Tagging the tjp1a Gene in Zebrafish with Monomeric Red Fluorescent Protein Using Biotin Homology Arms.

Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a. Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFPis86 line to examine the behavior and interactions between cells during vascular formation in zebrafish.

Optimisation of denaturing ion pair reversed phase HPLC for the purification of ssDNA in SELEX.

Aptamers have shown great promise as oligonucleotide-based affinity ligands for various medicinal and industrial applications. A critical step in the production of DNA aptamers via selective enhancement of ligands by exponential enrichment (SELEX) is the generation of ssDNA from dsDNA. There are a number of caveats associated with current methods for ssDNA generation, which can lower success rates of SELEX experiments. They often result in low yields thereby decreasing diversity or fail to eliminate parasitic PCR by-products leading to accumulation of by-products from round to round. Both contribute to the failure of SELEX protocols and therefore potentially limit the impact of aptamers compared to their peptide-based antibody counterparts. We have developed a novel method using ion pair reversed phase HPLC (IP RP HPLC) employed under denaturing conditions for the ssDNA re-generation stage of SELEX following PCR. We have utilised a range of 5' chemical modifications on PCR primers to amplify PCR fragments prior to separation and purification of the DNA strands using denaturing IP RP HPLC. We have optimised mobile phases to enable complete denaturation of the dsDNA at moderate temperatures that circumvents the requirement of high temperatures and results in separation of the ssDNA based on differences in their hydrophobicity. Validation of the ssDNA isolation and purity assessment was performed by interfacing the IP RP HPLC with mass spectrometry and fluorescence-based detection. The results show that using a 5' Texas Red modification on the reverse primer in the PCR stage enabled purification of the ssDNA from its complimentary strand via IP RP HPLC under denaturing conditions. Additionally, we have confirmed the purity of the ssDNA generated as well as the complete denaturation of the PCR product via the use of mass-spectrometry and fluorescence analysis therefore proving the selective elimination of PCR by-products and the unwanted complementary strand. Following lyophilisation, ssDNA yields of up to 80% were obtained. In comparison the streptavidin biotin affinity chromatography also generates pure ssDNA with a yield of 55%. The application of this method to rapidly generate and purify ssDNA of the correct size, offers the opportunity to improve the development of new aptamers via SELEX.

Biotinidase biochemical and molecular analyses: Experience at a large reference laboratory.

BACKGROUND: Biotinidase deficiency is caused by absent activity of the biotinidase, encoded by the biotinidase gene (BTD). Affected individuals cannot recycle the biotin, leading to heterogeneous symptoms that are primarily neurological and cutaneous. Early treatment with biotin supplementation can prevent irreversible neurological damage and is recommended for patients with profound deficiency, defined as enzyme activity <10% mean normal (MN). Molecular testing has been utilized along with biochemical analysis for diagnosis and management. In this study, our objective was to correlate biochemical phenotype/enzyme activity to BTD genotype in patients for whom both enzyme and molecular testing were performed at our lab, and to review how the correlations inform on variant severity. METHODS: We analyzed results of biotinidase enzyme analysis and BTD gene sequencing in 407 patients where samples were submitted to our laboratory from 2008 to 2020. RESULTS: We identified 84 BTD variants; the most common was c.1330G>C, and 19/84 were novel BTD variants. A total of 36 patients had enzyme activity <10% of MN and the most common variant found in this group was c.528G>T. No variant was reported in one patient in the profound deficiency group. The most common variant found in patients with enzyme activity more than 10% MN was c.1330G>C. CONCLUSIONS: Although enzyme activity alone may be adequate for diagnosing profound biotinidase deficiency, molecular testing is necessary for accurate carrier screening and in cases where the enzyme activity falls in the range where partial deficiency and carrier status cannot be discriminated.

Novel missense variants cause intermediate phenotypes in the phenotypic spectrum of SLC5A6-related disorders.

