Structure of the Major G-Quadruplex in the Human EGFR Oncogene Promoter Adopts a Unique Folding Topology with a Distinctive Snap-Back Loop.

EGFR tyrosine kinase inhibitors have made remarkable success in targeted cancer therapy. However, therapeutic resistance inevitably occurred and EGFR-targeting therapy has been demonstrated to have limited efficacy or utility in glioblastoma, colorectal cancer, and hepatocellular carcinoma. Therefore, there is a high demand for the development of new targets to inhibit EGFR signaling. Herein, we found that the EGFR oncogene proximal promoter sequence forms a unique type of snap-back loop containing G-quadruplex (G4), which can be targeted by small molecules. For the first time, we determined the NMR solution structure of this snap-back EGFR-G4, a three-tetrad-core, parallel-stranded G4 with naturally occurring flanking residues at both the 5'-end and 3'-end. The snap-back loop located at the 3'-end region forms a stable capping structure through two stacked G-triads connected by multiple potential hydrogen bonds. Notably, the flanking residues are consistently absent in reported snap-back G4s, raising the question of whether such structures truly exist under in vivo conditions. The resolved EGFR-G4 structure has eliminated the doubt and showed distinct structural features that distinguish it from the previously reported snap-back G4s, which lack the flanking residues. Furthermore, we found that the snap-back EGFR-G4 structure is highly stable and can form on an elongated DNA template to inhibit DNA polymerase. The unprecedented high-resolution EGFR-G4 structure has thus contributed a promising molecular target for developing alternative EGFR signaling inhibitors in cancer therapeutics. Meanwhile, the two stacked triads may provide an attractive site for specific small-molecule targeting.

[Value of metagenomic next-generation sequencing in children with hematological malignancies complicated with infections]. / å®åŸºå› ç»„äºŒä»£æµ‹åºåœ¨å„¿ç«¥è¡€æ¶²è‚¿ç˜¤åˆå¹¶æ„ŸæŸ“ä¸­çš„åº”ç”¨.

OBJECTIVES: To explore the value of metagenomic next-generation sequencing (mNGS) in the pathogen identification in children with hematological malignancies complicated with infections. METHODS: A retrospective analysis was conducted on clinical data and pathogenic test results of 43 children with hematological malignancies who underwent microbial culture and mNGS due to infections in the Third Xiangya Hospital of Central South University between June 2020 and July 2022. Differences in detection rates and characteristics of pathogenic microorganisms detected by mNGS and microbial culture were compared. RESULTS: A total of 54 specimens were examined, and the overall detection rate of pathogen by mNGS (80%, 43/54) was significantly higher than that by microbial culture (30%, 16/54) (P<0.001). The most commonly detected infection type by mNGS was viral infection, followed by fungal infection combined viral infection, while that by microbial culture was bacterial infection, followed by fungal infection. The detection rate of fungi by mNGS (33%, 18/54) was higher than that by microbial culture (6%, 3/54) (P<0.001). The detection rate of two or more pathogenic microorganisms by mNGS was higher at 48% compared to microbial culture at 9% (P<0.05). The detection rate of two or more types of pathogenic microorganisms by mNGS was also significantly higher at 33% compared to microbial culture at 2% (P<0.05). The most commonly detected bacteria and fungi by mNGS were Pseudomonas aeruginosa and Candida tropicalis, respectively, in peripheral blood, while Streptococcus pneumoniae and Pneumocystis jirovecii were most commonly detected in bronchoalveolar lavage fluid. Treatment adjustments based on mNGS results were beneficial for 35% (15/43) of the cases. CONCLUSIONS: mNGS has a higher detection rate than microbial culture and has obvious advantages in diagnosing mixed and fungal infections, making it a useful supplementary diagnostic method to microbial culture.

Oxidative Damage Induces a Vacancy G-Quadruplex That Binds Guanine Metabolites: Solution Structure of a cGMP Fill-in Vacancy G-Quadruplex in the Oxidized BLM Gene Promoter.

