A detoxification pathway initiated by a nuclear receptor TcHR96h in Tetranychus cinnabarinus (Boisduval).

Understanding the mechanism of detoxification initiation in arthropods after pesticide exposure is crucial. Although the identity of transcription factors that induce and regulate the expression of detoxification genes in response to pesticides is beginning to emerge, whether transcription factors directly interact with xenobiotics is unclear. The findings of this study revealed that a nuclear hormone receptor, Tetranychus cinnabarinus hormone receptor (HR) TcHR96h, regulates the overexpression of the detoxification gene TcGSTm02, which is involved in cyflumetofen resistance. The nuclear translocation of TcHR96h increased after cyflumetofen exposure, suggesting direct binding with cyflumetofen. The direct binding of TcHR96h and cyflumetofen was supported by several independent proteomic assays that quantify interactions with small molecules. Together, this study proposes a model for the initiation of xenobiotic detoxification in a polyphagous agricultural pest. These insights not only provide a better understanding of the mechanisms of xenobiotic detoxification and metabolism in arthropods, but also are crucial in understanding adaptation in polyphagous herbivores.

Exploring timescale-specific functional brain networks and their associations with aging and cognitive performance in a healthy cohort without dementia.

INTRODUCTION: Functional brain networks (FBNs) coordinate brain functions and are studied in fMRI using blood-oxygen-level-dependent (BOLD) signal correlations. Previous research links FBN changes to aging and cognitive decline, but various physiological factors influnce BOLD signals. Few studies have investigated the intrinsic components of the BOLD signal in different timescales using signal decomposition. This study aimed to explore differences between intrinsic FBNs and traditional BOLD-FBN, examining their associations with age and cognitive performance in a healthy cohort without dementia. MATERIALS AND METHODS: A total of 396 healthy participants without dementia (men = 157; women = 239; age range = 20-85 years) were enrolled in this study. The BOLD signal was decomposed into several intrinsic signals with different timescales using ensemble empirical mode decomposition, and FBNs were constructed based on both the BOLD and intrinsic signals. Subsequently, network features-global efficiency and local efficiency values-were estimated to determine their relationship with age and cognitive performance. RESULTS: The findings revealed that the global efficiency of traditional BOLD-FBN correlated significantly with age, with specific intrinsic FBNs contributing to these correlations. Moreover, local efficiency analysis demonstrated that intrinsic FBNs were more meaningful than traditional BOLD-FBN in identifying brain regions related to age and cognitive performance. CONCLUSIONS: These results underscore the importance of exploring timescales of BOLD signals when constructing FBN and highlight the relevance of specific intrinsic FBNs to aging and cognitive performance. Consequently, this decomposition-based FBN-building approach may offer valuable insights for future fMRI studies.

Rapid and Precise Differentiation and Authentication of Agricultural Products via Deep Learning-Assisted Multiplex SERS Fingerprinting.

Accurate and rapid differentiation and authentication of agricultural products based on their origin and quality are crucial to ensuring food safety and quality control. However, similar chemical compositions and complex matrices often hinder precise identification, particularly for adulterated samples. Herein, we propose a novel method combining multiplex surface-enhanced Raman scattering (SERS) fingerprinting with a one-dimensional convolutional neural network (1D-CNN), which enables the effective differentiation of the category, origin, and grade of agricultural products. This strategy leverages three different SERS-active nanoparticles as multiplex sensors, each tailored to selectively amplify the signals of preferentially adsorbed chemicals within the sample. By strategically combining SERS spectra from different NPs, a 'SERS super-fingerprint' is constructed, offering a more comprehensive representation of the characteristic information on agricultural products. Subsequently, utilizing a custom-designed 1D-CNN model for feature extraction from the 'super-fingerprint' significantly enhances the predictive accuracy for agricultural products. This strategy successfully identified various agricultural products and simulated adulterated samples with exceptional accuracy, reaching 97.7% and 94.8%, respectively. Notably, the entire identification process, encompassing sample preparation, SERS measurement, and deep learning analysis, takes only 35 min. This development of deep learning-assisted multiplex SERS fingerprinting establishes a rapid and reliable method for the identification and authentication of agricultural products.

