Northwest China accounts for more than 80% of total local water consumption, which is 1.23 times that of China. However, Northwest China is the most water-scarce place in China. Water scarcity in restricts crop growth and production. Reference crop evapotranspiration (ET0) is important for agricultural water management. Understanding the reason for ET0 change is helpful to provide a basis for rational planning of agricultural irrigation systems to conserve water. This study investigated the temporal and spatial variation characteristics of ET0 at 181 meteorological stations in Northwest China from 2000 to 2019. And the sensitive factors and dominant factors affecting ET0 change were quantitatively identified based on sensitivity analysis and contribution rate evaluation. Results showed that (1) a significant increase in maximum and minimum temperature (Tmax and Tmin), a significant decrease in sunshine duration (SD) and relative humidity (RH), and a slight increase in wind speed at 10 m height (U10) were observed. (2) Annual ET0 had an insignificant increasing trend. Spring and autumn ET0 contributed greatly to the growth of annual ET0, especially in March, May, September, October, and November. ET0 in HH (Yellow River Basin area) had decreased at annual scale, while other subregions were the opposite trend. Significant differences in monthly and seasonal changes in the spatial distribution of ET0. (3) U10 was the dominating contribution factor related to annual ET0 variability, followed by Tmin, RH, Tmax, and SD. In seasonal time scale, Tmin, SD, U10, and RH were the most dominant factors in spring, summer, autumn, and winter respectively. (4) Spatial distribution for contribution rates of various meteorological factors showed significant diversity among various subregions. The positive contribution of U10 was the major cause of the increase in ET0 in semi-arid grassland area (BGH), the southwest of "Qice line" (QCXXN), and the southeast of "Qice line" (QCXDN); the significant increase in Tmin contributed most in Qaidam Basin (CDM), Hexi inland river basin (HX), the northeast of "Qice line" (QCXDB), and the northwest of "Qice line" (QCXXB), while the contribution of decreasing SD offsets the positive effects of other factors, leading to the decrease in ET0 in HH. Our work illustrates that water management measures should be different at different spatial and temporal scales. The effect of U10 can be offset by covering, to reduce evaporation and maintain water in BGH, QCXXN, and QCXDN. And high-temperature resistant varieties are planted to adapt to temperature growth in CDM, HX, QCXDB, and QCXXB. Agricultural water management strategies should be formulated and selected according to local conditions.

Human-Centric Relation Segmentation: Dataset and Solution.

Vision and language understanding techniques have achieved remarkable progress, but currently it is still difficult to well handle problems involving very fine-grained details. For example, when the robot is told to "bring me the book in the girl's left hand", most existing methods would fail if the girl holds one book respectively in her left and right hand. In this work, we introduce a new task named human-centric relation segmentation (HRS), as a fine-grained case of HOI-det. HRS aims to predict the relations between the human and surrounding entities and identify the relation-correlated human parts, which are represented as pixel-level masks. For the above exemplar case, our HRS task produces results in the form of relation triplets 〈girl [left hand], hold, book 〉 and exacts segmentation masks of the book, with which the robot can easily accomplish the grabbing task. Correspondingly, we collect a new Person In Context (PIC) dataset for this new task, which contains 17,122 high-resolution images and densely annotated entity segmentation and relations, including 141 object categories, 23 relation categories and 25 semantic human parts. We also propose a Simultaneous Matching and Segmentation (SMS) framework as a solution to the HRS task. It contains three parallel branches for entity segmentation, subject object matching and human parsing respectively. Specifically, the entity segmentation branch obtains entity masks by dynamically-generated conditional convolutions; the subject object matching branch detects the existence of any relations, links the corresponding subjects and objects by displacement estimation and classifies the interacted human parts; and the human parsing branch generates the pixelwise human part labels. Outputs of the three branches are fused to produce the final HRS results. Extensive experiments on PIC and V-COCO datasets show that the proposed SMS method outperforms baselines with the 36 FPS inference speed. Notably, SMS outperforms the best performing baseline m-KERN with only 17.6 percent time cost. The dataset and code will be released at http://picdataset.com/challenge/index/.

Multiplex gene quantification as digital markers for extremely rapid evaluation of chemo-drug sensitivity.

Current administrations for precision drug uses are limited in evaluation speed. Here, we propose the use of multiplex gene-based digital markers for the extremely rapid personalized prediction of individual sensitivity to cancer drugs. We first screen the transcriptional profiles by applying two to three gene filters and scoring genes by their impact on drug sensitivity and finalize the gene lists by K-nearest neighbors cross-validation. The digital markers are cancer type dependent, are composed of tens to hundreds of gene expressions, and are rapidly quantified by reverse transcription quantitative real-time PCR (qRT-PCR) within 1-3 h after tumor sampling. The area under the receiver operating characteristic curve reached 0.88 when testing the performance of digital markers on organoids derived from colorectal cancer patient tumors. The algorithm and corresponding graphic user interface were developed to demonstrate the promise of digital markers for extremely rapid drug recommendation.

Optogenetically Controlled TrkA Activity Improves the Regenerative Capacity of Hair-Follicle-Derived Stem Cells to Differentiate into Neurons and Glia.