Guanine (G)-oxidation to 8-oxo-7,8-dihydroguanine (OG) by reactive oxygen species in genomic DNA has been implicated with various human diseases. G-quadruplex (G4)-forming sequences in gene promoters are highly susceptible to G-oxidation, which can subsequently cause gene activation. However, the underlying G4 structural changes that result from OG modifications remain poorly understood. Herein, we investigate the effect of G-oxidation on the BLM gene promoter G4. For the first time, we show that OG can induce a G-vacancy-containing G4 (vG4), which can be filled in and stabilized by guanine metabolites and derivatives. We determined the NMR solution structure of the cGMP-fill-in oxidized BLM promoter vG4. This is the first complex structure of an OG-induced vG4 from a human gene promoter sequence with a filled-in guanine metabolite. The high-resolution structure elucidates the structural features of the specific 5'-end cGMP-fill-in for the OG-induced vG4. Interestingly, the OG is removed from the G-core and becomes part of the 3'-end capping structure. A series of guanine metabolites and derivatives are evaluated for fill-in activity to the oxidation-induced vG4. Significantly, cellular guanine metabolites, such as cGMP and GTP, can bind and stabilize the OG-induced vG4, suggesting their potential regulatory role in response to oxidative damage in physiological and pathological processes. Our work thus provides exciting insights into how oxidative damage and cellular metabolites may work together through a G4-based epigenetic feature for gene regulation. Furthermore, the NMR structure can guide the rational design of small-molecule inhibitors that specifically target the oxidation-induced vG4s.

[Application value of metagenomic next-generation sequencing for pathogen detection in childhood agranulocytosis with fever]. / ç— åŽŸä½“å®åŸºå› ç»„äºŒä»£æµ‹åºåœ¨å„¿ç«¥ç²’ç»†èƒžç¼ºä¹ä¼´å‘çƒ­ä¸­çš„åº”ç”¨ä»·å€¼.

OBJECTIVES: To study the application value of metagenomic next-generation sequencing (mNGS) for pathogen detection in childhood agranulocytosis with fever. METHODS: A retrospective analysis was performed on the mNGS results of pathogen detection of 116 children with agranulocytosis with fever who were treated from January 2020 to December 2021. Among these children, 38 children with negative mNGS results were enrolled as the negative group, and 78 children with positive results were divided into a bacteria group (n=22), a fungal group (n=23), and a viral group (n=31). Clinical data were compared between groups. RESULTS: For the 116 children with agranulocytosis and fever, the median age was 8 years at diagnosis, the median turnaround time of mNGS results was 2 days, and the positive rate of mNGS testing was 67.2% (78/116). Compared with the negative group, the bacterial group had a higher procalcitonin level (P<0.05), the fungal group had higher level of C-reactive protein and positive rate of (1,3)-ß-D glucan test/galactomannan test (P<0.05), and the fungal group had a longer duration of fever (P<0.05). Among the 22 positive microbial culture specimens, 9 (41%) were consistent with the mNGS results. Among the 17 positive blood culture specimens, 8 (47%) were consistent with the mNGS results. Treatment was adjusted for 28 children (36%) with the mNGS results, among whom 26 were cured and discharged. CONCLUSIONS: The mNGS technique has a shorter turnaround time and a higher sensitivity for pathogen detection and can provide evidence for the pathogenic diagnosis of children with agranulocytosis and fever.

Anticandidal formyl phloroglucinol meroterpenoids: Biomimetic synthesis and in vitro evaluation.

Inspired by the diversity-oriented synthesis, some novel formyl phloroglucinol meroterpenoids were synthesized via biomimetic synthesis using essential oils. Eight of them were demonstrated with good in vitro fungicidal activity against Candida albicans and C. glabrata. Compound c2 showed the best anticandidal ability that was powerfully comparable to fluconazole when testing against several strains in vitro. The antibiofilm activity was also found for the c2 treating group which was evidenced to block the hyphal elongation and filamentation of C. albicans. Therefore, compound c2 is a promising candidate for further antifungal-based structure modification.

