Syndromic immune disorder caused by a viable hypomorphic allele of spliceosome component Snrnp40.

We report a new immunodeficiency disorder in mice caused by a viable hypomorphic mutation of Snrnp40, an essential gene encoding a subunit of the U5 small nuclear ribonucleoprotein (snRNP) complex of the spliceosome. Snrnp40 is ubiquitous but strongly expressed in lymphoid tissue. Homozygous mutant mice showed hypersusceptibility to infection by murine cytomegalovirus and multiple defects of lymphoid development, stability and function. Cell-intrinsic defects of hematopoietic stem cell differentiation also affected homozygous mutants. SNRNP40 deficiency in primary hematopoietic stem cells or T cells or the EL4 cell line increased the frequency of splicing errors, mostly intron retention, in several hundred messenger RNAs. Altered expression of proteins associated with immune cell function was also observed in Snrnp40-mutant cells. The immunological consequences of SNRNP40 deficiency presumably result from cumulative, moderate effects on processing of many different mRNA molecules and secondary reductions in the expression of critical immune proteins, yielding a syndromic immune disorder.

NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component.

The NLRP3 inflammasome responds to microbes and danger signals by processing and activating proinflammatory cytokines, including interleukin 1ß (IL-1ß) and IL-18. We found here that activation of the NLRP3 inflammasome was restricted to interphase of the cell cycle by NEK7, a serine-threonine kinase previously linked to mitosis. Activation of the NLRP3 inflammasome required NEK7, which bound to the leucine-rich repeat domain of NLRP3 in a kinase-independent manner downstream of the induction of mitochondrial reactive oxygen species (ROS). This interaction was necessary for the formation of a complex containing NLRP3 and the adaptor ASC, oligomerization of ASC and activation of caspase-1. NEK7 promoted the NLRP3-dependent cellular inflammatory response to intraperitoneal challenge with monosodium urate and the development of experimental autoimmune encephalitis in mice. Our findings suggest that NEK7 serves as a cellular switch that enforces mutual exclusivity of the inflammasome response and cell division.

An integrated multi-omics analysis of identifies distinct molecular characteristics in pulmonary infections of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) can cause severe acute infections, including pneumonia and sepsis, and cause chronic infections, commonly in patients with structural respiratory diseases. However, the molecular and pathophysiological mechanisms of P. aeruginosa respiratory infection are largely unknown. Here, we performed assays for transposase-accessible chromatin using sequencing (ATAC-seq), transcriptomics, and quantitative mass spectrometry-based proteomics and ubiquitin-proteomics in P. aeruginosa-infected lung tissues for multi-omics analysis, while ATAC-seq and transcriptomics were also examined in P. aeruginosa-infected mouse macrophages. To identify the pivotal factors that are involved in host immune defense, we integrated chromatin accessibility and gene expression to investigate molecular changes in P. aeruginosa-infected lung tissues combined with proteomics and ubiquitin-proteomics. Our multi-omics investigation discovered a significant concordance for innate immunological and inflammatory responses following P. aeruginosa infection between hosts and alveolar macrophages. Furthermore, we discovered that multi-omics changes in pioneer factors Stat1 and Stat3 play a crucial role in the immunological regulation of P. aeruginosa infection and that their downstream molecules (e.g., Fas) may be implicated in both immunosuppressive and inflammation-promoting processes. Taken together, these findings indicate that transcription factors and their downstream signaling molecules play a critical role in the mobilization and rebalancing of the host immune response against P. aeruginosa infection and may serve as potential targets for bacterial infections and inflammatory diseases, providing insights and resources for omics analyses.

Unusual enantioselective cytoplasm-to-nucleus translocation and photosensitization of the chiral Ru(II) cationic complex via simple ion-pairing with lipophilic weak acid counter-anions.