Rose Bengal diacetate-mediated antimicrobial photodynamic inactivation: potentiation by potassium iodide and acceleration of wound healing in MRSA-infected diabetic mice.

Previous studies have shown that antimicrobial photodynamic inactivation (aPDI) can be strongly potentiated by the addition of the non-toxic inorganic salt, potassium iodide (KI). This approach was shown to apply to many different photosensitizers, including the xanthene dye Rose Bengal (RB) excited by green light (540 nm). Rose Bengal diacetate (RBDA) is a lipophilic RB derivative that is easily taken up by cells and hydrolyzed to produce an active photosensitizer. Because KI is not taken up by microbial cells, it was of interest to see if aPDI mediated by RBDA could also be potentiated by KI. The addition of 100 mM KI strongly potentiated the killing of Gram-positive methicillin-resistant Staphylocccus aureus, Gram-negative Eschericia coli, and fungal yeast Candida albicans when treated with RBDA (up to 15 µM) for 2 hours followed by green light (540 nm, 10 J/cm2). Both RBDA aPDI regimens (400 µM RBDA with or without 400 mM KI followed by 20 J/cm2 green light) accelerated the healing of MRSA-infected excisional wounds in diabetic mice, without damaging the host tissue.

Design and experiment of high-field asymmetric waveform ion mobility spectrometry chip based on an integrated temperature control printed circuit board structure.

RATIONALE: During the detection of volatile organic compounds (VOC) using high-field asymmetric waveform ion mobility spectrometry (FAIMS), the ambient temperature significantly impacts the accuracy of planar FAIMS. To mitigate the influence of ambient temperature on detection accuracy and enhance resolution, a FAIMS system based on the inner impedance characteristics of a printed circuit board (PCB) was designed for temperature control. METHODS: This study, conducted under standard atmospheric pressure, aimed to assess the signal stability of a planar FAIMS instrument with and without temperature control, and the effect of temperature change on the detection ability of acetone, ethanol, and their mixture was studied using PCB self-heating. RESULTS: Experimental results demonstrated that the base noise in FAIMS with temperature control was 0.2 pA, whereas that in FAIMS without temperature control was 1.8 pA. Notably, with increasing temperature, the detection ability of FAIMS changes accordingly. The optimal relative detection ability of acetone was observed when the electrode plate was heated to 45°C under an electric field of 15 kV/cm. CONCLUSIONS: This study provides a novel approach to improve the resolving power of FAIMS systems and their signal-to-noise ratio. The utilization of a PCB-based temperature control proved effective in stabilizing FAIMS signal characteristics and optimizing detection capabilities, particularly for VOCs such as acetone. These findings have significant implications for improving the accuracy and resolving power of FAIMS systems in VOC detection applications.

A synthetically useful catalytic system for aliphatic C-H oxidation with a nonheme cobalt complex and m-CPBA.

We report herein a synthetically useful catalytic system for aliphatic C-H oxidation with a mononuclear nonheme cobalt(II) complex and m-chloroperbenzoic acid (m-CPBA). Preliminary mechanistic studies suggest that a high-valent cobalt-oxygen species (e.g., cobalt(IV)-oxo or cobalt(III)-oxyl) is the oxidant that effects C-H oxidation via a rate-determining hydrogen atom abstraction (HAA) step.

Transcriptome-wide identification of ARF gene family in medicinal plant Polygonatum kingianum and expression analysis of PkARF members in different tissues.

