An Enzymatically Activated and Catalytic Hairpin Assembly-Driven Intelligent AND-Gated DNA Network for Tumor Molecular Imaging.

Due to the potential off-tumor signal leakage and limited biomarker content, there is an urgent need for stimulus-responsive and amplification-based tumor molecular imaging strategies. Therefore, two tetrahedral framework DNA (tFNA-Hs), tFNA-H1AP, and tFNA-H2, were rationally engineered to form a polymeric tFNA network, termed an intelligent DNA network, in an AND-gated manner. The intelligent DNA network was designed for tumor-specific molecular imaging by leveraging the elevated expression of apurinic/apyrimidinic endonuclease 1 (APE1) in tumor cytoplasm instead of normal cells and the high expression of miRNA-21 in tumor cytoplasm. The activation of tFNA-H1AP can be achieved through specific recognition and cleavage by APE1, targeting the apurinic/apyrimidinic site (AP site) modified within the stem region of hairpin 1 (H1AP). Subsequently, miRNA-21 facilitates the hybridization of activated H1AP on tFNA-H1AP with hairpin 2 (H2) on tFNA-H2, triggering a catalytic hairpin assembly (CHA) reaction that opens the H1AP at the vertices of tFNA-H1AP to bind with H2 at the vertices of tFNA-H2 and generate fluorescence signals. Upon completion of hybridization, miRNA-21 is released, initiating the subsequent cycle of the CHA reaction. The AND-gated intelligent DNA network can achieve specific tumor molecular imaging in vivo and also enables risk stratification of neuroblastoma patients.

[Risk factors for embolism in children with refractory Mycoplasmapneumoniae pneumonia and construction of a nomogram model for prediction of embolism]. / éš¾æ²»æ€§è‚ºç‚Žæ”¯åŽŸä½“è‚ºç‚Žæ‚£å„¿åˆå¹¶æ “å¡žçš„å±é™©å› ç´ åˆ†æžåŠåˆ—çº¿å›¾é¢„æµ‹æ¨¡åž‹çš„æž„å»º.

OBJECTIVES: To study the risk factors for embolism in children with refractory Mycoplasma pneumoniae pneumonia (RMPP) and to construct a nomogram model for prediction of embolism. METHODS: This retrospective study included 175 children diagnosed with RMPP at Children's Hospital Affiliated toZhengzhou University from January 2019 to October 2023. They were divided into two groups based on the presence of embolism: the embolism group (n=62) and the non-embolism group (n=113). Multivariate logistic regression analysis was used to screen for risk factors of embolism in children with RMPP, and the R software was applied to construct the nomogram model for prediction of embolism. RESULTS: Multivariate logistic regression analysis indicated that higher levels of D-dimer, interleukin-6 (IL-6) and neutrophil to lymphocyte ratio (NLR), lung necrosis, and pleural effusion were risk factors for embolism in children with RMPP (P<0.05). The area under the curve of the nomogram model for prediction of embolism constructed based on the aforementioned risk factors was 0.912 (95%CI: 0.871-0.952, P<0.05). The Hosmer-Lemeshow goodness-of-fit test showed that the model had a good fit with the actual situation (P<0.05). Calibration and decision curve analysis indicated that the model had high predictive efficacy and clinical applicability. CONCLUSIONS: Higher levels of D-dimer, IL-6 and NLR, lung necrosis, and pleural effusion are risk factors for embolism in children with RMPP. The nomogram model based on these risk factors has high clinical value for predicting embolism in children with RMPP.

[Specific changes in gut microbiota and short-chain fatty acid levels in infants with cow's milk protein allergy]. / ç‰›å¥¶è›‹ç™½è¿‡æ•å©´å„¿è‚ é“èŒç¾¤ç‰¹å¼‚æ€§å˜åŒ–åŠçŸ­é“¾è„‚è‚ªé ¸æ°´å¹³åˆ†æž.

