Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Multichannel Multijunction Droplet Microfluidic Device to Synthesize Hydrogel Microcapsules with Different Core-Shell Structures and Adjustable Core Positions.

Core-shell hydrogel microcapsules have sparked great interest due to their unique characteristics and prospective applications in the medical, pharmaceutical, and cosmetic fields. However, complex synthetic procedures and expensive costs have limited their practical application. Herein, we designed and prepared several multichannel and multijunctional droplet microfluidic devices based on soft lithography for the effective synthesis of core-shell hydrogel microcapsules for different purposes. Additionally, two different cross-linking processes (ultraviolet (UV) exposure and interfacial polymerization) were used to synthesize different types of core-shell structured hydrogel microcapsules. Hydrogel microcapsules with gelatin methacryloyl (GelMA) as the core and polyacrylamide (PAM) as the thin shell were synthesized using UV cross-linking. Using an interfacial polymerization process, another core-shell structured microcapsule with GelMA as the core and Ca2+ cross-linked alginate with polyethylenimine (PEI) as the shell was constructed, and the core diameter and total droplet diameter were flexibly controlled by carving. Noteworthy, these hydrogel microcapsules exhibit stimuli-responsiveness and controlled release ability. Overall, a novel technique was developed to successfully synthesize various hydrogel microcapsules with core-shell microstructures. The hydrogel microcapsules possess a multilayered structure that facilitates the coassembly of cells and drugs, as well as the layered assembly of multiple drugs, to develop synergistic therapeutic regimens. These adaptable and controllable hydrogel microdroplets shall held great promise for multicell or multidrug administration as well as for high-throughput drug screening.

Preparation of strong, tough and conductive soy protein isolate/poly(vinyl alcohol)-based hydrogel via the synergy of biomineralization and salting out.

Conductive hydrogels have shown a great potential in the field of flexible electronic devices. However, conductive hydrogels prepare by traditional methods are difficult to combine high strength and toughness, which limits their application in various fields. In this study, a strategy for preparing conductive hydrogels with high strength and toughness by using the synergistic effect of biomineralization and salting-out was pioneered. In simple terms, by immersing the CaCl2 doped soy protein isolate/poly(vinyl alcohol)/dimethyl sulfoxide (SPI/PVA/DMSO) hydrogel in Na2CO3 and Na3Cit complex solution, the biomineralization aroused by Ca2+ and CO32-, and the salting-out effect of both NaCl and Na3Cit would enhance the mechanical properties of SPI/PVA/DMSO hydrogel. Meanwhile, the ionic conductivity of the hydrogel would also increase due the introduction of cation and anion. The mechanical and electrical properties of SPI/PVA/DMSO/CaCO3/Na3Cit hydrogels were significantly enhanced by the synergistic effect of biomineralization and salting-out. The optimum tensile strength, toughness, Young's modulus and ionic conductivity of the hydrogel were 1.4 ± 0.08 MPa, 0.51 ± 0.04 MPa and 1.46 ± 0.01 S/m, respectively. The SPI/PVA/DMSO/CaCO3/Na3Cit hydrogel was assembled into a strain sensor. The strain sensor had good sensitivity (GF = 3.18, strain in 20 %-500 %) and could be used to accurately detect various human movements.

High-sensitivity whole-genome recovery of single viral species in environmental samples.

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. This work presents a method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A novel gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. Validation experiments using a sewage sample spiked with two known viruses demonstrate the method's efficacy. We achieve 100% recovery of the spiked-in SV40 (Simian virus 40, 5243bp) genome sequence with uniform coverage distribution, and approximately 99.4% for the larger HAd5 genome (Human Adenovirus 5, 35938bp). Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables targeted characterizations of rare viral species and whole-genome amplification of single genomes for accessing the mutational profile in single virus genomes, contributing to an improved understanding of viral ecology.

Enhancing Anticancer Efficacy of Formononetin Microspheres via Microfluidic Fabrication.