Targeted and enantioselective delivery of chiral diagnostic-probes and therapeutics into specific compartments inside cells is of utmost importance in the improvement of disease detection and treatment. The classical DNA 'light-switch' ruthenium(II)-polypyridyl complex, [Ru(DIP)2(dppz)]Cl2 (DIP = 4,7-diphenyl-1,10-phenanthroline, dppz = dipyridophenazine) has been shown to be accumulated only in the cytoplasm and membrane, but excluded from its intended nuclear DNA target. In this study, the cationic [Ru(DIP)2(dppz)]2+ is found to be redirected into live-cell nucleus in the presence of lipophilic 3,5-dichlorophenolate or flufenamate counter-anions via ion-pairing mechanism, while maintaining its original DNA recognition characteristics. Interestingly and unexpectedly, further studies show that only the Δ-enantiomer is selectively translocated into nucleus while the Λ-enantiomer remains trapped in cytoplasm, which is found to be mainly due to their differential enantioselective binding affinities with cytoplasmic proteins and nuclear DNA. More importantly, only the nucleus-relocalized Δ-enantiomer can induce obvious DNA damage and cell apoptosis upon prolonged visible-light irradiation. Thus, the use of Δ-enantiomer can significantly reduce the dosage needed for maximal treatment effect. This represents the first report of enantioselective targeting and photosensitization of classical Ru(II) complex via simple ion-pairing with suitable weak acid counter-anions, which opens new opportunities for more effective enantioselective cancer treatment.

Unprecedented enantio-selective live-cell mitochondrial DNA super-resolution imaging and photo-sensitizing by the chiral ruthenium polypyridyl DNA "light-switch".

Mitochondrial DNA (mtDNA) is known to play a critical role in cellular functions. However, the fluorescent probe enantio-selectively targeting live-cell mtDNA is rare. We recently found that the well-known DNA 'light-switch' [Ru(phen)2dppz]Cl2 can image nuclear DNA in live-cells with chlorophenolic counter-anions via forming lipophilic ion-pairing complex. Interestingly, after washing with fresh-medium, [Ru(phen)2dppz]Cl2 was found to re-localize from nucleus to mitochondria via ABC transporter proteins. Intriguingly, the two enantiomers of [Ru(phen)2dppz]Cl2 were found to bind enantio-selectively with mtDNA in live-cells not only by super-resolution optical microscopy techniques (SIM, STED), but also by biochemical methods (mitochondrial membrane staining with Tomo20-dronpa). Using [Ru(phen)2dppz]Cl2 as the new mtDNA probe, we further found that each mitochondrion containing 1-8 mtDNA molecules are distributed throughout the entire mitochondrial matrix, and there are more nucleoids near nucleus. More interestingly, we found enantio-selective apoptotic cell death was induced by the two enantiomers by prolonged visible light irradiation, and in-situ self-monitoring apoptosis process can be achieved by using the unique 'photo-triggered nuclear translocation' property of the Ru complex. This is the first report on enantio-selective targeting and super-resolution imaging of live-cell mtDNA by a chiral Ru complex via formation and dissociation of ion-pairing complex with suitable counter-anions.

High-Fidelity Sensitive Tracing Circulating Tumor Cell Telomerase Activity.

Dynamic tracing of intracellular telomerase activity plays a crucial role in cancer cell recognition and correspondingly in earlier cancer diagnosis and personalized precision therapy. However, due to the complexity of the required reaction system and insufficient loading of reaction components into cells, achieving a high-fidelity determination of telomerase activity is still a challenge. Herein, an Aptamer-Liposome mediated Telomerase activated poly-Molecular beacon Arborescent Nanoassembly(ALTMAN) approach was described for direct high-fidelity visualization of telomerase activity. Briefly, intracellular telomerase activates molecular beacons, causing their hairpin structures to unfold and produce fluorescent signals. Furthermore, multiple molecular beacons can self-assemble, forming arborescent nanostructures and leading to exponential amplification of fluorescent signals. Integrating the enzyme-free isothermal signal amplification successfully increased the sensitivity and reduced interference by leveraging the skillful design of the molecular beacon and the extension of the telomerase-activated TTAGGG repeat sequence. The proposed approach enabled ultrasensitive visualization of activated telomerase exclusively with a prominent detection limit of 2 cells·µL-1 and realized real-time imaging of telomerase activity in living cancer cells including blood samples from breast cancer patients and urine samples from bladder cancer patients. This approach opens an avenue for establishing a telomerase activity determination and in situ monitoring technique that can facilitate both telomerase fundamental biological studies and cancer diagnostics.