SLC5A6 encodes the sodium-dependent multivitamin transporter, a transmembrane protein that uptakes biotin, pantothenic acid, and lipoic acid. Biallelic SLC5A6 variants cause sodium-dependent multivitamin transporter deficiency (SMVTD) and childhood-onset biotin-responsive peripheral motor neuropathy (COMNB), which both respond well to replacement therapy with the above three nutrients. SMVTD usually presents with various symptoms in multiple organs, such as gastrointestinal hemorrhage, brain atrophy, and global developmental delay, at birth or in infancy. Without nutrient replacement therapy, SMVTD can be lethal in early childhood. COMNB is clinically milder and has a later onset than SMVTD, at approximately 10 years of age. COMNB symptoms are mostly limited to peripheral motor neuropathy. Here we report three patients from one Japanese family harboring novel compound heterozygous missense variants in SLC5A6, namely NM_021095.4:c.[221C>T];[642G>C] p.[(Ser74Phe)];[(Gln214His)]. Both variants were predicted to be deleterious through multiple lines of evidence, including amino acid conservation, in silico predictions of pathogenicity, and protein structure considerations. Drosophila analysis also showed c.221C>T to be pathogenic. All three patients had congenital brain cysts on neonatal cranial imaging, but no other morphological abnormalities. They also had a mild motor developmental delay that almost completely resolved despite no treatment. In terms of severity, their phenotypes were intermediate between SMVTD and COMNB. From these findings we propose a new SLC5A6-related disorder, spontaneously remitting developmental delay with brain cysts (SRDDBC) whose phenotypic severity is between that of SMVTD and COMNB. Further clinical and genetic evidence is needed to support our suggestion.

Evaluation of clinical, laboratory, and molecular genetic features of patients with biotinidase deficiency.

Biotinidase deficiency (BD) is an autosomal recessive inherited metabolic disorder which results from the inability of biotin-dependent carboxylase enzymes to function due to the release and absorption of biotin, leading to neurological and cutaneous findings. In the present study, evaluation of demographic characteristics, clinical findings, laboratory results, molecular genetic characteristics, and genotype-phenotype correlations of cases with BD. Two hundred forty-seven cases were included in the study who were admitted to the Department of Pediatric Metabolism of Ankara Bilkent City Hospital after being identified with potential BD through the Newborn Screening Program (NBS), during family screening or based on suspicious clinical findings, or following the detection of a pathogenic variant in a BTD genetic analysis during the period of October 2020 and February 2022. The medical files of the cases were reviewed retrospectively. An analysis of the admission routes of all cases to our clinic revealed 89.5% NBS, 5.7% family screening, and 4.9% suspicious clinical findings suggestive of BD. Complete enzyme deficiency was identified in 19.8%, partial enzyme deficiency in 55.1%, and heterogenous enzyme deficiency in 9.7%. The most common pathogenic variants were c.1270G > C (p.Asp424His), c.410G > A (p.Arg137His), and c.38_44delGCGCTGinsTCC (p.Cys13Phefs*36) in BTD gene. The c.1270G > C variant was most common in patients with cutaneous symptoms. The c.410G > A and c.38_44delGCGCTGinsTCC variants were more common in the patients with neurological symptoms. The mean activity level in patients with the c.1270G > C homozygous variant was statistically significantly higher than the mean activity level in the c.1270G > C compound heterozygous patients and the activity level of patients without the c.1270G > C variant. The mean activity level in c.410G > A homozygous patients was statistically significantly lower than the mean activity level of the c.410G > A compound heterozygous patients and the activity level of patients without the c.410G > A variant. In the course of our study, four new pathogenic variants were detected, namely: c.190G > A (p.Glu64Lys), c.249 + 5G > T, c.228delA (p.Val77*), and c.682A > G (p.Ile228Val).     Conclusions: The present study has determined the clinical and genetic spectrum of a large group of patients with BD in a single center. The frequent mutations in our study were similar to those reported in literature, and four novel variants were also described. What is Known: • Biotinidase deficiency is an autosomal recessive, treatable inborn error of metabolism. Two hundred ninety-four pathogenic variants in the BTD gene have been identified and the c.1270G > C variant is the most frequent BTD gene mutation in both Turkey and around the world. What is New: • Four new pathogenic variants (c.190G > A, p.Glu64Lys; c.249 + 5G > T; c.228delA, p.Val77*; and c.682A > G, p.Ile228Val) have been identified. It is believed that the c.38_44delGCGGCTGinsTCC variant is more commonly seen in individuals with ocular issues; however, further genotype-phenotype correlations are needed.