[Management plan for prevention and control of novel coronavirus pneumonia among children in Xiangya Hospital of Central South University].

Since December 2019, an epidemic of novel coronavirus pneumonia (NCP) has occurred in China. How to effectively prevent and control NCP among children with limited resources is an urgent issue to be explored. Under the unified arrangement of the Xiangya Hospital of Central South University, the Department of Pediatrics has formulated an action plan with Xiangya unique model to prevent and control NCP among children according to the current epidemic situation and diagnostic and therapeutic program in China.

Ciliatasecones A-C, three rearranged limonoids from Toona ciliata var. yunnanensis.

Ciliatasecones A-C (1-3), three rearranged limonoids with a novel ring-seco model and an unprecedented cycle system, were isolated from the root bark of Toona ciliata var. yunnanensis. Ciliatasecones A-B (1-2) share a novel cyclopenta[b]furan ring C/D system through C-9/11-seco and C-11/14 ether linkage. Ciliatasecone C (3) was found to possess a rare rearranged six-membered lactone ring B between C-7 and C-9. Plausible biogenetic pathway speculation indicated that C-9/11 cleavage and oxygen bridge formation played the key roles in the framework rearrangement of 1-3.

[Research Advances in Lysine-specific Demethylase 6A and Its Application in Treating Leukemia].

Leukemia is a disease featured by the malignant proliferation of hematopoietic stem cells or progenitor cells in the blood system.While chemotherapy remains its mainstream treatment,disease relapse and drug resistance are still challenging problems.As one of the epigenetic mechanisms,histone methylation is involved in cell proliferation,differentiation,and apoptosis by regulating gene transcription.Recent studies have found that the histone demethylase lysine-specific demethylase 6A(KDM6A),also known as ubiquitously transcribed tetratricopeptide repeat on chromosome X(UTX),is closely related to the occurrence of a variety of tumors,especially leukemia.KDM6A activates gene expression by demethylating H3K27me3 to H3K27me2 or H3K27me1.Besides,KDM6A can regulate the activation of the target gene transcription through its non-demethylase functions.It can serve as the subunit of complex of proteins associated with Set1,thus getting involved in the regulation of H3K4me1.It can be combined with yeast mating type conversion/sucrose unfermented complex family to promote the formation of an open chromatin conformation.Finally,it can promote the production of H3K27ac.This article reviews the recent studies on the structure and biological activity of histone demethylase KDM6A(UTX)and its role in treating leukemia,thus providing a new research direction for targeted treatment of leukemia.

[Clinical and genetic analyses of juvenile myelomonocytic leukemia].

OBJECTIVE: To study the clinical and genetic features of juvenile myelomonocytic leukemia (JMML) and the association between genotype and prognosis. Methods The clinical data of 15 children who were diagnosed with JMML were collected. Next-generation sequencing was used to detect common gene mutations of JMML. RESULTS: The male/female ratio was 6.5:1, and the age of onset was 19 months (range 2-67 months). Of the 15 children, 11 (73%) experienced disease onset before the age of 4 years, with abdominal distension and pyrexia as initial symptoms. All children had hepatosplenomegaly and superficial lymphadenectasis, with a number of peripheral blood mononuclear cells of >1.0×109/L and a percentage of juvenile cells of 1%-7% in peripheral blood smear. The percentage of bone marrow blasts + juvenile cells was <20%, and the percentage of monoblasts + promonocytes was 1%-10%. Of the 15 children, 10 (67%) had a higher level of hemoglobin F than the normal level at the corresponding age, with the highest level of 62.5%. All 15 children had the absence of Philadelphia chromosome, and one child had chromosome 7 deletion. All 15 children had a negative result of BCR/ABL fusion gene detection. PTPN11 gene mutation was found in 5 children (33%), NF1 mutation in 4 children (27%), CBL mutation in 3 children (20%), and RAS mutation in 3 children (20%). No children received regular chemotherapy, and one child underwent hematopoietic stem cell transplantation. The median follow-up time of 15 children was 18 months (range 1-48 months). Among the 15 children, 8 died (among whom 4 had PTPN11 gene mutation, 3 had NF1 mutation, and 1 had RAS mutation) and 7 survived. The children with PTPN11 mutation had the worst prognosis and the highest mortality rate, and those with CBL or NRAS mutation had a relatively good prognosis. The level of hemoglobin F was negatively correlated with survival time (rs=-7.21, P=0.002). CONCLUSIONS: In children with JMML, the type of gene mutation is associated with prognosis. The children with PTPN11 mutation often have a poor prognosis, and those with CBL or NRAS mutation have a relatively good prognosis.