The anti-cancer drug candidate CBL0137 induced necroptosis via forming left-handed Z-DNA and its binding protein ZBP1 in liver cells.

CBL0137, a promising small molecular anti-cancer drug candidate, has been found to effectively induce apoptosis via activating p53 and suppressing nuclear factor-kappa B (NF-κB). However, it is still not clear whether CBL0137 can induce necroptosis in liver cancer; and if so, what is the underlying molecular mechanism. Here we found that CBL0137 could significantly induce left-handed double helix structure Z-DNA formation in HepG2 cells as shown by Z-DNA specific antibody assay, which was further confirmed by observing the expression of Z-DNA binding protein 1 (ZBP1) and adenosine deaminase acting on RNA 1 (ADAR1). Interestingly, we found that caspase inhibition significantly promoted CBL0137-induced necroptosis, which was further supported with the increase of the late apoptosis and necrosis assessed by the flow cytometry. Furthermore, we found that CBL0137 can also induce the expression of the three necroptosis-related proteins: receptor interacting serine/threonine kinase 1 (RIPK1), receptor interacting serine/threonine kinase 3 (RIPK3), and mixed lineage kinase domain-like (MLKL). Taken together, it was assumed that CBL0137-indued necroptosis in liver cells was due to induction of Z-DNA and ZBP1, which activated RIPK1/RIPK3/MLKL pathway. This represents the first report on the induction of the Z-DNA-mediated necroptosis by CBL0137 in the liver cancer cells, which should provide new perspectives for CBL0137 treatment of liver cancer.

An improvement on the electrocatalytic performance of ZIF-67 byin situself-growing CNTs on surface.

Efficient and robust oxygen reduction reaction (ORR) catalysts are essential for the development of high-performance anion-exchange membrane fuel cells (AEMFC). To enhance the electrochemical performance of metal-organic frameworks of cobalt-based zeolite imidazolium skeleton (ZIF-67), this study reported a novel ZIF-67-4@CNT byin situgrowing carbon nanotubes (CNTs) on the surface of ZIF-67 via a mild two-step pyrolysis/oxidation treatment. The electrochemical results showed that the as-prepared ZIF-67-4@CNT after CTAB modification exhibited excellent catalytic activity with good stability, with Eonset, E1/2, and Ilimit, respectively were 0.98 V (versus RHE), 0.87 V (versus RHE) and 6.04 mA cm-2@1600 rpm, and a current retention rate of about 94.21% after polarized at 0.80 V for 10 000 s, which were all superior to that of the commercial 20 wt% Pt/C. The excellent ORR catalytic performance was mainly attributed to the large amount of thein situgrowing CNTs on the surface, encapsulated with a wide range of valence states of metallic cobalt.

Thousands of induced germline mutations affecting immune cells identified by automated meiotic mapping coupled with machine learning.

Forward genetic studies use meiotic mapping to adduce evidence that a particular mutation, normally induced by a germline mutagen, is causative of a particular phenotype. Particularly in small pedigrees, cosegregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the identification of mutations causing immune phenotypes in mice by creating Candidate Explorer (CE), a machine-learning software program that integrates 67 features of genetic mapping data into a single numeric score, mathematically convertible to the probability of verification of any putative mutation-phenotype association. At this time, CE has evaluated putative mutation-phenotype associations arising from screening damaging mutations in ∼55% of mouse genes for effects on flow cytometry measurements of immune cells in the blood. CE has therefore identified more than half of genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.

Advanced supramolecular design for direct ink writing of soft materials.

