Assessment of Key Factors Impacting Variability in AAV Vector Genome Titration by Digital PCR.

Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze-thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.

Methodological Validation of Sedimentation Velocity Analytical Ultracentrifugation Method for Adeno-Associated Virus and Collaborative Calibration of System Suitability Substance.

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and high sedimentation coefficient substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF (high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the relative standard deviation (RSD) could be within 5%. Even for empty or partially less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4 × 1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235 nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Because of the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control) laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.

Photoionisation detection of a single Er3+ ion with sub-100-ns time resolution.

Efficient detection of single optical centres in solids is essential for quantum information processing, sensing and single-photon generation applications. In this work, we use radio-frequency (RF) reflectometry to electrically detect the photoionisation induced by a single Er3+ ion in Si. The high bandwidth and sensitivity of the RF reflectometry provide sub-100-ns time resolution for the photoionisation detection. With this technique, the optically excited state lifetime of a single Er3+ ion in a Si nano-transistor is measured for the first time to be [Formula: see text]s. Our results demonstrate an efficient approach for detecting a charge state change induced by Er excitation and relaxation. This approach could be used for fast readout of other single optical centres in solids and is attractive for large-scale integrated optical quantum systems thanks to the multi-channel RF reflectometry demonstrated with frequency multiplexing techniques.

Generation and utilization of endostatin-sensitive cell lines for assessing the biological activity of endostatin.

Recombinant proteins are gaining increasing popularity for treating human diseases. The clinical effectiveness of recombinant proteins is directly related to their biological activity, which is an important indicator in drug development and quality control. However, certain recombinant proteins have unclear or complex signal pathways, making detecting their activity in vitro difficult. For instance, recombinant human endostatin (endostatin), a new antitumor drug developed in China, lacks a sensitive and stable assay for its biological activity since being market approval. To address this issue, we performed a genome-wide screening of immortalized human umbilical vein endothelial cells (HUVECs) using a CRISPR/Cas9 knockout library containing 20,000 targeted genes. We identified two potential endostatin-resistant genes, NEPSPP and UTS2, and successfully constructed a highly sensitive cell line, HUVEC-UTS2-3#, by knocking down the UTS2 gene. Based on the optimized parameters of HUVEC-UTS2-3# cells, we established a new method for detecting the biological activity of endostatin. The method was validated, and it produced results consistent with primary HUVEC cells but with higher sensitivity and more stable data. The use of gene-editing technology provides a novel solution for detecting the biological activity of recombinant proteins that other methods cannot detect.

Andrographolide acts with dexamethasone to inhibit the growth of acute lymphoblastic leukemia CEM­C1 cells via the regulation of the autophagy­dependent PI3K/AKT/mTOR signaling pathway.

Acute lymphoblastic leukemia (ALL) is one of the most common malignant tumor types of the circulatory system. Dexamethasone (DEX) acts on the glucocorticoid (GC) receptor (GR) and is a first-line chemotherapy drug for ALL. However, long-term or high-dose applications of the drug can not only cause adverse reactions, such as osteoporosis and high blood pressure, but can also cause downregulation of GR and lead to drug resistance. In the present study, reverse transcription-quantitative PCR, western blotting and LysoTracker Red staining were used to observe the effects of DEX and andrographolide (AND; a botanical with antitumorigenic properties) combined treatment. It was found that AND enhanced the sensitivity of CEM-C1 cells, a GC-resistant cell line, to DEX, and synergistically upregulated GR both at the transcriptional and post-transcriptional level with DEX. The combination of AND with DEX synergistically alkalized lysosomal lumen and downregulated the expression of autophagy-related genes Beclin1 and microtubule-associated 1 protein light chain 3 (LC3), thereby inhibiting autophagy. Knocking down LC3 expression enhanced GR expression, suggesting that GR was regulated by autophagy. Furthermore, compared with the monotherapy group (AND or DEX in isolation), AND interacted with DEX to activate the autophagy-dependent PI3K/AKT/mTOR signaling pathway by enhancing the phosphorylation of PI3K, AKT and mTOR, thereby decreasing GR degradation and increasing the sensitivity of cells to GCs. In conclusion, the present study demonstrated that AND exhibited a synergistic anti-ALL effect with DEX via upregulation of GR, which was orchestrated by the autophagy-related PI3K/AKT/mTOR signaling pathway. The results of the present study therefore provided novel research avenues and strategies for the treatment of ALL.