Hair-follicle-derived stem cells (HSCs) originating from the bulge region of the mouse vibrissa hair follicle are able to differentiate into neuronal and glial lineage cells. The tropomyosin receptor kinase A (TrkA) receptor that is expressed on these cells plays key roles in mediating the survival and differentiation of neural progenitors as well as in the regulation of the growth and regeneration of different neural systems. In this study, the OptoTrkA system is introduced, which is able to stimulate TrkA activity via blue-light illumination in HSCs. This allows to determine whether TrkA signaling is capable of influencing the proliferation, migration, and neural differentiation of these somatic stem cells. It is found that OptoTrkA is able to activate downstream molecules such as ERK and AKT with blue-light illumination, and subsequently able to terminate this kinase activity in the dark. HSCs with OptoTrkA activity show an increased ability for proliferation and migration and also exhibited accelerated neuronal and glial cell differentiation. These findings suggest that the precise control of TrkA activity using optogenetic tools is a viable strategy for the regeneration of neurons from HSCs, and also provides a novel insight into the clinical application of optogenetic tools in cell-transplantation therapy.

Decoupled molecular and inorganic framework dynamics in CH3NH3PbCl3.

The organic-inorganic lead-halide perovskites are composed of organic molecules imbedded in an inorganic framework. The compounds with general formula CH3NH3PbX 3 (MAPbX 3) display large photovoltaic efficiencies for halogens X = Cl, Br, and I in a wide variety of sample geometries and preparation methods. The organic cation and inorganic framework are bound by hydrogen bonds that tether the molecules to the halide anions, and this has been suggested to be important to the optoelectronic properties. We have studied the effects of this bonding using time-of-flight neutron spectroscopy to measure the molecular dynamics in CH3NH3PbCl3 (MAPbCl3). Low-energy/high-resolution neutron backscattering reveals thermally activated molecular dynamics with a characteristic temperature of ~95 K. At this same temperature, higher-energy neutron spectroscopy indicates the presence of an anomalous broadening in energy (reduced lifetime) associated with the molecular vibrations. By contrast, neutron powder diffraction shows that a spatially long-range structural phase transitions occurs at 178 K (cubic → tetragonal) and 173 K (tetragonal → orthorhombic). The large difference between these two temperature scales suggests that the molecular and inorganic lattice dynamics in MAPbCl3 are actually decoupled. With the assumption that underlying physical mechanisms do not change with differing halogens in the organic-inorganic perovskites, we speculate that the energy scale most relevant to the photovoltaic properties of the lead-halogen perovskites is set by the lead-halide bond, not by the hydrogen bond.

Identifying Key Drivers of Return Reversal with Dynamical Bayesian Factor Graph.

In the stock market, return reversal occurs when investors sell overbought stocks and buy oversold stocks, reversing the stocks' price trends. In this paper, we develop a new method to identify key drivers of return reversal by incorporating a comprehensive set of factors derived from different economic theories into one unified dynamical Bayesian factor graph. We then use the model to depict factor relationships and their dynamics, from which we make some interesting discoveries about the mechanism behind return reversals. Through extensive experiments on the US stock market, we conclude that among the various factors, the liquidity factors consistently emerge as key drivers of return reversal, which is in support of the theory of liquidity effect. Specifically, we find that stocks with high turnover rates or high Amihud illiquidity measures have a greater probability of experiencing return reversals. Apart from the consistent drivers, we find other drivers of return reversal that generally change from year to year, and they serve as important characteristics for evaluating the trends of stock returns. Besides, we also identify some seldom discussed yet enlightening inter-factor relationships, one of which shows that stocks in Finance and Insurance industry are more likely to have high Amihud illiquidity measures in comparison with those in other industries. These conclusions are robust for return reversals under different thresholds.

Generating a long DNA fragment of the target ncRNA for quantitative polymerase chain reaction by combining ncRNA-oligos hybridization and oligos ligation.

The poor reproducibility of the reverse transcription combined with quantitative polymerase chain reaction (RT-qPCR) results in an unacceptable reliability of publications based on these data. We established a novel method, in which two short complementary DNA oligos were hybridized with target ncRNA molecules and linked by DNA ligase to obtain a long DNA strand (HL-DNA) replacing cDNA for qPCR detection (HL-qPCR). A series of diluted samples prepared from the same total RNA resource were measured by HL-qPCR and RT-qPCR respectively to acquire their relative concentration of RNU4-1, AK026510 and SNORA73B. For every tested sample, the relative concentration of RNU4-1, AK026510 and SNORA73B obtained by HL-qPCR instead of RT-qPCR is closer to its corresponding true value without significant difference, demonstrating that HL-qPCR exhibits higher accuracy compared with RT-qPCR. With three independent repeats, no significant difference was observed among AK026510/RNU4-1 values of four samples diluted from the same RNA resource, by employing HL-qPCR but not RT-qPCR. It strongly suggests that the good reproducibility of HL-qPCR results from the stable efficiency of HL-DNA production regardless of the concentration and individual features of ncRNA. The novel HL-qPCR could be applied for the regular relative ncRNA concentration detection in the future.

N-Heterocyclic Carbene-Catalyzed Oxidative Annulations of α,ß-Unsaturated Aldehydes with Hydrazones: Selective Synthesis of Optically Active 4,5-Dihydropyridazin-3-ones and Pyridazin-3-ones.

A novel and efficient method for the highly enantioselective synthesis of chiral 4,5-dihydropyridazin-3-one derivatives has been developed based on the chiral N-heterocyclic carbene-catalyzed oxidative annulation between α,ß-unsaturated aldehydes and hydrazones. Meanwhile, the selective synthesis of either 4,5-dihydropyridazin-3-ones or pyridazin-3-one derivatives from the same reactants has been achieved by simply varying catalytic and reaction conditions.