OBJECTIVES: To explore the changes in gut microbiota and levels of short-chain fatty acids (SCFA) in infants with cow's milk protein allergy (CMPA), and to clarify their role in CMPA. METHODS: A total of 25 infants diagnosed with CMPA at Children's Hospital Affiliated to Zhengzhou University from August 2019 to August 2020 were enrolled as the CMPA group, and 25 healthy infants were selected as the control group. Fecal samples (200 mg) were collected from both groups and subjected to 16S rDNA high-throughput sequencing technology and liquid chromatography-mass spectrometry to analyze the changes in gut microbial composition and metabolites. Microbial diversity was analyzed in conjunction with metabolites. RESULTS: Compared to the control group, the CMPA group showed altered gut microbial structure and significantly increased α-diversity (P<0.001). The abundance of Firmicutes, Clostridiales and Bacteroidetes was significantly decreased, while the abundance of Sphingomonadaceae, Clostridiaceae_1 and Mycoplasmataceae was significantly increased in the CMPA group compared to the control group (P<0.001). Metabolomic analysis revealed reduced levels of acetic acid, butyric acid, and isovaleric acid in the CMPA group compared to the control group, and the levels of the metabolites were positively correlated with the abundance of SCFA-producing bacteria such as Faecalibacterium and Roseburia (P<0.05). CONCLUSIONS: CMPA infants have alterations in gut microbial structure, increased microbial diversity, and decreased levels of SCFA, which may contribute to increased intestinal inflammation.

Impairment of intestinal barrier associated with the alternation of intestinal flora and its metabolites in cow's milk protein allergy.

Cow's milk protein allergy (CMPA), one of the most prevalent food allergies, seriously affects the growth and development of infants and children with the rising incidence and prevalence. The dysbiosis of intestinal flora acts to promote disease including allergic disease. Therefore, studying the role of intestinal flora in allergic diseases holds great promise for developing effective strategies to mitigate the risk of food allergies. This study aims to elucidate the role of disrupted intestinal flora and its metabolites in children with CMPA.16S rDNA sequence analysis was applied to characterize the changes in the composition of intestinal flora. The findings revealed heightened diversity of intestinal flora in CMPA, marked by decreased abundance of Firmicutes and Bacteroidetes, and increased abundance of Proteobacteria and Actinobacteria. Furthermore, metabolite analysis identified a total of 1245 differential metabolites in children with CMPA compared to those in healthy children. Among these, 765 metabolites were down-regulated, while 480 were up-regulated. Notably, there were 10 negative differential metabolites identified as bile acids and derivatives, including second bile acids, such as deoxycholic acid, ursodeoxycholic acid and isoursodexycholic acid. The intestinal barrier was further analyzed and showed that the enterocytes proliferation and the expression of Claudin-1, Claudin-3 and MUC2 were down-regulated with the invasion of biofilm community members in the CMPA group. In summary, these findings provide compelling evidence that food allergies disrupt intestinal flora and its metabolites, consequently damaging the intestinal barrier's integrity to increase intestinal permeability and immune response.

[Clinical characteristics and genetic analysis of two children with Tuberous sclerosis complex].

OBJECTIVE: To explore the clinical characteristics and genetic variants in two children with Tuberous sclerosis complex (TSC). METHODS: Two children who had presented at the Children's Hospital Affiliated to Zhengzhou University respectively in June 2020 and July 2021 were selected as the study subjects. Clinical data of the children were collected, and potential pathogenic variants were screened by whole exome sequencing (WES). Candidate variants were verified by Sanger sequencing of their family members. RESULTS: Child 1 was a 7-month-and-29-day-old male, and child 2 was a 2-year-and-6-month-old male. Both children had shown symptoms of epileptic seizures and multiple hypomelanotic macules. Genetic testing revealed that both children had harbored de novo variants of the TSC2 gene, namely c.3239_3240insA and c.3330delC, which were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were rated as pathogenic (PVS1+PS2+PM2_Supporting). CONCLUSION: This study has uncovered the genetic etiology for two children with TSC. Above findings have also enriched the phenotypic and mutational spectrum of TSC in the Chinese population.

[Application of fluorescence in situ hybridization combined with chromosomal karyotyping analysis in children with disorders of sex development due to sex chromosome abnormalities].