Field-programmable-gate-array based hardware platform for nitrogen-vacancy center based fast magnetic imaging.

A nitrogen-vacancy center based scanning magnetic microscope can be used to characterize magnetics at the nanoscale with high sensitivity. This paper reports a field-programmable-gate-array based hardware system that is designed to realize control and signal readout for fast scanning magnetic imaging with a nitrogen-vacancy center. A 10-channel 1 Msps @ 20 bit analog signal generator, a 12-channel 50 ps resolution pulse generator, a 300 Msps @ 16 bit lock-in amplifier with proportional integral derivative control function, and a 4-channel 200 Msps counter are integrated on the platform. A customized acceleration algorithm is realized with the re-configurable field-programmable-gate-array chip to accelerate the imaging speed of the nitrogen-vacancy system, and the experimental results prove that the imaging efficiency can be accelerated by five times compared to the system without the acceleration algorithm. The platform has considerable potential for future applications of fast scanning magnetic imaging.

Protein-guided biomimetic nanomaterials: a versatile theranostic nanoplatform for biomedical applications.

Over the years, bioinspired mineralization-based approaches have been applied to synthesize multifunctional organic-inorganic nanocomposites. These nanocomposites can address the growing demands of modern biomedical applications. Proteins, serving as vital biological templates, play a pivotal role in the nucleation and growth processes of various organic-inorganic nanocomposites. Protein-mineralized nanomaterials (PMNMs) have attracted significant interest from researchers due to their facile and convenient preparation, strong physiological activity, stability, impressive biocompatibility, and biodegradability. Nevertheless, few comprehensive reviews have expounded on the progress of these nanomaterials in biomedicine. This article systematically reviews the principles and strategies for constructing nanomaterials using protein-directed biomineralization and biomimetic mineralization techniques. Subsequently, we focus on their recent applications in the biomedical field, encompassing areas such as bioimaging, as well as anti-tumor, anti-bacterial, and anti-inflammatory therapies. Furthermore, we discuss the challenges encountered in practical applications of these materials and explore their potential in future applications. This review aspired to catalyze the continued development of these bioinspired nanomaterials in drug development and clinical diagnosis, ultimately contributing to the fields of precision medicine and translational medicine.

Characterization of Adeno-Associated Virus Capsid Proteins by Microflow Liquid Chromatography Coupled with Mass Spectrometry.

Adeno-associated virus (AAV) has been widely used to treat various human diseases as an important delivery vector for gene therapy due to its low immunogenicity and safety. AAV capsids proteins are comprised of three capsid viral proteins (VP; VP1, VP2, VP3). The capsid proteins play a key role in viral vector infectivity and transduction efficiency. To ensure the safety and efficacy of AAV gene therapy products, the quality of AAV vector capsid proteins during development and production should be carefully monitored and controlled. Microflow liquid chromatography coupled with mass spectrometry provides superior sensitivity and fast analysis capability. It showed significant advantages in the analysis of low- concentration and large numbers of AAV samples. The intact mass of capsid protein can be accurately determined using high-resolution mass spectrometry (MS). And MS also provides highly confident confirmation of sequence coverage and post-translational modifications site identification and quantitation. In this study, we used microflow liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the characterization of AAV2 capsid protein. we obtained nearly 100% sequence coverage of low-concentration AAV2 capsid protein (8 × 1011 GC/mL). More than 30 post-translational modifications (PTMs) sites were identified, the PTMs types included deamidation, oxidation and acetylation. From this study, the proposed microflow LC-MS/MS method provides a sensitive and high throughput approach in the characterization of AAVs and other biological products with low abundance.

Revealing active constituents within traditional Chinese Medicine used for treating bacterial pneumonia, with emphasis on the mechanism of baicalein against multi-drug resistant Klebsiella pneumoniae.