Evolution of Wolbachia reproductive and nutritional mutualism: insights from the genomes of two novel strains that double infect the pollinator of dioecious Ficus hirta.

Wolbachia is a genus of maternally inherited endosymbionts that can affect reproduction of their hosts and influence metabolic processes. The pollinator, Valisia javana, is common in the male syconium of the dioecious fig Ficus hirta. Based on a high-quality chromosome-level V. javana genome with PacBio long-read and Illumina short-read sequencing, we discovered a sizeable proportion of Wolbachia sequences and used these to assemble two novel Wolbachia strains belonging to supergroup A. We explored its phylogenetic relationship with described Wolbachia strains based on MLST sequences and the possibility of induction of CI (cytoplasmic incompatibility) in this strain by examining the presence of cif genes known to be responsible for CI in other insects. We also identified mobile genetic elements including prophages and insertion sequences, genes related to biotin synthesis and metabolism. A total of two prophages and 256 insertion sequences were found. The prophage WOjav1 is cryptic (structure incomplete) and WOjav2 is relatively intact. IS5 is the dominant transposon family. At least three pairs of type I cif genes with three copies were found which may cause strong CI although this needs experimental verification; we also considered possible nutritional effects of the Wolbachia by identifying genes related to biotin production, absorption and metabolism. This study provides a resource for further studies of Wolbachia-pollinator-host plant interactions.

Effect of Biotin Starvation on Gene Expression in Komagataella phaffii Cells.

Methylotrophic yeast Komagataella phaffii is widely used in biotechnology for recombinant protein production. Due to the practical significance of these yeasts, it is extremely important to properly select cultivation conditions and optimize the media composition. In this study the effect of biotin starvation on gene expression in K. phaffii at transcriptome level was investigated. It was demonstrated, that the response of K. phaffii cell to biotin deficiency strongly depends on the carbon source in the medium. In the media containing glycerol, biotin deficiency led to activation of the genes involved in biotin metabolism, glyoxylate cycle, and synthesis of acetyl-CoA in cytoplasm, as well as repression of the genes, involved in lipo- and gluconeogenesis. In the methanol-containing media, biotin deficiency primarily led to repression of the genes, involved in protein synthesis, and activation of cell response to oxidative stress.

The prevalence of Wolbachia in multiple cockroach species and its implication for urban insect management.

Cockroach management relies heavily on the use of conventional insecticides in urban settings, which no longer provide the anticipated level of control. Knowledge of cockroach endosymbionts, like Wolbachia, might provide novel avenues for control. Therefore, we screened 16 cockroach species belonging to 3 families (Ectobiidae, Blattidae, and Blaberidae) for the presence of Wolbachia. We mapped the evolution of Wolbachia-cockroach relationships based on maximum likelihood phylogeny and phylogenetic species clustering on a multi-loci sequence dataset (i.e., coxA, virD4, hcpA, and gatB) of Wolbachia genes. We confirmed the previous report of Wolbachia in 1 Ectobiid species; Supella longipalpa (Fab.), and detected the presence of Wolbachia in 2 Ectobiid species; Balta notulata (Stål) and Pseudomops septentrionalis Hebard, and 1 Blaberid species; Gromphadorhina portentosa (Schaum). All cockroach-associated Wolbachia herein detected were clustered with the ancestor of F clade Wolbachia of Cimex lectularius L. (bed bugs). Since Wolbachia provision C. lectularius with biotin vitamins that confer reproductive fitness, we screened the cockroach-associated Wolbachia for the presence of biotin genes. In toto, our results reveal 2 important findings: (i) Wolbachia is relatively uncommon among cockroach species infecting about 25% of species investigated, and (ii) cockroach-associated Wolbachia have biotin genes that likely provide nutritional benefits to their hosts. Thus, we discuss the potential of exploring Wolbachia as a tool for urban insect management.