BACKGROUND: Polygonatum kingianum holds significant importance in Traditional Chinese Medicine due to its medicinal properties, characterized by its diverse chemical constituents including polysaccharides, terpenoids, flavonoids, phenols, and phenylpropanoids. The Auxin Response Factor (ARF) is a pivotal transcription factor known for its regulatory role in both primary and secondary metabolite synthesis. However, our understanding of the ARF gene family in P. kingianum remains limited. METHODS AND RESULTS: We employed RNA-Seq to sequence three distinct tissues (leaf, root, and stem) of P. kingianum. The analysis revealed a total of 31,558 differentially expressed genes (DEGs), with 43 species of transcription factors annotated among them. Analyses via gene ontology and the Kyoto Encyclopedia of Genes and Genomes demonstrated that these DEGs were predominantly enriched in metabolic pathways and secondary metabolite biosynthesis. The proposed temporal expression analysis categorized the DEGs into nine clusters, suggesting the same expression trends that may be coordinated in multiple biological processes across the three tissues. Additionally, we conducted screening and expression pattern analysis of the ARF gene family, identifying 12 significantly expressed PkARF genes in P. kingianum roots. This discovery lays the groundwork for investigations into the role of PkARF genes in root growth, development, and secondary metabolism regulation. CONCLUSION: The obtained data and insights serve as a focal point for further research studies, centred on genetic manipulation of growth and secondary metabolism in P. kingianum. Furthermore, these findings contribute to the understanding of functional genomics in P. kingianum, offering valuable genetic resources.

Targeting cytohesin-1 suppresses acute myeloid leukemia progression and overcomes resistance to ABT-199.

Adhesion molecules play essential roles in the homeostatic regulation and malignant transformation of hematopoietic cells. The dysregulated expression of adhesion molecules in leukemic cells accelerates disease progression and the development of drug resistance. Thus, targeting adhesion molecules represents an attractive anti-leukemic therapeutic strategy. In this study, we investigated the prognostic role and functional significance of cytohesin-1 (CYTH1) in acute myeloid leukemia (AML). Analysis of AML patient data from the GEPIA and BloodSpot databases revealed that CYTH1 was significantly overexpressed in AML and independently correlated with prognosis. Functional assays using AML cell lines and an AML xenograft mouse model confirmed that CYTH1 depletion significantly inhibited the adhesion, migration, homing, and engraftment of leukemic cells, delaying disease progression and prolonging animal survival. The CYTH1 inhibitor SecinH3 exerted in vitro and in vivo anti-leukemic effects by disrupting leukemic adhesion and survival programs. In line with the CYTH1 knockdown results, targeting CYTH1 by SecinH3 suppressed integrin-associated adhesion signaling by reducing ITGB2 expression. SecinH3 treatment efficiently induced the apoptosis and inhibited the growth of a panel of AML cell lines (MOLM-13, MV4-11 and THP-1) with mixed-lineage leukemia gene rearrangement, partly by reducing the expression of the anti-apoptotic protein MCL1. Moreover, we showed that SecinH3 synergized with the BCL2-selective inhibitor ABT-199 (venetoclax) to inhibit the proliferation and promote the apoptosis of ABT-199-resistant leukemic cells. Taken together, our results not only shed light on the role of CYTH1 in cell-adhesion-mediated leukemogenesis but also propose a novel combination treatment strategy for AML.

Lethal pulmonary thromboembolism in mice induced by intravenous human umbilical cord mesenchymal stem cell-derived large extracellular vesicles in a dose- and tissue factor-dependent manner.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have obvious advantages over MSC therapy. But the strong procoagulant properties of MSC-EVs pose a potential risk of thromboembolism, an issue that remains insufficiently explored. In this study, we systematically investigated the procoagulant activity of large EVs derived from human umbilical cord MSCs (UC-EVs) both in vitro and in vivo. UC-EVs were isolated from cell culture supernatants. Mice were injected with UC-EVs (0.125, 0.25, 0.5, 1, 2, 4 µg/g body weight) in 100 µL PBS via the tail vein. Behavior and mortality were monitored for 30 min after injection. We showed that these UC-EVs activated coagulation in a dose- and tissue factor-dependent manner. UC-EVs-induced coagulation in vitro could be inhibited by addition of tissue factor pathway inhibitor. Notably, intravenous administration of high doses of the UC-EVs (1 µg/g body weight or higher) led to rapid mortality due to multiple thrombus formations in lung tissue, platelets, and fibrinogen depletion, and prolonged prothrombin and activated partial thromboplastin times. Importantly, we demonstrated that pulmonary thromboembolism induced by the UC-EVs could be prevented by either reducing the infusion rate or by pre-injection of heparin, a known anticoagulant. In conclusion, this study elucidates the procoagulant characteristics and mechanisms of large UC-EVs, details the associated coagulation risk during intravenous delivery, sets a safe upper limit for intravenous dose, and offers effective strategies to prevent such mortal risks when high doses of large UC-EVs are needed for optimal therapeutic effects, with implications for the development and application of large UC-EV-based as well as other MSC-EV-based therapies.