OBJECTIVE: To retrospectively analyze sex chromosomal abnormalities and clinical manifestations of children with disorders of sex development (DSD). METHODS: A total of 14 857 children with clinical features of DSD including short stature, cryptorchidism, hypospadia, buried penis and developmental delay were recruited from Zhengzhou Children's Hospital from January 2013 to March 2022. Fluorescence in situ hybridization (FISH) and chromosomal karyotyping were carried out for such children. RESULTS: In total 423 children were found to harbor sex chromosome abnormalities, which has yielded a detection rate of 2.85%. There were 327 cases (77.30%) with Turner syndrome and a 45,X karyotype or its mosaicism. Among these, 325 were females with short stature as the main clinical manifestation, 2 were males with short stature, cryptorchidism and hypospadia as the main manifestations. Sixty-two children (14.66%) had a 47,XXY karyotype or its mosaicism, and showed characteristics of Klinefelter syndrome (KS) including cryptorchidism, buried penis and hypospadia. Nineteen cases (4.49%) had sex chromosome mosaicisms (XO/XY), which included 11 females with short stature, 8 males with hypospadia, and 6 cases with cryptorchidism, buried penis, testicular torsion and hypospadia. The remainder 15 cases (3.55%) included 9 children with a XYY karyotype or mosaicisms, with main clinical manifestations including cryptorchidisms and hypospadia, 4 children with a 47,XXX karyotype and clinical manifestations including short stature and labial adhesion, 1 child with a 46,XX/46,XY karyotype and clinical manifestations including micropenis, hypospadia, syndactyly and polydactyly, and 1 case with XXXX syndrome and clinical manifestations including growth retardation. CONCLUSION: Among children with DSD due to sex chromosomal abnormalities, sex chromosome characteristics consistent with Turner syndrome was most common, among which mosaicism (XO/XX) was the commonest. In terms of clinical manifestations, the females mainly featured short stature, while males mainly featured external genital abnormalities. Early diagnosis and treatment are particularly important for improving the quality of life in such children.

[Analysis of CLCN4 gene variant in a child with Raynaud-Claes syndrome].

OBJECTIVE: To analyze the clinical phenotype and genetic variant in a child with Raynaud-Claes syndrome (RCS). METHODS: A child who was diagnosed with RCS at the Children's Hospital Affiliated to Zhengzhou University for delayed language and motor development in August 2022 was selected as the study subject. Clinical data of the child were collected, and potential genetic variant was detected by next-generation sequencing and Sanger sequencing. The pathogenicity of the candidate variant was analyzed. RESULTS: The child, a 4-year-and-4-month-old male, has manifested global developmental delay, speech disorders, special facial features and behavioral abnormalities. Genetic testing revealed that he has harbored a hemizygous c.1174C>T (p.Gln392Ter) variant of the CLCN4 gene, which was not detected in either of his parents. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was rated as pathogenic (PVS1+PS2+PM2_Supporting). CONCLUSION: The c.1174C>T (p.Gln392Ter) variant of the CLCN4 gene probably underlay the PCS in this child. Above finding has expanded the mutational spectrum of the CLCN4 gene and enabled genetic counseling and prenatal diagnosis for his family.

Dual isothermal amplification all-in-one approach for rapid and highly sensitive quantification of plasma circulating MYCN gene of neuroblastoma.

A dual isothermal amplification assay with dual fluorescence signal detection strategy, named dual isothermal amplification all-in-one approach, was developed for rapid, one-step, highly sensitive quantification of plasma circulating MYCN copy number of neuroblastoma (NB). The developed strategy consisted of rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) on a real-time PCR system using highly specific probe, molecular beacon (MB), as detection probe. The developed strategy possessing a broad linear dynamic range of 10 aM to 1 pM for both target gene (MYCN) and reference gene (NAGK). The ratio of the MYCN copy number to NAGK copy number (M/N ratio) was detected by the developed approach in cell lines, NB tumor tissues, hepatoblastoma tumor tissues and Wilms' tumor tissues, to which the M/N ratios were consistent with previous reports. In particular, the M/N ratio in NB clinical tissue specimens and NB plasma specimens detected with the developed approach were in keeping with the standard RT-PCR approach. More importantly, the M/N ratio in NB tissue samples and corresponding plasma samples of NB patients were consistent with each other with a correlation coefficient of 0.9690, indicating that plasma circulating MYCN is a promising indicator for the risk classification of NB.

Molecular beacon based real-time PCR p1 gene genotyping, macrolide resistance mutation detection and clinical characteristics analysis of Mycoplasma pneumoniae infections in children.