Formononetin is a flavonoid compound with anti-tumor and anti-inflammatory properties. However, its low solubility limits its clinical use. We employed microfluidic technology to prepare formononetin-loaded PLGA-PEGDA microspheres (Degradable polymer PLGA, Crosslinking agent PEGDA), which can encapsulate and release drugs in a controlled manner. We optimized and characterized the microspheres, and evaluated their antitumor effects. The microspheres had uniform size, high drug loading efficiency, high encapsulation efficiency, and stable release for 35 days. They also inhibited the proliferation, migration, and apoptosis. The antitumor mechanism involved the induction of reactive oxygen species and modulation of Bcl-2 family proteins. These findings suggested that formononetin-loaded PLGA-PEGDA microspheres, created using microfluidic technology, could be a novel drug delivery system that can overcome the limitations of formononetin and enhance its antitumor activity.

Synergistic Adsorption and In Situ Catalytic Conversion of SO2 by Transformed Bimetal-Phenolic Functionalized Biomass.

SO2 removal is critical to flue gas purification. However, based on performance and cost, materials under development are hardly adequate substitutes for active carbon-based materials. Here, we engineered biomass-derived nanostructured carbon nanofibers integrated with highly dispersed bimetallic Ti/CoOx nanoparticles through the thermal transition of metal-phenolic functionalized industrial leather wastes for synergistic SO2 adsorption and in situ catalytic conversion. The generation of surface-SO32- and peroxide species (O22-) by Ti/CoOx achieved catalytic conversion of adsorbed SO2 into value-added liquid H2SO4, which can be discharged from porous nanofibers. This approach can also avoid the accumulation of the adsorbed SO2, thereby achieving high desulfurization activity and a long operating life over 6000 min, preceding current state-of-the-art active carbon-based desulfurization materials. Combined with the techno-economic and carbon footprint analysis from 36 areas in China, we demonstrated an economically viable and scalable solution for real-world SO2 removal on the industrial scale.

Photothermal Therapy Mediated Hybrid Membrane Derived Nano-formulation for Enhanced Cancer Therapy.

Emodin is applied as an antitumor drug in many tumor therapies. However, its pharmacology performances are limited due to its low solubility. Herein, we fused erythrocyte and macrophage to form a hybrid membrane (EMHM) and encapsulated emodin to form hybrid membrane-coated nanoparticles. We employed glycyrrhizin to increase the solubility of emodin first and prepared the hybrid membrane nanoparticle-coated emodin and glycyrrhizin (EG@EMHM NPs) which exhibited an average particle size of 170 ± 20 nm and encapsulation efficiency of 98.13 ± 0.67%. The half-inhibitory concentrations (IC50) of EG@EMHM NPs were 1.166 µg/mL, which is half of the free emodin. Based on the photosensitivity of emodin, the reactive oxygen species (ROS) results disclosed that ROS levels of the photodynamic therapy (PDT) section were higher than the normal section (P < 0.05). Compared to the normal section, PDT-mediated EG@EMHM NPs could induce an early stage of apoptosis of B16. The western blot and flow cytometry results verified that PDT-mediated EG@EMHM NPs can significantly improve the solubility of emodin and perform a remarkably antitumor effect on melanoma via BAX and BCL-2 pathway. The application of the combined chemical and PDT therapy could provide an improving target therapy for cutaneous melanoma and also may offer an idea for other insoluble components sources of traditional Chinese medicine. Schematic of EG@EMHM NPs formulation.

Microfluidic fabricated bisdemethoxycurcumin thermosensitive liposome with enhanced antitumor effect.

Bisdemethoxycurcumin (BDMC) is the main active ingredient that is isolated from Zingiberaceae plants, wherein it has excellent anti-tumor effects. However, insolubility in water limits its clinical application. Herein, we reported a microfluidic chip device that can load BDMC into the lipid bilayer to form BDMC thermosensitive liposome (BDMC TSL). The natural active ingredient glycyrrhizin was selected as the surfactant to improve solubility of BDMC. Particles of BDMC TSL had small size, homogenous size distribution, and enhanced cultimulative release in vitro. The anti-tumor effect of BDMC TSL on human hepatocellular carcinomas was investigated via 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, live/dead staining, and flowcytometry. These results showed that the formulated liposome had a strong cancer cell inhibitory, and presented a dose-dependent inhibitory effect on migration. Further mechanistic studies showed that BDMC TSL combined with mild local hyperthermia could significantly upregulate B cell lymphoma 2 associated X protein levels and decrease B cell lymphoma 2 protein levels, thereby inducing cell apoptosis. The BDMC TSL that was fabricated via microfluidic device were decomposed under mild local hyperthermia, which could beneficially enhance the anti-tumor effect of raw insoluble materials and promote translation of liposome.