[Value of S100A8 in evaluating the prognosis of children with acute lymphoblastic leukemia].

OBJECTIVE: To study the association between S100A8 expression and prognosis in children with acute lymphoblastic leukemia (ALL). METHODS: The clinical data of 377 children with ALL who were treated with the CCLG-2008-ALL regimen were retrospectively reviewed. ELISA and PCR were used to measure serum protein levels and mRNA expression of S100A8. The Kaplan-Meier method was used for survival analysis and a Cox regression analysis was also performed. RESULTS: The children were followed up for 56 months, and the overall survival rate of the 377 children was 89.1%. The prednisone good response group had significantly lower S100A8 protein and mRNA levels than the prednisone poor response group (P<0.01). In the children with standard or median risk, both S100A8 protein and mRNA levels were associated with event-free survival rate (P<0.05). There were significant differences in S100A8 protein and mRNA levels between the children with different risk stratifications (P<0.01). The children who experienced events had significantly higher S100A8 protein and mRNA levels than those who did not (P<0.01). The Kaplan-Meier survival analysis and the Cox regression model suggested that S100A8 overexpression was an independent risk factor for the prognosis of children with ALL. CONCLUSIONS: High S100A8 expression may be associated with the poor prognosis of children with ALL and is promising as a new marker for individualized precise treatment of children with ALL.

Multicenter randomized trial of arsenic trioxide and Realgar-Indigo naturalis formula in pediatric patients with acute promyelocytic leukemia: Interim results of the SCCLG-APL clinical study.

Intravenous arsenic trioxide (ATO) has been adopted as the first-line treatment for acute promyelocytic leukemia (APL). Another arsenic compound named the Realgar-Indigo naturalis formula (RIF), an oral traditional Chinese medicine containing As4 S4 , has been shown to be highly effective in treating adult APL. In the treatment of pediatric APL, the safety and efficacy of RIF remains to be confirmed. This randomized, multicenter, and noninferiority trial was conducted to determine whether intravenous ATO can be substituted by oral RIF in the treatment of pediatric APL. From September 2011 to January 2017, among 92 patients who were 16 years old or younger with newly diagnosed PML-RARa positive APL, 82 met eligible criteria and were randomly assigned to ATO (n = 42) or RIF (n = 40) group. The remaining 10 patients did not fulfilled eligible criteria because five did not accept randomization, four died and one had hemiplegia prior to arsenic randomization due to intracranial hemorrhage or cerebral thrombosis. Induction and consolidation treatment contained ATO or RIF, all-trans-retinoic acid and low intensity chemotherapy. End points included event-free survival (EFS), adverse events and hospital days. After a median 3-year follow-up, the estimated 5-year EFS was 100% in both groups, and adverse events were mild. However, patients in the RIF group had significantly less hospital stay than those in the ATO group. This interim analysis shows that oral RIF is as effective and safe as intravenous ATO for the treatment of pediatric APL, with the advantage of reducing hospital stay. Final trial analysis will reveal mature outcome data.

Hypoxia-Protective Azaphilone Adducts from Peyronellaea glomerata.