BACKGROUND: Mycoplasma pneumoniae can be divided into different subtypes on the basis of the sequence differences of adhesive protein P1, but the relationship between different subtypes, macrolide resistance and clinical manifestations are still unclear. In the present study, we established a molecular beacon based real-time polymerase chain reaction (real-time PCR) p1 gene genotyping method, analyzed the macrolide resistance gene mutations and the relationship of clinical characteristics with the genotypes. METHODS: A molecular beacon based real-time PCR p1 gene genotyping method was established, the mutation sites of macrolide resistance genes were analyzed by PCR and sequenced, and the relationship of clinical characteristics with the genotypes was analyzed. RESULTS: The detection limit was 1-100 copies/reaction. No cross-reactivity was observed in the two subtypes. In total, samples from 100 patients with positive M. pneumoniae detection results in 2019 and 2021 were genotyped using the beacon based real-time PCR method and P1-1 M. pneumoniae accounted for 69.0%. All the patients had the A2063G mutation in the macrolide resistance related 23S rRNA gene. Novel mutations were also found, which were C2622T, C2150A, C2202G and C2443A mutations. The relationship between p1 gene genotyping and the clinical characteristics were not statistically related. CONCLUSION: A rapid and easy clinical application molecular beacon based real-time PCR genotyping method targeting the p1 gene was established. A shift from type 1 to type 2 was found and 100.0% macrolide resistance was detected. Our study provided an efficient method for genotyping M. pneumoniae, valuable epidemiological monitoring information and clinical treatment guidance to control high macrolide resistance.

Comparative Genomic Analysis of Streptococcus pneumoniae Strains: Penicillin Non-susceptible Multi-drug-Resistant Serotype 19A Isolates.

Streptococcus pneumoniae can cause several diseases including otitis media, sinusitis, pneumonia, sepsis and meningitis. The introduction of pneumococcal vaccines has changed the molecular epidemiological and antibiotic resistance profiles of related diseases. Analysis of molecular patterns and genome sequences of clinical strains may facilitate the identification of novel drug resistance mechanism. Three multidrug resistance 19A isolates were verified, serotyped and the complete genomes were sequenced combining the Pacific Biosciences and the Illumina Miseq platform. Genomic annotation revealed that similar central networks were found in the clinical isolates, and Mauve alignments indicated high similarity between different strains. The pan-genome analysis showed the shared and unique cluster in the strains. Mobile elements were predicted in the isolates including prophages and CRISPER systems, which may participate in the virulence and antibiotic resistance of the strains. The presence of 31 virulence factor genes was predicted from other pathogens for PRSP 19339 and 19343, while 30 for PRSP 19087. Meanwhile, 33 genes antibiotic resistance genes were predicted including antibiotic resistance genes, antibiotic-target genes and antibiotic biosynthesis genes. Further analysis of the antibiotic resistance genes revealed new mutations in the isolates. By comparative genomic analysis, we contributed to the understanding of resistance mechanism of the clinical isolates with other serotype strains, which could facilitate the concrete drug resistance mechanism study.

Metabolomic variation in wild and cultured cordyceps and mycelia of Isaria cicadae.

Isaria cicadae is one of the fungi used in traditional Chinese medicine with the longest tradition. It is used not only as a herbal medicine but also as a health food in Asia, together with cultured cordyceps and mycelia of the fungus used as substitute. However, the differences in their metabolite are unknown. Using a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based metabolomic method, we found that the fungus varies in its metabolism during growth on wild insects, artificially raised insects and artificial medium. There were 109 discriminatory metabolites detected in the samples by orthogonal projection to latent structure discriminant analysis and one-way ANOVA. High level of nonribosomal peptides (NRPs) only existed in the insect portions of the wild cordyceps (WI) and cultured cordyceps (CI), revealing that immunostimulation of the host insects enhanced the synthesis of NRPs in the fungus. The finding of a significantly higher level of sphingolipids in both the insect portions (WI, CI) and the coremia of the wild cordyceps (WC) and cultured cordyceps (CC) but not in cultured mycelia (CM) of I. cicadae implies that the immunostimulation of the live insects can induce the fungus to produce more sphingolipids, and this enhanced ability is probably heritable. Apart from NRPs and sphingolipids, the insect portions also contained higher levels of bioactive compounds such as lateritin, anisomycin, streptimidone and ustiloxins. In contrast, the coremium groups (WC, CC) and CM contained 10-fold less NRP but much higher levels of sanative metabolites such as tocotrienol, 3'-deoxy-hanasanagin, γ-aminobutyric acid and phospholipids than the insect portions. The significantly higher content of antioxidants in WC, CC and CM than in WI and CI suggests that environmental oxygen has a significant effect on the metabolites. The temperature stress which the wild cordyceps encounters during growth is responsible for the relatively high content of trehalose. These findings indicate that the immunity of the host insect and growth environment have a strong impact on the metabolomic variation in Isaria cicadae. The variation in metabolites suggests differential utilization value for the insect portions, coremia and mycelia of the fungus.