ETHNOPHARMACOLOGICAL RELEVANCE: The emergence of antibiotic-resistant bacteria has rendered it more challenging to treat bacterial pneumonia. Traditional Chinese medicine (TCM) has superior efficacy in the treatment of pneumonia, and it has the unique advantage of antibacterial resistance against multi-drug resistant (MDR) bacteria, but the medication rule and pharmacological mechanism of its antibacterial activity are not clear. AIM OF THE STUDY: This study aims to reveal Chinese medication patterns in treating bacterial pneumonia to select bioactive constituents in core herbs, predict their pharmacological mechanisms and further explore their antibacterial ability against clinically isolated MDR Klebsiella pneumoniae (KP) and their antibacterial mechanisms. MATERIALS AND METHODS: The high-frequency medicinal herbs to treat lung diseases were first screened from Pharmacopoeia of the People's Republic of China (ChP.), and then bioactive compounds in core herbs and targets for compounds and disease were collected. Potential targets, signaling pathways, and drugs' core components were determined by constructing protein-protein interaction network, enrichment analysis and "component-target-pathway-disease" network were mapped by Cytoscape 3.8.2, and the potential therapeutic value of selected core components was verified by comparing the disease targets in the GEO database with the herbal component targets in the ITCM database. The clinically isolated KP were screened by drug sensitivity tests with meropenem (MEM), polymyxin E (PE), and tigecycline and biofilm-forming assay; broth microdilution, chessboard methods and biofilm morphology and permeability experiments were employed to determine the antibacterial, bactericidal and biofilm inhibition ability of selected bioactive constituents alone and in combination with antibiotics; The mechanism of bioactive components on quorum sensing (QS) genes LuxS and LuxR was predicted by molecular docking and tested by RT-PCR. RESULTS: The 13 core Chinese medicines were obtained by mining ChP., and 615 potential targets of core herbal medicine were screened, and the PI3K-Akt signaling pathway might play crucial roles in the therapeutic process. In-vitro experiments revealed that the selected core compounds, including forsythoside B, baicalin, baicalein, and forsythin, all have antibacterial activity, in which baicalein had the strongest ability and a synergistic effect in combination with MEM or PE. Their synergy exhibited a stronger effect on biofilms of MDR KP, inhibiting biofilm formation, disrupting formed biofilms, and removing the residual structures of dead bacteria. Baicalein was predicted to have stable binding capacity to LuxS and LuxR genes by molecular docking, and RT-PCR results verified that the combination of baicalein with MEM or PE was effective in inhibiting the expression of QS genes (LuxS and LuxR) and consequently suppressing biofilm formation. CONCLUSION: The core Chinese herbal medicine in the ChP. to treat lung diseases has a multi-component, multi-target, and multi-pathway synergy to improve bacterial pneumonia. Experimental studies have confirmed that the bioactive compound baicalein was able to combat MDR KP alone and synergistic with MEM or PE, inhibited and disrupted biofilms via regulating LuxS and LuxR genes, and further disturbed quorum sensing system to promote the therapeutic efficacy, which provides a new pathway and rationale for treating MDR KP-induced bacterial pneumonia.

RNA m6A reader YTHDF2 facilitates precursor miR-126 maturation to promote acute myeloid leukemia progression.

As the most common internal modification of mRNA, N6-methyladenosine (m6A) and its regulators modulate gene expression and play critical roles in various biological and pathological processes including tumorigenesis. It was reported previously that m6A methyltransferase (writer), methyltransferase-like 3 (METTL3) adds m6A in primary microRNAs (pri-miRNAs) and facilitates its processing into precursor miRNAs (pre-miRNAs). However, it is unknown whether m6A modification also plays a role in the maturation process of pre-miRNAs and (if so) whether such a function contributes to tumorigenesis. Here, we found that YTHDF2 is aberrantly overexpressed in acute myeloid leukemia (AML) patients, especially in relapsed patients, and plays an oncogenic role in AML. Moreover, YTHDF2 promotes expression of miR-126-3p (also known as miR-126, as it is the main product of precursor miR-126 (pre-miR-126)), a miRNA that was reported as an oncomiRNA in AML, through facilitating the processing of pre-miR-126 into mature miR-126. Mechanistically, YTHDF2 recognizes m6A modification in pre-miR-126 and recruits AGO2, a regulator of pre-miRNA processing, to promote the maturation of pre-miR-126. YTHDF2 positively and negatively correlates with miR-126 and miR-126's downstream target genes, respectively, in AML patients, and forced expression of miR-126 could largely rescue YTHDF2/Ythdf2 depletion-mediated suppression on AML cell growth/proliferation and leukemogenesis, indicating that miR-126 is a functionally important target of YTHDF2 in AML. Overall, our studies not only reveal a previously unappreciated YTHDF2/miR-126 axis in AML and highlight the therapeutic potential of targeting this axis for AML treatment, but also suggest that m6A plays a role in pre-miRNA processing that contributes to tumorigenesis.