The exciting advancements in 3D-printing of soft materials are changing the landscape of materials development and fabrication. Among various 3D-printers that are designed for soft materials fabrication, the direct ink writing (DIW) system is particularly attractive for chemists and materials scientists due to the mild fabrication conditions, compatibility with a wide range of organic and inorganic materials, and the ease of multi-materials 3D-printing. Inks for DIW need to possess suitable viscoelastic properties to allow for smooth extrusion and be self-supportive after printing, but molecularly facilitating 3D printability to functional materials remains nontrivial. While supramolecular binding motifs have been increasingly used for 3D-printing, these inks are largely optimized empirically for DIW. Hence, this review aims to establish a clear connection between the molecular understanding of the supramolecularly bound motifs and their viscoelastic properties at bulk. Herein, extrudable (but not self-supportive) and 3D-printable (self-supportive) polymeric materials that utilize noncovalent interactions, including hydrogen bonding, host-guest inclusion, metal-ligand coordination, micro-crystallization, and van der Waals interaction, have been discussed in detail. In particular, the rheological distinctions between extrudable and 3D-printable inks have been discussed from a supramolecular design perspective. Examples shown in this review also highlight the exciting macroscale functions amplified from the molecular design. Challenges associated with the hierarchical control and characterization of supramolecularly designed DIW inks are also outlined. The perspective of utilizing supramolecular binding motifs in soft materials DIW printing has been discussed. This review serves to connect researchers across disciplines to develop innovative solutions that connect top-down 3D-printing and bottom-up supramolecular design to accelerate the development of 3D-print soft materials for a sustainable future.

Tripodal Organic Cages with Unconventional CH···O Interactions for Perchlorate Remediation in Water.

Perchlorate anions used in industry are harmful pollutants in groundwater. Therefore, selectively binding perchlorate provides solutions for environmental remediation. Here, we synthesized a series of tripodal organic cages with highly preorganized Csp3-H bonds that exhibit selectively binding to perchlorate in organic solvents and water. These cages demonstrated binding affinities to perchlorate of 105-106 M-1 at room temperature, along with high selectivity over competing anions, such as iodide and nitrate. Through single crystal structure analysis and density functional theory calculations, we identified unconventional Csp3-H···O interactions as the primary driving force for perchlorate binding. Additionally, we successfully incorporated this cage into a 3D-printable polymer network, showcasing its efficacy in removing perchlorate from water.

A label-free fluorescent sensor for rapid and sensitive detection of ctDNA based on fluorescent PDA nanoparticles.

Technological advances in the detection of circulating tumor DNA (ctDNA) have made new options available for diagnosis, classification, biological studies, and treatment selection. However, effective and practical methods for analyzing this emerging class of biomarkers are still lacking. In this work, a fluorescent biosensor was designed for the label-free detection of ctDNA (EGFR 19 del for non-small cell lung cancer, NSCLC). The biosensor was based on the fact that MnO2 nanosheets (MnO2 NSs) have stronger affinity towards single-stranded DNA (ssDNA), as compared with double-stranded DNA (dsDNA). As a high-performance nanoenzyme, MnO2 NSs could oxidize dopamine (DA) into fluorescent polydopamine nanoparticles (FL-PDA NPs), which could be used as a fluorescence signal. The probe ssDNA could be adsorbed on the surface of MnO2 NSs through π-π stacking, and the active site would be masked, causing a lower fluorescence. After the targets were recognized by probe ssDNA to form dsDNA, its affinity for MnO2 NSs decreased and the active site recovered, causing a restored fluorescence. It was verified that Mn ions, •OH radicals and electron transfer were the important factors in the catalytic oxidation of DA. Under the optimal experimental conditions, this biosensor exhibited a detection limit of 380 pM and a linear range of 25-125 nM, providing reliable readout in a short time (45 min). This sensor exhibited outstanding specificity, stability and reproducibility. In addition, this sensor was applied to the detection of ctDNA in serum samples and cell lysates. It is demonstrated that FL-PDA NPs can be used as a fluorescence signal for easy, rapid and label-free detection of ctDNA without any other amplification strategies, and the proposed strategy has great potential for biomarker detection in the field of liquid biopsy.

Essential requirement for nicastrin in marginal zone and B-1 B cell development.