[Holocarboxylase synthetase deficiency induced by HLCS gene mutations: a rare disease study]. / ç½•è§ç— ç ”ç©¶ï¼šHLCSåŸºå› çªå˜è‡´å ¨ç¾§åŒ–é ¶åˆæˆé ¶ç¼ºä¹ç—‡.

A boy, aged 16 months, attended the hospital due to head and facial erythema for 15 months and vulva erythema for 10 months with aggravation for 5 days. The boy developed perioral and periocular erythema in the neonatal period and had erythema and papules with desquamation and erosion in the neck, armpit, and trigone of vulva in infancy. Blood gas analysis showed metabolic acidosis; the analysis of amino acid and acylcarnitine profiles for inherited metabolic diseases and the analysis of organic acid in urine suggested multiple carboxylase deficiency; genetic testing showed a homozygous mutation of c.1522C>T(p.R508W) in the HLCS gene. Finally the boy was diagnosed with holocarboxylase synthetase deficiency and achieved a good clinical outcome after oral biotin treatment. This article analyzes the clinical data of a child with holocarboxylase synthetase deficiency and summarizes the etiology, diagnosis, and treatment of this child, so as to provide ideas for clinicians to diagnose this rare disease.

CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.

AIMS: To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis. MATERIALS AND RESULTS: This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5 days. With the guidance of truncated crRNA containing 16 bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously. CONCLUSIONS: This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.

Genome-wide identification of biotin carboxyl carrier subunits of acetyl-CoA carboxylase in Brassica and their role in stress tolerance in oilseed Brassica napus.

BACKGROUND: Biotin carboxyl carrier protein (BCCP) is a subunit of Acetyl CoA-carboxylase (ACCase) which catalyzes the conversion of acetyl-CoA to malonyl-CoA in a committed step during the de novo biosynthesis of fatty acids. Lipids, lipid metabolites, lipid-metabolizing and -modifying enzymes are known to play a role in biotic and abiotic stress tolerance in plants. In this regard, an understanding of the Brassica napus BCCP genes will aid in the improvement of biotic and abiotic stress tolerance in canola. RESULTS: In this study, we identified 43 BCCP genes in five Brassica species based on published genome data. Among them, Brassica rapa, Brassica oleracea, Brassica nigra, Brassica napus and Brassica juncea had six, seven, seven, 10 and 13 BCCP homologs, respectively. Phylogenetic analysis categorized them into five classes, each with unique conserved domains. The promoter regions of all BCCP genes contained stress-related cis-acting elements as determined by cis-element analysis. We identified four and three duplicated gene pairs (segmental) in B. napus and B. juncea respectively, indicating the role of segmental duplication in the expansion of this gene family. The Ka/Ks ratios of orthologous gene pairs between Arabidopsis thaliana and five Brassica species were mostly less than 1.0, implying that purifying selection, i.e., selective removal of deleterious alleles, played a role during the evolution of Brassica genomes. Analysis of 10 BnaBCCP genes using qRT-PCR showed a different pattern of expression because of exposure of the plants to biotic stresses, such as clubroot and sclerotinia diseases, and abiotic stresses such as drought, low temperature and salinity stresses. CONCLUSIONS: The identification and functional analysis of the Brassica BCCPs demonstrated that some of these genes might play important roles in biotic and abiotic stress responses. Results from this study could lay the foundation for a better understanding of these genes for the improvement of Brassica crops for stress tolerance.