Comparative chloroplast genome analyses provide insights into evolutionary history of Rhizophoraceae mangroves.

Background: The Rhizophoraceae family comprises crucial mangrove plants that inhabit intertidal environments. In China, eight Rhizophoraceae mangrove species exist. Although complete chloroplast (Cp) genomes of four Rhizophoraceae mangrove plants have been reported, the Cp genomes of the remaining four species remain unclear, impeding a comprehensive understanding of the evolutionary history of this family. Methods: Illumina high-throughput sequencing was employed to obtain the DNA sequences of Rhizophoraceae species. Cp genomes were assembled by NOVOPlasty and annotated using CpGAVAS software. Phylogenetic and divergence time analyses were conducted using MEGA and BEAST 2 software. Results: Four novel Cp genomes of Rhizophoraceae mangrove species (Bruguiera sexangula, Bruguiera gymnorrhiza, Bruguiera × rhynchopetala and Rhizophora apiculata) were successfully assembled. The four Cp genomes ranged in length from 163,310 to 164,560 bp, with gene numbers varying from 124 to 128. The average nucleotide diversity (Pi) value of the eight Rhizophoraceae Cp genomes was 0.00596. Phylogenetic trees constructed based on the complete Cp genomes supported the monophyletic origin of Rhizophoraceae. Divergence time estimation based on the Cp genomes of representative species from Malpighiales showed that the origin of Rhizophoraceae occurred at approximately 58.54-50.02 million years ago (Mya). The divergence time within the genus Rhizophora (∼4.51 Mya) was much earlier than the divergence time within the genus Bruguiera (∼1.41 Mya), suggesting recent speciation processes in these genera. Our data provides new insights into phylogenetic relationship and evolutionary history of Rhizophoraceae mangrove plants.

Refraction and ocular biometric parameters of preschool children in the Beijing whole childhood eye study: the first-year report.

BACKGROUND: Prevention of myopia should begin before school age. However, few population-based cohort studies have investigated refractive status in preschool children with cycloplegia. This study aimed to investigate the post-COVID-19 refraction and ocular biometric parameters of preschool children in Beijing Tongzhou District. METHODS: A population-based cohort study of kindergarten children in Tongzhou District, Beijing, commenced in November 2021. The present study reports data from the first year of the aforementioned population-based study. We selected children aged 3-6 years from nine kindergartens. Biometric parameters, including axial length (AL), anterior chamber depth (ACD), and corneal radius of curvature (CR), were collected before cycloplegia. Cycloplegic refraction was also measured. The spherical equivalent (SE), lens power (LP), and AL-to-CR ratio were calculated. Multiple linear regression analysis was used to analyse the correlation between refraction and ocular biometric parameters. RESULTS: A total of 1,505 children completed the examination, and a mean SE of 1.24 ± 0.91 D was found. The overall prevalence of myopia was 1.93%. The mean AL, ACD, CR, LP, and AL-to-CR ratio were 22.24 ± 0.70 mm, 3.28 ± 0.26 mm, 7.77 ± 0.26 mm, 26.01 ± 1.56 D, and 2.86 ± 0.07, respectively. Longer AL, deeper ACD, larger AL-to-CR ratio, and lower LP were associated with older age; the CR was not significantly different among different ages. In the multiple linear regression analysis, after adjusting for sex and age, the model that included AL, CR, and LP explained 87% of the SE variation. No differences were observed in the prevalence of myopia or the SE in this particular age range. CONCLUSION: The findings of this study suggest that a large proportion of preschool children in Beijing are mildly hyperopic, with a considerably low prevalence of myopia. In preschool children, refractive development was found to present mild hyperopia rather than emmetropia or myopia, a phenomenon that is characteristic of this age range.

Design of Plasmonic Photonic Crystal Fiber for Highly Sensitive Magnetic Field and Temperature Simultaneous Measurement.