The Synergy between Deep Learning and Organs-on-Chips for High-Throughput Drug Screening: A Review.

Organs-on-chips (OoCs) are miniature microfluidic systems that have arguably become a class of advanced in vitro models. Deep learning, as an emerging topic in machine learning, has the ability to extract a hidden statistical relationship from the input data. Recently, these two areas have become integrated to achieve synergy for accelerating drug screening. This review provides a brief description of the basic concepts of deep learning used in OoCs and exemplifies the successful use cases for different types of OoCs. These microfluidic chips are of potential to be assembled as highly potent human-on-chips with complex physiological or pathological functions. Finally, we discuss the future supply with perspectives and potential challenges in terms of combining OoCs and deep learning for image processing and automation designs.

Microfluidic Generation of Near-Infrared Photothermal Vitexin/ICG Liposome with Amplified Photodynamic Therapy.

Glioma, in which a malignant tumor cell occurs in neural mesenchymal cells, has a rapid progression and poor prognosis, which is still far from desirable in clinical treatments. We developed a lab-on-a-chip (LOC) device for the rapid and efficient preparation of vitexin/indocyanine green (ICG) liposomes. Vitexin could be released from liposome to kill cancer cell, which can potentially improve the glioma therapeutic effect and reduce the treatment time through synergistic photodynamic/photothermal therapies (PDT/PTT). The vitexin/ICG liposome was fabricated via LOC and its physicochemical property and release in vitro were evaluated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and live/dead staining were used to examine the enhanced antitumor effect of vitexin/ICG liposome in cooperation with PDT/PTT, while the related mechanism was explored by flow cytometry and western blot. The results were as follows: (1) The prepared vitexin/ICG liposome was smaller in size, homogenous in particle size distribution with significant low polydispersity index (PDI), and enhanced cumulative release in vitro. (2) We found that the formulated liposome presented strong cancer cell inhibition and suppression of its migration in a dose-dependent manner. (3) Further mechanistic studies showed that liposome combined with near-infrared irradiation could significantly upregulate levels of B cell lymphoma 2-associated X (Bax) protein and decrease B cell lymphoma 2 (Bcl-2) at protein levels. The vitexin/ICG liposomes prepared based on a simple LOC platform can effectively enhance the solubility of insoluble drugs, and the combined effect of PTT/PDT can effectively increase their antitumor effect, which provides a simple and valid method for the clinical translation of liposomes.

Supplementary Feeding of Grazing Inner Mongolian Cashmere Goats during Pregnancy-Based on "Nutrient Requirements of Cashmere Goats".

This study aimed to conduct precise supplementation for pregnant cashmere goats under grazing based on the feeding standard. Eight Inner Mongolian pregnant cashmere goats of near-average body weight were selected at early gestation (44.41 ± 4.03 kg) and late gestation (46.54 ± 4.02 kg) to measure their nutrient intake. Then, two pregnant cashmere goat flocks, No. 10 (control group, on-farm supplement) and No. 11 (supplemented group, supplement based on standard), with the same goat herd structure and grassland type, were chosen to conduct the supplemental feeding experiment. The results showed that pregnant cashmere goats lacked daily the intake of dry matter, digestive energy, crude protein and most essential mineral elements under grazing. After supplemental feeding, the supplementation based on the feeding standard increased the cashmere length and cashmere length growth volume and decreased the cashmere fineness, with no statistical significance. The goat cashmere yield, goat weight after shearing, single and twin-birth kid weight and kids' mature secondary hair follicle density were significantly higher in the supplemented group (p < 0.05). In conclusion, supplementation in accordance with "Nutrient Requirements of Cashmere Goats" can enhance pregnant cashmere goats' fiber production, growth performance, fertility and kids' secondary hair follicles development, which is of great importance for the healthy and precise nutrition and management of cashmere goats.