γ-secretase is an intramembrane protease complex that catalyzes the proteolytic cleavage of amyloid precursor protein and Notch. Impaired γ-secretase function is associated with the development of Alzheimer's disease and familial acne inversa in humans. In a forward genetic screen of mice with N-ethyl-N-nitrosourea-induced mutations for defects in adaptive immunity, we identified animals within a single pedigree exhibiting both hypopigmentation of the fur and diminished T cell-independent (TI) antibody responses. The causative mutation was in Ncstn, an essential gene encoding the protein nicastrin (NCSTN), a member of the γ-secretase complex that functions to recruit substrates for proteolysis. The missense mutation severely limits the glycosylation of NCSTN to its mature form and impairs the integrity of the γ-secretase complex as well as its catalytic activity toward its substrate Notch, a critical regulator of B cell and T cell development. Strikingly, however, this missense mutation affects B cell development but not thymocyte or T cell development. The Ncstn allele uncovered in these studies reveals an essential requirement for NCSTN during the type 2 transitional-marginal zone precursor stage and peritoneal B-1 B cell development, the TI antibody response, fur pigmentation, and intestinal homeostasis in mice.

Genetic and structural studies of RABL3 reveal an essential role in lymphoid development and function.

The small GTPase RABL3 is an oncogene of unknown physiological function. Homozygous knockout alleles of mouse Rabl3 were embryonic lethal, but a viable hypomorphic allele (xiamen [xm]) causing in-frame deletion of four amino acids from the interswitch region resulted in profound defects in lymphopoiesis. Impaired lymphoid progenitor development led to deficiencies of B cells, T cells, and natural killer (NK) cells in Rabl3xm/xm mice. T cells and NK cells exhibited impaired cytolytic activity, and mice infected with mouse cytomegalovirus (MCMV) displayed elevated titers in the spleen. Myeloid cells were normal in number and function. Biophysical and crystallographic studies demonstrated that RABL3 formed a homodimer in solution via interactions between the effector binding surfaces on each subunit; monomers adopted a typical small G protein fold. RABL3xm displayed a large compensatory alteration in switch I, which adopted a ß-strand configuration normally provided by the deleted interswitch residues, thereby permitting homodimer formation. Dysregulated effector binding due to conformational changes in the switch I-interswitch-switch II module likely underlies the xm phenotype. One such effector may be GPR89, putatively an ion channel or G protein-coupled receptor (GPCR). RABL3, but not RABL3xm, strongly associated with and stabilized GPR89, and an N-ethyl-N-nitrosourea (ENU)-induced mutation (explorer) in Gpr89 phenocopied Rabl3xm.

Forward genetic analysis using OCT screening identifies Sfxn3 mutations leading to progressive outer retinal degeneration in mice.

Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Forward genetics provides an excellent tool to find, in an unbiased manner, genes that are essential to these processes. Using N-ethyl-N-nitrosourea mutagenesis in mice in combination with a screening protocol using optical coherence tomography (OCT) and automated meiotic mapping, we identified 11 mutations presumably causative of retinal phenotypes in genes previously known to be essential for retinal integrity. In addition, we found multiple statistically significant gene-phenotype associations that have not been reported previously and decided to target one of these genes, Sfxn3 (encoding sideroflexin-3), using CRISPR/Cas9 technology. We demonstrate, using OCT, light microscopy, and electroretinography, that two Sfxn3-/- mouse lines developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium and disruption of the external limiting membrane. Using single-cell RNA sequencing of retinal cells isolated from C57BL/6J mice, we demonstrate that Sfxn3 is expressed in several bipolar cell subtypes, retinal ganglion cells, and some amacrine cell subtypes but not significantly in Müller cells or photoreceptors. In situ hybridization confirmed these findings. Furthermore, pathway analysis suggests that Sfxn3 may be associated with synaptic homeostasis. Importantly, electron microscopy analysis showed disruption of synapses and synaptic ribbons in the outer plexiform layer of Sfxn3-/- mice. Our work describes a previously unknown requirement for Sfxn3 in retinal function.