Transcriptomic landscape reveals germline potential of porcine skin-derived multipotent dermal fibroblast progenitors.

According to estimations, approximately about 15% of couples worldwide suffer from infertility, in which individuals with azoospermia or oocyte abnormalities cannot be treated with assisted reproductive technology. The skin-derived stem cells (SDSCs) differentiation into primordial germ cell-like cells (PGCLCs) is one of the major breakthroughs in the field of stem cells intervention for infertility treatment in recent years. However, the cellular origin of SDSCs and their dynamic changes in transcription profile during differentiation into PGCLCs in vitro remain largely undissected. Here, the results of single-cell RNA sequencing indicated that porcine SDSCs are mainly derived from multipotent dermal fibroblast progenitors (MDFPs), which are regulated by growth factors (EGF/bFGF). Importantly, porcine SDSCs exhibit pluripotency for differentiating into three germ layers and can effectively differentiate into PGCLCs through complex transcriptional regulation involving histone modification. Moreover, this study also highlights that porcine SDSC-derived PGCLCs specification exhibit conservation with the human primordial germ cells lineage and that its proliferation is mediated by the MAPK signaling pathway. Our findings provide substantial novel insights into the field of regenerative medicine in which stem cells differentiate into germ cells in vitro, as well as potential therapeutic effects in individuals with azoospermia and/or defective oocytes.

LSM14B controls oocyte mRNA storage and stability to ensure female fertility.

Controlled mRNA storage and stability is essential for oocyte meiosis and early embryonic development. However, how to regulate mRNA storage and stability in mammalian oogenesis remains elusive. Here we showed that LSM14B, a component of membraneless compartments including P-body-like granules and mitochondria-associated ribonucleoprotein domain (MARDO) in germ cell, is indispensable for female fertility. To reveal loss of LSM14B disrupted primordial follicle assembly and caused mRNA reduction in non-growing oocytes, which was concomitant with the impaired assembly of P-body-like granules. 10× Genomics single-cell RNA-sequencing and immunostaining were performed. Meanwhile, we conducted RNA-seq analysis of GV-stage oocytes and found that Lsm14b deficiency not only impaired the maternal mRNA accumulation but also disrupted the translation in fully grown oocytes, which was closely associated with dissolution of MARDO components. Moreover, Lsm14b-deficient oocytes reassembled a pronucleus containing decondensed chromatin after extrusion of the first polar body, through compromising the activation of maturation promoting factor, while the defects were restored via WEE1/2 inhibitor. Together, our findings reveal that Lsm14b plays a pivotal role in mammalian oogenesis by specifically controlling of oocyte mRNA storage and stability.

Comparison of 755-nm picosecond alexandrite laser versus 1064-nm Q-switched Nd:YAG laser for melasma: A randomized, split-face controlled, 2-year follow-up study.