CircJag1 promotes apoptosis of ethylene thiourea-exposed anorectal malformations through sponging miR-137-3p by regulating Sox9 and suppressing Wnt/ß-catenin pathway during the hindgut development of rat embryos.

Anorectal malformations (ARMs) are common birth defects involving congenital structural anomalies of the gastrointestinal tract. As an important component of non-coding RNAs, circular RNAs (circRNAs) widely participate in the digestive system development; however, the specific molecular mechanism of their involvement in ARM occurrence remains obscure. Herein, we generated rat models of ARMs induced by ethylene thiourea. A novel circRNA (circJag1) was screened and identified by RNA-Seq, which is remarkably upregulated in hindgut tissues of ARM rat embryos. In vivo experiments, colocation analysis via fluorescence in situ hybridization, and immunofluorescence further demonstrated that the disordered circJag1/miR-137-3p/Sox9 expression caused a spatiotemporal imbalance in the urorectal septum (URS) of ARMs. In vitro, functional assays confirmed that circJag1 upregulation resulted in the degradation of nuclear ß-catenin, C-myc, and Cyclin D1 in rat intestinal epithelial cells, as well as the promotion of apoptosis and suppression of cell proliferation. Mechanistically, dual-luciferase reporter assay and RNA immunoprecipitation assay indicated that circJag1 acted as a miR-137-3p sponge, thereby inhibiting its repressive effect on its target Sox9. Further experiments showed that a loss of Sox9 abolished the circJag1-mediated increase in apoptosis. In conclusion, aberrantly high circJag1 expression promotes epithelial apoptosis by suppressing the canonical Wnt/ß-catenin pathway via the miR-137-3p/Sox9 axis, which leads to fusion failure of the URS and cloacal membrane, and eventually contributed to ARMs. Our achievements might boost the comprehension of ARM pathogenesis and could provide a novel candidate target for the development of therapies for ARMs to complement surgical treatment.

Research progress on the application of wearable devices in foot monitoring and management of diabetic patients / 中国实用护理杂志

As the focus of public health work in the world, diabetic foot disease has aroused high public concern. This paper introduces the application of the diabetic foot wearable monitoring equipment types, including plantar pressure monitoring, temperature monitoring, monitoring of the biomechanics and multimode monitoring, and wearable devices application status in patients with diabetes, puts forward the existing problems and prospect, in order to carry out domestic related to diabetic foot wearable monitoring equipment research to provide the reference.

RGS16 regulated by let-7c-5p promotes glioma progression by activating PI3K-AKT pathway / 医学前沿

Gliomas are the most common central nervous system tumours; they are highly aggressive and have a poor prognosis. RGS16 belongs to the regulator of G-protein signalling (RGS) protein family, which plays an important role in promoting various cancers, such as breast cancer, pancreatic cancer, and colorectal cancer. Moreover, previous studies confirmed that let-7c-5p, a well-known microRNA, can act as a tumour suppressor to regulate the progression of various tumours by inhibiting the expression of its target genes. However, whether RGS16 can promote the progression of glioma and whether it is regulated by miR let-7c-5p are still unknown. Here, we confirmed that RGS16 is upregulated in glioma tissues and that high expression of RGS16 is associated with poor survival. Ectopic deletion of RGS16 significantly suppressed glioma cell proliferation and migration both in vitro and in vivo. Moreover, RGS16 was validated as a direct target gene of miR let-7c-5p. The overexpression of miR let-7c-5p obviously downregulated the expression of RGS16, and knocking down miR let-7c-5p had the opposite effect. Thus, we suggest that the suppression of RGS16 by miR let-7c-5p can promote glioma progression and may serve as a potential prognostic biomarker and therapeutic target in glioma.

miR-141-3p affects ß-catenin signaling and apoptosis by targeting Ubtd2 in rats with anorectal malformations.