Research on Safety Helmet Detection Algorithm Based on Improved YOLOv5s.

Safety helmets are essential in various indoor and outdoor workplaces, such as metallurgical high-temperature operations and high-rise building construction, to avoid injuries and ensure safety in production. However, manual supervision is costly and prone to lack of enforcement and interference from other human factors. Moreover, small target object detection frequently lacks precision. Improving safety helmets based on the helmet detection algorithm can address these issues and is a promising approach. In this study, we proposed a modified version of the YOLOv5s network, a lightweight deep learning-based object identification network model. The proposed model extends the YOLOv5s network model and enhances its performance by recalculating the prediction frames, utilizing the IoU metric for clustering, and modifying the anchor frames with the K-means++ method. The global attention mechanism (GAM) and the convolutional block attention module (CBAM) were added to the YOLOv5s network to improve its backbone and neck networks. By minimizing information feature loss and enhancing the representation of global interactions, these attention processes enhance deep learning neural networks' capacity for feature extraction. Furthermore, the CBAM is integrated into the CSP module to improve target feature extraction while minimizing computation for model operation. In order to significantly increase the efficiency and precision of the prediction box regression, the proposed model additionally makes use of the most recent SIoU (SCYLLA-IoU LOSS) as the bounding box loss function. Based on the improved YOLOv5s model, knowledge distillation technology is leveraged to realize the light weight of the network model, thereby reducing the computational workload of the model and improving the detection speed to meet the needs of real-time monitoring. The experimental results demonstrate that the proposed model outperforms the original YOLOv5s network model in terms of accuracy (Precision), recall rate (Recall), and mean average precision (mAP). The proposed model may more effectively identify helmet use in low-light situations and at a variety of distances.

Effects of C10orf10 on growth and prognosis of glioma under hypoxia. / 低氧环境下C10orf10对胶质瘤生长和预后的影响.

OBJECTIVES: Glioma is the most common malignant tumor in the central nervous system, and the hypoxic microenvironment is prevalent in solid tumors. This study aims to investigate the up-regulation of genes under the condition of hypoxia and their roles in glioma growth, as well as their impact on glioma prognosis. METHODS: The hypoxia-related dataset with glioma was screened in the Gene Expression Omnibus database (GEO), and the differentially expressed genes were analyzed between hypoxia and normoxia through bioinformatics, and chromosome 10 open reading frame 10 (C10orf10) was verified and screened in hypoxia-treated cells through real-time PCR and Western blotting. The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) datasets were downloaded to analyze the mRNA expression of C10orf10 in different grades of glioma and its impact on prognosis. The glioma specimens and follow-up data of 68 gliomas who underwent surgical treatment in Xiangya Hospital of Central South University from March 2017 to January 2021 were collected, and real-time PCR was used to detect the mRNA expression of C10orf10 in different grades of glioma, and the Kaplan-Meier method was used to analyze the relationship between the expression C10orf10 and prognosis. The glioma cells, which could interfere the expression of C10orf10, were constructed, and the effect of C10orf10 on the proliferation of glioma cells was evaluated by cell counting kit-8 (CCK-8) and colony formation assays. RESULTS: Compared with the condition of normoxia, the expression levels of C10orf10 mRNA and protein were significantly up-regulated in glioma cells under hypoxia (P<0.001), and the mRNA expression level of C10orf10 in glioma tissues was up-regulated with the increase of WHO grade in glioma (P<0.001). Based on Kaplan-Meier survival analysis, the higher the mRNA expression level of C10orf10 was, the shorter the survival time of the patient was (P<0.05). And the expression of C10orf10 mRNA was higher in recurrent gliomas than that in primary gliomas in the CGGA database (P<0.001). Knockdown of C10orf10 could significantly inhibit the growth of glioma cells both under hypoxia and normoxia (both P<0.001). CONCLUSIONS: The expression level of C10orf10 can promote the proliferation and prognosis of glioma, which is expected to become a prognostic marker and therapeutic target for glioma.