A high-sensitivity plasmonic photonic crystal fiber (PCF) sensor is designed and a metal thin film is embedded for achieving surface plasmon resonance (SPR), which can detect the magnetic field and temperature simultaneously. Within the plasmonic PCF sensor, the SPR sensing is accomplished by coating both the upper sensing channel (Ch1) and the lower sensing channel (Ch2) with gold film. In addition, the temperature-sensitive medium polydimethylsiloxane (PDMS) is chosen to fill in Ch1, allowing the sensor to respond to the temperature. The magnetic field-sensitive medium magnetic fluid (MF) is chosen to fill in Ch2, allowing this sensor to respond to the magnetic field. During these processes, this proposed SPR-PCF sensor can achieve dual-parameter sensing. The paper also investigates the electrical field characteristics, structural parameters and sensing performance using COMSOL. Finally, under the magnetic field range of 50-130 Oe, this sensor has magnetic field sensing sensitivities of 0 pm/Oe (Ch1) and 235 pm/Oe (Ch2). In addition, this paper also investigates the response of temperature. Under the temperature range of 20-40 °C, Ch1 and Ch2 have temperature sensitivities of -2000 pm/°C and 0 pm/°C, respectively. It is noteworthy that the two sensing channels respond to only a single physical parameter; this sensing performance is not common in dual-parameter sensing. Due to this sensing performance, it can be found that the magnetic field and temperature can be detected by this designed SPR-PCF sensor simultaneously without founding and calculating a sensing matrix. This sensing performance can solve the cross-sensitivity problem of magnetic field and temperature, thus reducing the measurement error. Since it can sense without a matrix, it further can solve the ill-conditioned matrix and nonlinear change in sensitivity problems in dual-parameter sensing. These excellent sensing capabilities are very important for carrying out multiparameter sensing in complicated environments.

ZnGeSe2 monolayer: water-splitting photocatalyst with ultrahigh solar conversion efficiency.

Hydrogen production through solar water-splitting offers a clean and renewable solution to tackle the ongoing issues of energy scarcity and environmental pollution. Here, the solar water-splitting performance of the ZnGeSe2 monolayer was explored via first-principles calculations. Our calculated results reveal that the ZnGeSe2 monolayer embodies stable configurations and semiconducting properties with direct bandgaps ranging from 1.23 to 1.60 eV under the biaxial strain from -1% to +2%. The generated holes and electrons of the ZnGeSe2 monolayer are separately distributed because of the intrinsic dipole. The calculated band edges of the ZnGeSe2 monolayer are demonstrated to be favorable for solar water-splitting. Additionally, the ZnGeSe2 monolayer exhibits strong optical absorption in the whole visible region. The hydrogen and oxygen evolution reactions can be accomplished without cocatalysts. Of particular significance, the solar to hydrogen (STH) efficiency of the ZnGeSe2 monolayer reaches up to 32%, far exceeding the economic value (10%). In light of these hallmarks, the ZnGeSe2 monolayer is demonstrated as an excellent water-splitting photocatalyst.

The use of fluorescence laparoscopy in the resection of splenic tissue replantation in the right lobe of the liver: A case report and literature review.

BACKGROUND: Ectopic replantation and regeneration of splenic tissue fragments following splenic trauma or splenectomy is known as replantation of splenic tissue. It typically takes place in the abdominal cavity, however, splenic tissue replantation in the liver is extremely rare and difficult to diagnose. It is often misdiagnosed as a liver tumor and removed. CASE PRESENTATION: We present the case of a patient with a history of traumatic splenectomy 15 years prior to the replantation of splenic tissue in the liver. A 4 cm mass in the liver was found during the most recent physical examination, and a computed tomography scan indicated the possibility of a malignant tumor. The tumor was then removed using fluorescence laparoscopy. CONCLUSION: There is a possibility of intrahepatic replantation of splenic tissue in patients who have had a splenectomy in the past, have recently discovered an intrahepatic space-occupying lesion, and do not have any high-risk factors for liver cancer. Unnecessary surgery can be avoided if 99mTc-labeled red blood cells imaging using mass puncture or radionuclide examination provides a clear preoperative diagnosis. Globally, there are no reports of the use of fluorescence laparoscopy in resecting replanted splenic tissue in the liver. Specifically, in the current case, there was no indocyanine green uptake in the mass, and only a small amount was found in the normally functioning liver tissue surrounding the tumor.

QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

Phosphorylation stabilized TET1 acts as an oncoprotein and therapeutic target in B cell acute lymphoblastic leukemia.

Although the overall survival rate of B cell acute lymphoblastic leukemia (B-ALL) in childhood is more than 80%, it is merely 30% in refractory/relapsed and adult patients with B-ALL. This demonstrates a need for improved therapy targeting this subgroup of B-ALL. Here, we show that the ten-eleven translocation 1 (TET1) protein, a dioxygenase involved in DNA demethylation, is overexpressed and plays a crucial oncogenic role independent of its catalytic activity in B-ALL. Consistent with its oncogenic role in B-ALL, overexpression of TET1 alone in normal precursor B cells is sufficient to transform the cells and cause B-ALL in mice within 3 to 4 months. We found that TET1 protein is stabilized and overexpressed because of its phosphorylation mediated by protein kinase C epsilon (PRKCE) and ATM serine/threonine kinase (ATM), which are also overexpressed in B-ALL. Mechanistically, TET1 recruits STAT5B to the promoters of CD72 and JCHAIN and promotes their transcription, which in turn promotes B-ALL development. Destabilization of TET1 protein by treatment with PKC or ATM inhibitors (staurosporine or AZD0156; both tested in clinical trials), or by pharmacological targeting of STAT5B, greatly decreases B-ALL cell viability and inhibits B-ALL progression in vitro and in vivo. The combination of AZD0156 with staurosporine or vincristine exhibits a synergistic effect on inhibition of refractory/relapsed B-ALL cell survival and leukemia progression in PDX models. Collectively, our study reveals an oncogenic role of the phosphorylated TET1 protein in B-ALL independent of its catalytic activity and highlights the therapeutic potential of targeting TET1 signaling for the treatment of refractory/relapsed B-ALL.

A customized control and readout device for vector magnetometers based on nitrogen-vacancy centers.

A customized control and readout device, which is developed to perform real-time measurement for vector magnetometers based on nitrogen-vacancy centers, is presented in this paper. A dual-channel analog-to-digital-converter chip, which has a 25 MSa/s sampling rate and a 16 bits amplitude resolution, is integrated for analog signal acquisition. The data processing and the system control are realized using a Xilinx Kirtex-7 field-programmable-gate-array chip. Eight independent lock-in modules, a four-channel proportional-integral-derivative controller, a reference generator, and a vector field reconstruction module are integrated with the Kirtex-7 device in order to perform the real-time vector magnetic field measurement. The device has a bright future to be applied in practical applications.

METTL16 drives leukemogenesis and leukemia stem cell self-renewal by reprogramming BCAA metabolism.

N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, is involved in many pathological processes. METTL16 is a recently identified m6A methyltransferase. However, its role in leukemia has yet to be investigated. Here, we show that METTL16 is a highly essential gene for the survival of acute myeloid leukemia (AML) cells via CRISPR-Cas9 screening and experimental validation. METTL16 is aberrantly overexpressed in human AML cells, especially in leukemia stem cells (LSCs) and leukemia-initiating cells (LICs). Genetic depletion of METTL16 dramatically suppresses AML initiation/development and maintenance and significantly attenuates LSC/LIC self-renewal, while moderately influencing normal hematopoiesis in mice. Mechanistically, METTL16 exerts its oncogenic role by promoting expression of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) and BCAT2 in an m6A-dependent manner and reprogramming BCAA metabolism in AML. Collectively, our results characterize the METTL16/m6A/BCAT1-2/BCAA axis in leukemogenesis and highlight the essential role of METTL16-mediated m6A epitranscriptome and BCAA metabolism reprograming in leukemogenesis and LSC/LIC maintenance.

Curved π-Conjugated Helical Carbon Frameworks: Syntheses, Structural Analyses, and Properties.

Two enantiomers with helical carbon frameworks (M-HCFa and P-HCFa) and their conformational isomers (M-HCFb and P-HCFb) have been synthesized and characterized. The single-crystal analysis revealed the novel structures in which three propeller blades spiro-fused on two central benzene rings. The optical properties were further investigated, and stable bipolar electrochemiluminescence emissions were discovered for the first time existing in helical carbon frameworks, which provide new insights into the future development of high-performance molecular luminescent devices.