Anorectal malformations (ARMs) are the most common gastrointestinal malformations. miR-141-3p was obtained from whole-transcriptome sequencing, and Ub domain-containing protein 2 (Ubtd2) was predicted as the target gene. An ARM rat model was induced using ethylenethiourea. Fluorescence in situ hybridization and immunofluorescence were used to detect the spatiotemporal expression of miR-141-3p and Ubtd2, respectively. A dual-luciferase reporter assay confirmed their targeting relationship, and cell proliferation and apoptosis were investigated after transfection in the intestinal epithelium (IEC-6). Additionally, western blotting and co-immunoprecipitation were used to examine the protein levels and the endogenous binding relationship. miR-141-3p was downregulated in the ARM group, whereas Ubtd2 increased and colocalized with TUNEL-positive cells. After miR-141-3p inhibition, protein expression of USP5 and ß-catenin was affected via Ubtd2, and USP5 could bind to both Ubtd2 and ß-catenin. Flow cytometry analysis and caspase 3/7 staining demonstrated that downregulated miR-141-3p promoted cell apoptosis through Ubtd2. In summary, targeting Ubtd2 decreased in miR-141-3p and promoted apoptosis of intestinal epithelium and regulated ß-catenin expression. This may cause aberrant apoptosis during hindgut development and mediate the imbalance of ß-catenin signaling in the cloaca, further affecting the occurrence of ARMs.

[A case report of neonatal severe coronavirus disease 2019]. / æ–°ç”Ÿå„¿é‡åž‹æ–°åž‹å† çŠ¶ç— æ¯’è‚ºç‚Ž1例报告.

A 7-day-old male neonate was admitted due to testing positive for SARS-CoV-2. The neonate was born through cesarian section at 40 weeks and 2 days of gestation. His mother was diagnosed with coronavirus disease 2019 (COVID-19) caused by Omicron variant infection 1 day before delivery. The neonate was separated from his mother after birth and was taken care of by his father. Three days after the neonate was born, his father was also diagnosed with COVID-19. The neonate was diagnosed with COVID-19 on day 7 of life. The neonate presented with hyperpyrexia, dyspnea, hypoxia, and feeding difficulties, and the chest CT showed the coexistence of consolidation and ground glass-like changes mainly located below the posterior pleura. He was given symptomatic support treatment such as low flow oxygen therapy and posture management after admission. He was cured and discharged after 10 days of hospitalization. This is the first reported case of neonatal severe COVID-19 caused by Omicron variant infection in China. It is necessary to take appropriate protective measures for the neonate to prevent infection when the mother or caregiver of the neonate is a suspected or confirmed cases of COVID-19.

Efficacy and safety of esophagectomy via left thoracic approach versus via right thoracic approach for middle and lower thoracic esophageal cancer: a multicenter randomized clinical trial (NST1501).

Background: Left thoracic approach (LTA) has been a favorable selection in surgical treatment for esophageal cancer (EC) patients in China before minimally invasive esophagectomy (MIE) is popular. This study aimed to demonstrate whether right thoracic approach (RTA) is superior to LTA in the surgical treatment of middle and lower thoracic esophageal squamous cell carcinoma (TESCC). Methods: Superiority clinical trial design was used for this multicenter randomized controlled two-parallel group study. Between April 2015 and December 2018, cT1b-3N0-1M0 TESCC patients from 14 centers were recruited and randomized by a central stratified block randomization program into LTA or RTA groups. All enrolled patients were followed up every three months after surgery. The software SPSS 20.0 and R 3.6.2. were used for statistical analysis. Efficacy and safety outcomes, 3-year overall survival (OS) and disease-free survival (DFS) were calculated and compared using the Kaplan-Meier method and the log-rank test. Results: A total of 861 patients without suspected upper mediastinal lymph nodes (umLN) were finally enrolled in the study after 95 ineligible patients were excluded. 833 cases (98.7%) were successfully followed up until June 1, 2020. Esophagectomies were performed via LTA in 453 cases, and via RTA in 408 cases. Compared with the LTA group, the RTA group required longer operating time (274.48±78.92 vs. 205.34±51.47 min, P<0.001); had more complications (33.8% vs. 26.3% P=0.016); harvested more lymph nodes (LNs) (23.61±10.09 vs. 21.92±10.26, P=0.015); achieved a significantly improved OS in stage IIIa patients (67.8% vs. 51.8%, P=0.022). The 3-year OS and DFS were 68.7% and 64.3% in LTA arm versus 71.3% and 63.7% in RTA arm (P=0.20; P=0.96). Conclusions: Esophagectomies via both LTA and RTA can achieve similar outcomes in middle or lower TESCC patients without suspected umLN. RTA is superior to LTA and recommended for the surgical treatment of more advanced stage TESCC due to more complete lymphadenectomy. Trial Registration: ClinicalTrials.gov NCT02448979.