Mini-XT, a miniaturized tagmentation-based protocol for efficient sequencing of SARS-CoV-2.

BACKGROUND: The COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost 1 million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. RESULTS: We present the 'Mini-XT' miniaturized tagmentation-based library preparation protocol available on protocols.io ( https://doi.org/10.17504/protocols.io.bvntn5en ). SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. The final version of Mini-XT has been used to sequence 4384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. CONCLUSIONS: The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes eightfold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.

GRB10 is a novel oncogene associated with cell proliferation and prognosis in glioma.

BACKGROUND: Glioma is the most common malignant tumor of the central nervous system and is associated with a poor prognosis. This study aimed to explore the function of growth factor receptor-bound protein 10(GRB 10) in glioma. METHODS: The expression of GRB10 in glioma was determined based on the glioma transcriptome profile downloaded from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), and Gene Expression Omnibus (GEO) databases. RT-qPCR was performed to detect the expression of GRB10 in tissue samples obtained from 68 glioma patients. The patients were followed up via telephone or in-person outpatient visits to determine survival. Kaplan-Meier survival analyses were used to evaluate the effect of GRB10 on the prognosis of glioma patients. Further, we constructed GRB10 knockdown cell lines were constructed to investigate the effect of GRB10 on glioma. The cell growth, colony formation, cell cycle assay, EdU assay, and tumor formation in xenograft were performed. RESULTS: The expression level of GRB10 was positively correlated to the histological grades of gliomas. In addition, Kaplan-Meier survival curves revealed that glioma patients with lower expression of GRB10 had more prolonged survival. The knockdown of GRB10 was shown to inhibit cell proliferation, colony formation, and tumor formation in the xenograft models. Cell cycle assay revealed that the knockdown of GRB10 can inhibit the cells entering the G2/M phase from the S phase. The analysis of GSEA suggests that the expression of GRB10 was positively correlated with the hypoxia and EMT signaling pathway. CONCLUSIONS: Our data revealed that GRB10 regulated tumorigenesis in glioma and played a vital role in promoting the glioma progression, which indicated that GRB10 could be used as a potential prognostic marker.

Hyaluronic Acid-PEG-Based Diels-Alder In Situ Forming Hydrogels for Sustained Intraocular Delivery of Bevacizumab.

Retinal diseases are the leading cause of visual impairment worldwide. The effectiveness of antibodies for the treatment of retinal diseases has been demonstrated. Despite the clinical success, achieving sufficiently high concentrations of these protein therapeutics at the target tissue for an extended period is challenging. Patients suffering from macular degeneration often receive injections once per month. Therefore, there is a growing need for suitable systems that can help reduce the number of injections and adverse effects while improving patient complacency. This study systematically characterized degradable "in situ" forming hydrogels that can be easily injected into the vitreous cavity using a small needle (29G). After intravitreal injection, the formulation is designed to undergo a sol-gel phase transition at the administration site to obtain an intraocular depot system for long-term sustained release of bioactives. A Diels-Alder reaction was exploited to crosslink hyaluronic acid-bearing furan groups (HAFU) with 4 arm-PEG10K-maleimide (4APM), yielding stable hydrogels. Here, a systematic investigation of the effects of polymer composition and the ratio between functional groups on the physicochemical properties of hydrogels was performed to select the most suitable formulation for protein delivery. Rheological analysis showed rapid hydrogel formation, with the fastest gel formation within 5 min after mixing the hydrogel precursors. In this study, the mechanical properties of an ex vivo intravitreally formed hydrogel were investigated and compared to the in vitro fabricated samples. Swelling and degradation studies showed that the hydrogels are biodegradable by the retro-Diels-Alder reaction under physiological conditions. The 4APM-HAFU (ratio 1:5) hydrogel formulation showed sustained release of bevacizumab > 400 days by a combination of diffusion, swelling, and degradation. A bioassay showed that the released bevacizumab remained bioactive. The hydrogel platform described in this study offers high potential for the sustained release of therapeutic antibodies to treat ocular diseases.