OBJECTIVES: Pulsed laser treatment of melasma has shown some promising results. To compare the effectiveness and safety of 755-nm picosecond alexandrite laser (PSAL) fitted with diffractive lens array (DLA) versus 1064-nm Q-switched neodynimum:yttrium aluminum garnet laser (QSNYL) for the treatment of melasma. METHODS: We conducted a randomized, split face controlled, 2-year follow-up study. Each face was divided into two parts, each side receiving three treatments with either PSAL or QSNYL at 1 month intervals. Modified Melasma Area Severity Index scores (mMASI), pain scores, patient satisfaction and adverse events were recorded. In vivo reflectance confocal microscopy (RCM) images were acquired. RESULTS: Twenty subjects were enrolled and three dropped out. At 6 months, mMASI scores were significantly lower than baseline for QSNYL sides (p = 0.022), with no statistically significant difference between PSAL sides before and after treatment, PSAL sides versus QSNYL sides, or patient satisfaction scores. QSNYL treatment was associated with less pain (p = 0.014). No serious adverse events were reported. In the PSAL sides RCM showed a large number of dendritic melanocytes infiltrated in the dermis at 2 weeks and 4 weeks after treatment. Ten patients (58.82%) reported recurrence or exacerbation at 2-year follow-up with no statistically significant difference between the two lasers. CONCLUSIONS: QSNYL demonstrated short term clinical efficacy for melasma, but did not provide any additional benefit compared to PSAL with DLA. QSNYL was associated with less pain. There was a high recurrence rate at 2-year follow-up. RCM allowed the detection of cellular changes in melasma lesions.

Histological evaluation of monopolar and bipolar radiofrequency microneedling treatment in a porcine model.

BACKGROUND AND OBJECTIVE: Fractional radiofrequency microneedling (FRM) is widely used as an option for skin rejuvenation, however there is a lack of histological evidence for the various energy delivery systems available. The objective was to assess thermal denaturation of tissue and the wound healing response in monopolar mode versus bipolar mode. Histological analysis was performed to demonstrate the efficacy of automatic impedance feedback system in monopolar mode. STUDY DESIGN AND METHODS: In this study, the acute thermal effects caused by monopolar FRM treatment to the dorsal skin of pigs were assessed histologically by hematoxylin & eosin (H&E) staining. Then, one session of either monopolar or bipolar FRM was used to treat one or the other side of the pig using varying power levels and pulse widths. The acute and chronic tissue reactions were assessed using H&E, immunofluorescence, and western blot analysis at 0, 14, 30, and 90 days after treatment. The efficacy of the impedance feedback system was also monitored histologically. RESULTS: High-energy FRM treatment produced tissue loss and necrosis. The power level and pulse duration significantly affected the coagulation amount. Histopathology at 0, 14, 30, and 90 days showed that the skin tissue reaction was more pronounced for bipolar compared to monopolar FRM. Immunofluorescence showed the expression of TGF-ß, Ki67, MMP3, and elastin increased dramatically with both modes, but were higher in the bipolar FRM treated side. The automatic impedance feedback system could effectively adjust the output energy. CONCLUSIONS: We found that bipolar FRM produced greater thermal effects, more collagen coagulation, and more pronounced molecular changes compared with monopolar mode in a porcine animal model.

Plastome structure, phylogeny and evolution of plastid genes in Reevesia (Helicteroideae, Malvaceae).

Reevesia is an eastern Asian-eastern North American disjunction genus in the family Malvaceae s.l. and comprises approximately 25 species. The relationships within the genus are not well understood. Here, 15 plastomes representing 12 Reevesia species were compared, with the aim of better understanding the species circumscription and phylogenetic relationships within the genus and among genera in the family Malvaceae s.l. The 11 newly sequenced plastomes range between 161,532 and 161, 945 bp in length. The genomes contain 114 unique genes, 18 of which are duplicated in the inverted repeats (IRs). Gene content of these plastomes is nearly identical. All the protein-coding genes are under purifying selection in the Reevesia plastomes compared. The top ten hypervariable regions, SSRs, and the long repeats identified are potential molecular markers for future population genetic and phylogenetic studies. Phylogenetic analysis based on the whole plastomes confirmed the monophyly of Reevesia and a close relationship with Durio (traditional Bombacaceae) in subfamily Helicteroideae, but not with the morphologically similar genera Pterospermum and Sterculia (both of traditional Sterculiaceae). Phylogenetic relationships within Reevesia suggested that two species, R. pubescens and R. thyrsoidea, as newly defined, are not monophyletic. Six taxa, R. membranacea, R. xuefengensis, R. botingensis, R. lofouensis, R. longipetiolata and R. pycnantha, are suggested to be recognized.