Comprehensive analysis of transcriptome and metabolome analysis reveal new targets of Glaesserella parasuis glucose-specific enzyme IIBC (PtsG).

The ptsG (hpIIBCGlc) gene, belonging to the glucose-specific phosphotransferase system, encodes the bacterial glucose-specific enzyme IIBC. In this study, the effects of a deletion of the ptsG gene were investigated by metabolome and transcriptome analyses. At the transcriptional level, we identified 970 differentially expressed genes between ΔptsG and sc1401 (Padj<0.05) and 2072 co-expressed genes. Among these genes, those involved in methane metabolism, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, pyruvate metabolism, phosphotransferase system (PTS), biotin metabolism, Two-component system and Terpenoid backbone biosynthesis showed significant changes in the ΔptsG mutant strain. Metabolome analysis revealed that a total of 310 metabolites were identified, including 20 different metabolites (p < 0.05). Among them, 15 metabolites were upregulated and 5 were downregulated in ΔptsG mutant strain. Statistical analysis revealed there were 115 individual metabolites having correlation, of which 89 were positive and 26 negative. These metabolites include amino acids, phosphates, amines, esters, nucleotides, benzoic acid and adenosine, among which amino acids and phosphate metabolites dominate. However, not all of these changes were attributable to changes in mRNA levels and must also be caused by post-transcriptional regulatory processes. The knowledge gained from this lays the foundation for further study on the role of ptsG in the pathogenic process of Glaesserella parasuis (G.parasuis).

Comparative genome analysis of commensal segmented filamentous bacteria (SFB) from turkey and murine hosts reveals distinct metabolic features.

BACKGROUND: Segmented filamentous bacteria (SFB) are intestinal commensal microorganisms that have been demonstrated to induce the innate and adaptive immune responses in mouse and rat hosts. SFB are Gram-positive, spore-forming bacteria that fail to grow optimally under in vitro conditions due to unique metabolic requirements. Recently, SFB have been implicated in improved health and growth outcomes in commercial turkey flocks. To assess the nature and variations in SFB of turkeys and how they may differ from mammalian-associated SFB, the genome of turkey-associated SFB was compared with six representative genomes from murine hosts using an in silico approach. RESULTS: The SFB-turkey genome is 1.6 Mb with a G + C content of 26.14% and contains 1,604 coding sequences (CDS). Comparative genome analyses revealed that all the seven SFB strain possesses a common set of metabolic deficiencies and auxotrophies. Specifically, the inability of all the SFB strains to synthesize most of the amino acids, nucleotides and cofactors, emphasizing the importance of metabolite acquisition from the host intestinal environment. Among the seven SFB genomes, the SFB-turkey genome is the largest and contains the highest number of 1,604 predicted CDS. The SFB-turkey genome possesses cellular metabolism genes that are absent in the rodent SFB strains, including catabolic pathways for sucrose, stachyose, raffinose and other complex glycans. Other unique genes associated with SFB-turkey genome is loci for the biosynthesis of biotin, and degradation enzymes to recycle primary bile acids, both of which may play an important role to help turkey associated SFB survive and secure mutualism with its avian host. CONCLUSIONS: Comparative genomic analysis of seven SFB genomes revealed that each strain have a core set of metabolic capabilities and deficiencies that make these bacteria challenging to culture under ex vivo conditions. When compared to the murine-associated strains, turkey-associated SFB serves as a phylogenetic outgroup and a unique member among all the sequenced strains of SFB. This turkey-associated SFB strain is the first reported non-mammalian SFB genome, and highlights the impact of host specificity and the evolution of metabolic capabilities.

Genome-centric metagenomics insights into functional divergence and horizontal gene transfer of denitrifying bacteria in anammox consortia.