Enzyme-free isothermal target-recycled amplification combined with PAGE for direct detection of microRNA-21.

MicroRNA-21 (miR-21) has been regarded as a kind of potential biomarker in several types of cancers. Herein, in this study, a simple, sensitive and cost-effective miR-21 approach was developed utilizing the isothermal target-recycled enzyme-free amplification strategy and polyacrylamide gel electrophoresis (PAGE). In the target-recycled enzyme-free amplification strategy, two rational designed hairpin probes (HPs, HP1 and HP2) can form into HP1-HP2 duplex in the presence of miR-21 under the isothermal condition, producing target signal in PAGE. The sensitivity and the linear range of miR-21 approach were demonstrated by the in vitro detection of miR-21, with the detection limit of 10 pM and the linear range of 50 pM to 8 nM. In particular, the contents of miR-21 in cell extractions of different cell lines were successfully detected through miR-21 approach and the relative expression was highly coincident with the results of stem-loop RT-PCR approach. In summary, the developed approach can detect miR-21 sensitively and easily.

Highly specific real-time quantification of diverse microRNAs in human samples using universal primer set frame.

In this study, one group of universal primer set frame, composed by one reverse transcription (RT) primer frame and a pair of quantitative real-time polymerase chain reaction (qRT-PCR) primer frames, was elaborately screened and designed by homebuilt software for sensitive and specific quantification of diverse miRNAs. The universal primer set frame can be applied for multiplex miRNAs detection by simply changing the RT-X part of RT primer frame and RP-Y part of qRT-PCR reverse primer frame based on target sequence. The maximum similarity of RT-Y, RT-Z and qRT-PCR forward primer to the human genome and human transcriptome is less than 76%, ensuring the high specificity in human sample detection. The high sensitivity and broad dynamic linear range of the developed approaches by using designed primer set frame were demonstrated on the in vitro detection of miR-21 and miR-155, with dynamic range of 10 fM to 10 nM and detection limit of 3.74 × 10-15 M and 5.81 × 10-15 M for miR-21 and miR-155, respectively. In particular, the developed assays also have high sequence specificity which could clearly discriminate a single base difference in miRNA sequence. The contents of miR-21 and miR-155 in tissue and serum samples have been successfully detected using the developed assays. Results indicated that miR-21 and miR-155 were elevated in cancer tissue and serum specimens than that of normal samples, implying the developed assays hold a great promise for further application in biomedical research and early clinical diagnosis. More importantly, the primer set frame can be universally used in any miRNA investigation.

Metabolomic comparison between wild Ophiocordyceps sinensis and artificial cultured Cordyceps militaris.

A systematic study on the metabolome differences between wild Ophiocordyceps sinensis and artificial cultured Cordyceps militaris was conducted using liquid chromatography-mass spectrometry. Principal component analysis and orthogonal projection on latent structure-discriminant analysis results showed that C. militaris grown on solid rice medium (R-CM) and C. militaris grown on tussah pupa (T-CM) evidently separated and individually separated from wild O. sinensis, indicating metabolome difference among wild O. sinensis, R-CM and T-CM. The metabolome differences between R-CM and T-CM indicated that C. militaris could accommodate to culture medium by differential metabolic regulation. Hierarchical clustering analysis was further performed to cluster the differential metabolites and samples based on their metabolic similarity. The higher content of amino acids (pyroglutamic acid, glutamic acid, histidine, phenylalanine and arginine), unsaturated fatty acid (linolenic acid and linoleic acid), peptides, mannitol, adenosine and succinoadenosine in O. sinensis make it as an excellent choice as a traditional Chinese medicine for invigoration or nutritional supplementation. Similar compositions with O. sinensis and easy cultivation make artificially cultured C. militaris a possible alternative to O. sinensis.