The role of microcomedones in acne: Moving from a description to treatment target?

A major factor in the pathogenesis of acne is ductal hyperproliferation in the pilosebaceous glands. This takes the form of invisible microcomedones and leads to the subsequent formation of both inflammatory and non-inflammatory clinical lesions. Microcomedones are the initial stage in the cyclical development of acne, so called comedogenesis. Microcomedones can be detected using cyanoacrylate skin surface stripping, electron microscopy, reflection confocal microscopy and other techniques. It has been proposed that the density and the size of microcomedones are positively correlated with acne severity. Thus, the purpose of this review is to summarize the root causes of acne, and suggest that treatment of microcomedones could, at least in part, resolve acne lesions and prevent relapse.

The energy metabolism of Balantidium polyvacuolum inhabiting the hindgut of Xenocypris davidi.

Anaerobic parasitic ciliates are a specialized group of ciliates that are adapted to anoxic and oxygen-depleted habitats. Among them, Balantidium polyvacuolum, which inhabits the hindgut of Xenocyprinae fishes, has received very limited scientific attention, so the molecular mechanism of its adaptation to the digestive tract microenvironment is still unclear. In this study, transmission electron microscopy (TEM) and single-cell transcriptome analysis were used to uncover the metabolism of B. polyvacuolum. Starch granules, endosymbiotic bacteria, and multiple specialized mitochondrion-related organelles (MROs) of various shapes were observed. The MROs may have completely lost the electron transport chain (ETC) complexes I, III, IV, and V and only retained succinate dehydrogenase subunit A (SDHA) of complex II. The tricarboxylic acid (TCA) cycle was also incomplete. It can be inferred that the hypoxic intestinal environment has led to the specialization of the mitochondria in B. polyvacuolum. Moreover, carbohydrate-active enzymes (CAZymes), including carbohydrate esterases, enzymes with a carbohydrate-binding module, glycoside hydrolases, and glycosyltransferases, were identified, which may constitute evidence that B. polyvacuolum is able to digest carbohydrates and starch. These findings can improve our knowledge of the energy metabolism and adaptive mechanisms of B. polyvacuolum.

TREM2 deficiency inhibits microglial activation and aggravates demyelinating injury in neuromyelitis optica spectrum disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disorder of the central nervous system (CNS) triggered by autoimmune mechanisms. Microglia are activated and play a pivotal role in response to tissue injury. Triggering receptor expressed on myeloid cells 2 (TREM2) is expressed by microglia and promotes microglial activation, survival and phagocytosis. Here, we identify a critical role for TREM2 in microglial activation and function during AQP4-IgG and complement-induced demyelination. TREM2-deficient mice had more severe tissue damage and neurological impairment, as well as fewer oligodendrocytes with suppressed proliferation and maturation. The number of microglia clustering in NMOSD lesions and their proliferation were reduced in TREM2-deficient mice. Moreover, morphology analysis and expression of classic markers showed compromised activation of microglia in TREM2-deficient mice, which was accompanied by suppressed phagocytosis and degradation of myelin debris by microglia. These results overall indicate that TREM2 is a key regulator of microglial activation and exert neuroprotective effects in NMOSD demyelination.

Single-cell RNA sequencing of the Mongolia sheep testis reveals a conserved and divergent transcriptome landscape of mammalian spermatogenesis.