Denitrifying bacteria with high abundances in anammox communities play crucial roles in achieving stable anammox-based systems. Despite the relative constant composition of denitrifying bacteria, their functional diversity remains to be explored in anammox communities. Herein, a total of 77 high-quality metagenome-assembled genomes (MAGs) of denitrifying bacteria were recovered from the anammox community in a full-scale swine wastewater treatment plant. Among these microbes, a total of 26 MAGs were affiliated with the seven dominant denitrifying genera that have total abundances higher than 1%. A meta-analysis of these species suggested that external organics reduced the abundances of genus Ignavibacterium and species MAG.305 of UTPRO2 in anammox communities. Comparative genome analysis revealed functional divergence across different denitrifying bacteria, largely owing to their distinct capabilities for carbohydrate (including endogenous and exogenous) utilization and vitamin (e.g., pantothenate and thiamine) biosynthesis. Serval microbes in this system contained fewer genes encoding biotin, pantothenate and methionine biosynthesis compared with their related species from other habitats. In addition, the genes encoding energy production and conversion (73 genes) and inorganic ion transport (53 genes) putatively transferred from other species to denitrifying bacteria, while these denitrifying bacteria (especially genera UTPRO2 and SCN-69-89) likely donated the genes encoding nutrients (e.g., inorganic ion and amino acid) transporter (64 genes) for other members to utilize new metabolites. Collectively, these findings highlighted the functional divergence of these denitrifying bacteria and speculated that the genetic interactions within anammox communities through horizontal gene transfer may be one of the reasons for their functional divergence.

Next-generation sequencing of Tunisian Leigh syndrome patients reveals novel variations: impact for diagnosis and treatment.

Mitochondrial cytopathies, among which the Leigh syndrome (LS), are caused by variants either in the mitochondrial or the nuclear genome, affecting the oxidative phosphorylation process. The aim of the present study consisted in defining the molecular diagnosis of a group of Tunisian patients with LS. Six children, belonging to five Tunisian families, with clinical and imaging presentations suggestive of LS were recruited. Whole mitochondrial DNA and targeted next-generation sequencing of a panel of 281 nuclear genes involved in mitochondrial physiology were performed. Bioinformatic analyses were achieved in order to identify deleterious variations. A single m.10197G>A (p.Ala47Thr) variant was found in the mitochondrial MT-ND3 gene in one patient, while the others were related to autosomal homozygous variants: two c.1412delA (p.Gln471ArgfsTer42) and c.1264A>G (p.Thr422Ala) in SLC19A3, one c.454C>G (p.Pro152Ala) in SLC25A19 and one c.122G>A (p.Gly41Asp) in ETHE1. Our findings demonstrate the usefulness of genomic investigations to improve LS diagnosis in consanguineous populations and further allow for treating the patients harboring variants in SLC19A3 and SLC25A19 that contribute to thiamine transport, by thiamine and biotin supplementation. Considering the Tunisian genetic background, the newly identified variants could be screened in patients with similar clinical presentation in related populations.

Circular RNA hsa_circ_0000915 promotes propranolol resistance of hemangioma stem cells in infantile haemangiomas.