[Identification of spore germination and virulence related biomarkers from Beauveria bassiana using an LC-MS-based metabolomic technique].

OBJECTIVE: To identify biomarkers associated with germination and virulence of Beauveria bassiana. METHODS: Spore germination rate and virulence of seven B. bassiana isolates against Euproctis pseudoconspersa were determined, and an LC-MS-based metabolomic analysis was applied to identify the biomarkers from mycelia and conidial extracts associated with spore germination and virulence. RESULTS: The metabolites of carnitine, hercynine, acetylcarnitine, alpha, alpha-trehalose; Octa-Me, arg-arg-gln, phosphatidylethanolamine (PE(18:2/0:0)), phosphotidylcholine (PC(18:3/0:0)) and PC(18:2/0:0)) were higher in the mycelia of highly virulent isolates than those less virulent strains. Conidia of isolates with a high germination rate were characterized by containing higher levels of 2, 3-dimethylmaleate, acetylcarnitine, propionyl-carnitine and PC(18:2/0:0). Histamine, 2,5-pyrrolidinedicarboxylic acid; Diamide, carnitine, acetylcarnitine, propionyl-carnitine, butyrylcarnitine, PE(18:2/0:0), PC(16:1/0:0) and PC(18: 3/0:0) were higher in the conidia of highly virulent isolates. Furthermore, relative content comparison of insecticidal cyclopeptides, such as beauverolides, beauvericins and bassianolide in mycelia showed that the content of a single peptide was not highly related to fungal virulence. However, the contents of 9 peptides were found higher in the highly virulent isolate Bb1898, suggesting that they might exert synergetic effects against insect hosts. CONCLUSION: The common biomarkers related to fungal virulence and germination are acyl carnitine and phospholipid which may play roles in maintaining appressorium turgor pressure and providing energy for penetrating the host cuticle.

Antibiotics-associated pseudomembranous colitis: a disproportionality analysis of the US food and drug administration adverse event reporting system (FAERS) database.

BACKGROUND: Evaluating antibiotics most commonly associated with pseudomembranous colitis (PMC) based on the real-world data is of great significance. RESEARCH DESIGN AND METHODS: We used the data from FAERS to evaluate the potential association between antibiotics and PMC by disproportionality analyzes. RESULTS: Eighty-one antibiotics which met the three algorithms simultaneously were enrolled. There were 1683 reports of PMC associated with the enrolled antibiotics. In the top 24 antibiotics, cefoxitin, streptomycin, fosfomycin, and micafungin had a high risk of PMC, but there were few reports in the literature. CONCLUSIONS: This study was of great significance for healthcare professionals to realize the potential PMC risks of antibiotics.

An efficient and universal In silico screening strategy for acquisition of high-affinity Aptamer and its application in analytical utility.

Numerous aptamers against various targets have been identified through the technology of systematic evolution of ligands by exponential enrichment (SELEX), but the affinity of these aptamers are often insufficient due to the limitations of SELEX. Therefore, a more rational in silico screening strategy (ISS) was developed for efficient screening of high affinity aptamers, which took shape complementarity and thermodynamic stability into consideration. Neuron specific enolase (NSE), a tumor marker, was selected as the target molecule. In the screening process, three aptamer candidates with good shape complementarity, lower ΔG values, and higher ZDOCK scores were produced. The dissociation constant (Kd) of these candidates to NSE was determined to be 10.13 nM, 14.82 nM, and 2.76 nM, respectively. Each of them exhibited higher affinity to NSE than the parent aptamer (Kd = 23.83 nM). Finally, an antibody-free fluorescence aptasensor assay, based on the aptamer with the highest affinity, P-5C8G, was conducted, resulting in a limit of detection (LOD) value of 1.8 nM, which was much lower than the parental aptamer (P, LOD = 12.6 nM). The proposed ISS approach provided an efficient and universal strategy to improve the aptamer to have a high affinity and good analytical utility.