Spermatogenesis is a highly coordinated and complex process, and is pivotal for transmitting genetic information between mammalian generations. In this study, we investigated the conservation, differences, and biological functions of homologous genes during spermatogenesis in Mongolia sheep, humans, cynomolgus monkey, and mice using single-cell RNA sequencing technology. We compared X chromosome meiotic inactivation events in Mongolia sheep, humans, cynomolgus monkey, and mice to uncover the concerted activity of X chromosome genes. Subsequently, we focused on the dynamics of gene expression, key biological functions, and signaling pathways at various stages of spermatogenesis in Mongolia sheep and humans. Additionally, the ligand-receptor networks of Mongolia sheep and humans in testicular somatic and germ cells at different developmental stages were mapped to reveal conserved germ cell-soma communication using single-cell resolution. These datasets provided novel information and insights to unravel the molecular regulatory mechanisms of Mongolia sheep spermatogenesis and highlight conservation in gene expression during spermatogenesis between Mongolia sheep and humans, providing a foundation for the establishment of a large mammalian disease model of male infertility.

Single-cell transcriptome landscape of ovarian cells during primordial follicle assembly in mice.

Primordial follicle assembly in the mouse occurs during perinatal ages and largely determines the ovarian reserve that will be available to support the reproductive life span. The development of primordial follicles is controlled by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time series on murine ovaries, coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to postnatal day (PD) 3 were reported. Along with confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified 5 distinct cell clusters associated with germ cells and 6 with granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. By building single-cell pseudotemporal trajectories, 3 states and 1 branch point of fate transition for the germ cells were revealed, as well as for the granulosa cells. Moreover, Gene Ontology (GO) term enrichment enabled identification of the biological process most represented in germ cells and granulosa cells or common to both cell types at each specific stage, and the interactions of germ cells and granulosa cells basing on known and novel pathway were presented. Finally, by using single-cell regulatory network inference and clustering (SCENIC) algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell-specific transcription programs throughout the period of investigation. Above all, this study provides the whole transcriptome landscape of ovarian cells and unearths new insights during primordial follicle assembly in mice.

Detection of NPM1 Mutations in Acute Myeloid Leukemia by using Drop-Off Droplet Digital PCR and its Clinical Application.

BACKGROUND: Nucleophosmin 1 (NPM1) mutations, which occur in 25 - 30% of acute myeloid leukemia (AML) and 50 - 60% of AML with normal karyotype, have been identified as an important marker for stratification of prog-nosis in AML. This study aimed to establish a new quantitative polymerase chain reaction (PCR) technique, the drop-off droplet digital PCR (ddPCR), for rapid and sensitive detection of NPM1 mutations in AML. METHODS: We established the drop-off ddPCR system and verified its performance. NPM1 mutations were screened in 130 AML patients by drop-off ddPCR and were validated by Sanger sequencing and next-generation sequencing (NGS). Then, the NPM1 mutation burden was dynamically monitored in five patients. RESULTS: The limit of blank (LOB) of drop-off ddPCR established for NPM1 mutation was 3.36 copies/µL, and the limit of detection (LOD) was 5.00 - 5.37 copies/µL in 50 ng DNA, and the sensitivity was about 0.05%, which had good linearity. Drop-off ddPCR identified 33/130 (25.4%) NPM1 mutated cases, consistent with Sanger sequencing. In 18 NPM1 positive cases selected randomly, NGS identified fourteen with type A mutation, two with type D mutation, and two with rare type mutations. The mutation burden of NPM1 mutation analyzed by NGS was consistent with the drop-off ddPCR. The sequential samples were detected for measurable residual disease (MRD) monitoring in 5 patients showed that the NPM1 mutation burden was consistent with clinical remission and recurrence. Compared with traditional ddPCR, drop-off ddPCR was also suitable for MRD monitoring. CONCLUSIONS: In this study, we established a drop-off ddPCR method for detecting three common mutations in AML with good sensitivity and repeatability, which can be used to screen mutations in newly diagnosed AML patients and for MRD monitoring after remission to guide treatment.