BACKGROUND: Propranolol is a first-line clinical drug for infantile haemangiomas (IH) therapy. Nevertheless, resistance to propranolol is observed in some patients with IH. Circular RNAs (circRNAs) has been increasingly reported to act as a pivotal regulator in tumor progression. However, the underlying mechanism of circRNAs in IH remains unclear. METHODS: Quantitative real-time polymerase chain reaction was performed to detect Circ_0000915, miR-890 and RNF187 expression. Protein levels were determined using western blot. CCK-8 assay was used to measure cell proliferation. Caspase-3 activity assay and flow cytometry were conducted to determine cell apoptosis. Luciferase reporter assay was carried out to assess the interaction between miR-890 and Circ_0000915 or RNF187. Chromatin immunoprecipitation assay was performed to detect the interaction between STAT3 and Circ_0000915 promoter. Biotin pull-down assay was used to detect the direct interaction between miR-890 and Circ_0000915. In vivo experiments were performed to measure tumor formation. RESULTS: Here, we discovered depletion of Circ_0000915 increased propranolol sensitivity of haemangioma derived stem cells (HemSCs) both in vitro and in vivo, whereas forced expression of Circ_0000915 exhibited opposite effects. Mechanistically, Circ_0000915, transcriptionally induced by IL-6/STAT3 pathway, competed with RNF187 for the biding site in miR-890, led to upregulation of RNF187 by acting as a miR-890 "sponge". Furthermore, silence of miR-890 reversed increased propranolol sensitivity of HemSCs due to Circ_0000915 ablation. Moreover, increased Circ_0000915 and RNF187 levels were observed in IH tissues and positively associated with propranolol resistance, miR-890 exhibited an inverse expression pattern. CONCLUSION: We thereby uncover the activation of IL-6/STAT3/Circ_0000915/miR-890/RNF187 axis in propranolol resistance of IH, and provide therapeutic implications for patients of IH with propranolol resistance.

Paper Device Combining CRISPR/Cas12a and Reverse-Transcription Loop-Mediated Isothermal Amplification for SARS-CoV-2 Detection in Wastewater.

Wastewater-based surveillance of the COVID-19 pandemic holds great promise; however, a point-of-use detection method for SARS-CoV-2 in wastewater is lacking. Here, a portable paper device based on CRISPR/Cas12a and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with excellent sensitivity and specificity was developed for SARS-CoV-2 detection in wastewater. Three primer sets of RT-LAMP and guide RNAs (gRNAs) that could lead Cas12a to recognize target genes via base pairing were used to perform the high-fidelity RT-LAMP to detect the N, E, and S genes of SARS-CoV-2. Due to the trans-cleavage activity of CRISPR/Cas12a after high-fidelity amplicon recognition, carboxyfluorescein-ssDNA-Black Hole Quencher-1 and carboxyfluorescein-ssDNA-biotin probes were adopted to realize different visualization pathways via a fluorescence or lateral flow analysis, respectively. The reactions were integrated into a paper device for simultaneously detecting the N, E, and S genes with limits of detection (LODs) of 25, 310, and 10 copies/mL, respectively. The device achieved a semiquantitative analysis from 0 to 310 copies/mL due to the different LODs of the three genes. Blind experiments demonstrated that the device was suitable for wastewater analysis with 97.7% sensitivity and 82% semiquantitative accuracy. This is the first semiquantitative endpoint detection of SARS-CoV-2 in wastewater via different LODs, demonstrating a promising point-of-use method for wastewater-based surveillance.

Measuring Cytological Proximity of Chromosomal Loci to Defined Nuclear Compartments with TSA-seq.

Distinct nuclear structures and bodies are involved in genome intranuclear positioning. Measuring proximity and relative distances of genomic loci to these nuclear compartments, and correlating this chromosome intranuclear positioning with epigenetic marks and functional readouts genome-wide, will be required to appreciate the true extent to which this nuclear compartmentalization contributes to regulation of genome functions. Here we present detailed protocols for TSA-seq, the first sequencing-based method for estimation of cytological proximity of chromosomal loci to spatially discrete nuclear structures, such as nuclear bodies or the nuclear lamina. TSA-seq uses Tyramide Signal Amplification (TSA) of immunostained cells to create a concentration gradient of tyramide-biotin free radicals which decays exponentially as a function of distance from a point-source target. Reaction of these free radicals with DNA deposits tyramide-biotin onto DNA as a function of distance from the point source. The relative enrichment of this tyramide-labeled DNA versus input DNA, revealed by DNA sequencing, can then be used as a "cytological ruler" to infer relative, or even absolute, mean chromosomal distances from immunostained nuclear compartments. TSA-seq mapping is highly reproducible and largely independent of the target protein or antibody choice for labeling a particular nuclear compartment. Our protocols include variations in TSA labeling conditions to provide varying spatial resolution as well as enhanced sensitivity. Our most streamlined protocol produces TSA-seq spatial mapping over a distance range of ~1 micron from major nuclear compartments using ~10-20 million cells.