[Detection of NPM1 Mutation in Acute Myeloid Leukemia by Droplet Digital PCR and Its Clinical Application Value].

OBJECTIVE: To establish the droplet digital PCR (ddPCR) assay for the detection of NPM1 type A mutation in patients with acute myeloid leukemia (AML), and to evaluate its specificity, sensitivity and its value in clinical application. METHODS: NPM1 mutant and wildtype plasmids were used to verify the performance of ddPCR. Both ddPCR and Sanger sequencing were used to detect the bone marrow samples of 87 AML patients, which were confirmed by next generation sequencing (NGS). Moreover, NPM1 mutation burden was dynamically monitored in five patients by ddPCR. RESULTS: The limit of blank (LOB) of ddPCR established for NPM1 mutation detection was 1.1 copies/µl, and the limit of detection (LOD) was 2.43 copies/µl, which had good linearity. Among the 87 newly diagnosed AML patients, ddPCR identified seventeen cases positive for NPM1 mutation (19.5%)， which was consistent with Sanger sequencing. NGS confirmed 12 positive cases, including 8 of type A mutations, 2 of type D mutations, and 2 of rare type mutations. The results of dynamic monitoring of NPM1 mutation burden in 5 patients showed that the NPM1 mutation burden decreased obviously even close to 0, when patients achieve complete remission after chemotherapy. However, the mutation burden was increased again at the time of relapse. CONCLUSION: In this study, we established a ddPCR method for detection of NPM1 mutation with good sensitivity and repeatability, which can be used for screening NPM1 mutation in newly diagnosed AML patients and for minimal residual disease monitoring after remission in positive AML patients to guide treatment.

A real-time pluripotency reporter for the long-term and real-time monitoring of pluripotency changes in induced pluripotent stem cells.

To master the technology of reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs), which will lay a good foundation for setting up a technology platform on reprogramming human cancer cells into iPSCs. Mouse iPSCs (i.e., Oct4-GFP miPSCs) was successfully generated from mouse embryonic fibroblasts (MEFs) harboring Oct4-EGFP transgene by introducing four factors, Oct4, Sox2, c-Myc and Klf4, under mESC (Murine embryonic stem cells) culture conditions. Oct4-GFP miPSCs were similar to mESCs in morphology, proliferation, mESC-specific surface antigens and gene expression. Additionally, Oct4-GFP miPSCs could be cultured in suspension to form embryoid bodies (EBs) and differentiate into cell types of the three germ layers in vitro. Moreover, Oct4-GFP miPSCs could develop to teratoma and chimera in vivo. Unlike cell cycle distribution of MEFs, Oct4-GFP miPSCs are similar to mESCs in the cell cycle structure which consists of higher S phase and lower G1 phase. More importantly, our data demonstrated that MEFs harboring Oct4-EGFP transgene did not express GFP, until they were reprogrammed to the pluripotent stage (iPSCs), while the GFP expression was progressively lost when these pluripotent Oct4-GFP miPSCs exposed to EB-mediated differentiation conditions, suggesting the pluripotency of Oct4-GFP miPSCs can be real-time monitored over long periods of time via GFP assay. Altogether, our findings demonstrate that Oct4-GFP miPSC line is successfully established, which will lay a solid foundation for setting up a technology platform on reprogramming cancer cells into iPSCs. Furthermore, this pluripotency reporter system permits the long-term real-time monitoring of pluripotency changes in a live single-cell, and its progeny.