Hydroxymethylation-Specific Ligation-Mediated Single Quantum Dot-Based Nanosensors for Sensitive Detection of 5-Hydroxymethylcytosine in Cancer Cells.

5-Hydroxymethylcytosine (5hmC) modification is a key epigenetic regulator of cellular processes in mammalian cells, and its misregulation may lead to various diseases. Herein, we develop a hydroxymethylation-specific ligation-mediated single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor for sensitive quantification of 5hmC modification in cancer cells. We design a Cy5-modified signal probe and a biotinylated capture probe for the recognition of specific 5hmC-containing genes. 5hmC in target DNA can be selectively converted by T4 ß-glucosyltransferase to produce a glycosyl-modified 5hmC, which cannot be cleaved by methylation-insensitive restriction enzyme MspI. The glycosylated 5hmC DNA may act as a template to ligate a signal probe and a capture probe, initiating hydroxymethylation-specific ligation to generate large amounts of biotin-/Cy5-modified single-stranded DNAs (ssDNAs). The assembly of biotin-/Cy5-modified ssDNAs onto a single QD through streptavidin-biotin interaction results in FRET and consequently the generation of a Cy5 signal. The nanosensor is very simple without the need for bisulfite treatment, radioactive reagents, and 5hmC-specific antibodies. Owing to excellent specificity and high amplification efficiency of hydroxymethylation-specific ligation and near-zero background of a single QD-based FRET, this nanosensor can quantify 5hmC DNA with a limit of detection of 33.61 aM and a wider linear range of 7 orders of magnitude, and it may discriminate the single-nucleotide difference among 5hmC, 5-methylcytosine, and unmodified cytosine. Moreover, this nanosensor can distinguish as low as a 0.001% 5hmC DNA in complex mixtures, and it can monitor the cellular 5hmC level and discriminate cancer cells from normal cells, holding great potential in biomedical research and clinical diagnostics.

NEAT1 variant 1 weakens the genome-wide effect of miR-3122 on blocking H3K79me3 in bladder cancer.

Nuclear-enriched abundant transcript 1 (NEAT1) is one of the most well-studied long non-coding RNAs (lncRNAs) in multiple human carcinoma. Two distinct variants of NEAT1, however, are never illuminated their specific functions and mechanisms underlying carcinogenesis. In this study, biotin-labelled NEAT1 variants were generated to incubate with cell lysate of bladder cancer cell T24 cells, and fished a batch of RNA substances. Here, we observed that NEAT1.1 (the short transcript) could capture 122 microRNAs (miRNAs), 36 small nucleolar RNAs (snoRNAs), 55 lncRNAs and 38 mRNAs while NEAT1.2 (the long transcript) could obtain 142 miRNAs, 51 snoRNAs, 72 lncRNAs and 41 mRNAs. Furthermore, we also found that the distinctions of RNA binding substances between these two variants were mainly expressed in nucleus rather than cytoplasm. GO analysis indicated that these non-coding RNAs governed histone modification, nucleosome assembly and chromosome organization. We picked up miRNA miR-3122, which substantially interacted with NEAT1.1, and found that histone H3K79me3 was reduced in bladder cancer T24, BIU-87 and EJ-1 cells after miR-3122 overexpression, and rescued by NEAT1.1 additional compensation. Nonetheless, we failed to find that miR-3122 could interfere with expression of H3K79 methyltransferase disruptor of telomeric silencing-1 like (DOT1L). Interestingly, we harvested histone 3 fished by biotin-labelled miR-3122, and validated this intercrossing using RNA immunoprecipitation. Taken together, we demonstrated that NEAT1.1 weakened the effect of miR-3122 on H3K79me3 suppression in bladder cancer.