Peyronellones A and B (1 and 2), a pair of rare tetracyclic caged adducts of azaphilone with pyruvic acid, along with four new analogues (3-6), were isolated from solid cultures of the endophytic fungus Peyronellaea glomerata. Their structures were elucidated through spectroscopic analysis, and their absolute configurations were unambiguously determined by a combination of single-crystal X-ray crystallography, Rh2(OCOCF3)4-induced ECD experiments, ECD calculations, and modified Mosher methods. Compound 2 (5 µM) was found to have a significant hypoxia-protective effect that improved the survival rate of hypoxia/reoxygenation-treated human umbilical vein endothelial cells from 35% to 70%, which was equal to the potency of the positive control, verapamil. Flow cytometry analysis suggested 2 could inhibit H/R-induced late-stage apoptosis of this cell line.

Protective effects of Araloside C against myocardial ischaemia/reperfusion injury: potential involvement of heat shock protein 90.

The present study was designed to investigate whether Araloside C, one of the major triterpenoid compounds isolated from Aralia elata known to be cardioprotective, can improve heart function following ischaemia/reperfusion (I/R) injury and elucidate its underlying mechanisms. We observed that Araloside C concentration-dependently improved cardiac function and depressed oxidative stress induced by I/R. Similar protection was confirmed in isolated cardiomyocytes characterized by maintaining Ca2+ transients and cell shortening against I/R. Moreover, the potential targets of Araloside C were predicted using the DDI-CPI server and Discovery Studio software. Molecular docking analysis revealed that Araloside C could be stably docked into the ATP/ADP-binding domain of the heat shock protein 90 (Hsp90) protein via the formation of hydrogen bonds. The binding affinity of Hsp90 to Araloside C was detected using nanopore optical interferometry and yielded KD values of 29 µM. Araloside C also up-regulated the expression levels of Hsp90 and improved cell viability in hypoxia/reoxygenation-treated H9c2 cardiomyocytes, whereas the addition of 17-AAG, a pharmacologic inhibitor of Hsp90, attenuated Araloside C-induced cardioprotective effect. These findings reveal that Araloside C can efficiently attenuate myocardial I/R injury by reducing I/R-induced oxidative stress and [Ca2+ ]i overload, which was possibly related to its binding to the Hsp90 protein.

In Vitro Antibiofilm Activity of Eucarobustol E against Candida albicans.

Formyl-phloroglucinol meroterpenoids (FPMs) are important types of natural products with various bioactivities. Our antifungal susceptibility assay showed that one of the Eucalyptus robusta-derived FPMs, eucarobustol E (EE), exerted a strong inhibitory effect against Candida albicans biofilms at a concentration of 16 µg/ml. EE was found to block the yeast-to-hypha transition and reduce the cellular surface hydrophobicity of the biofilm cells. RNA sequencing and real-time reverse transcription-PCR analysis showed that exposure to 16 µg/ml of EE resulted in marked reductions in the levels of expressions of genes involved in hyphal growth (EFG1, CPH1, TEC1, EED1, UME6, and HGC1) and cell surface protein genes (ALS3, HWP1, and SAP5). Interestingly, in response to EE, genes involved in ergosterol biosynthesis were downregulated, while the farnesol-encoding gene (DPP3) was upregulated, and these findings were in agreement with those from the quantification of ergosterol and farnesol. Combined with the obvious elevation of negative regulator genes (TUP1, NRG1), we speculated that EE's inhibition of carbon flow to ergosterol triggered the mechanisms of the negative regulation of hyphal growth and eventually led to biofilm inhibition.

Aerobic thiyl radical addition/cyclization of N-methacryloyl benzamides for the synthesis of isoquinoline-1,3(2H,4H)-dione derivatives.

A highly effective oxidative thiyl radical addition/cyclization of N-methacryloylbenzamides was explored using dioxygen as the sole terminal oxidant without the use of precious and/or toxic transition-metal catalysts. This method provides convenient access to a variety of useful sulfide-containing 4,4-disubstituted isoquinoline-1,3-diones by constructing C-S and C-C bonds in one step.

Isolation, Structure Elucidation, and Absolute Configuration of Syncarpic Acid-Conjugated Terpenoids from Rhodomyrtus tomentosa.

Three new syncarpic acid-conjugated sesquiterpenoids, tomentodiones E-G (1-3), and six new syncarpic acid-conjugated monoterpenoids, tomentodiones H-M (4-9), were isolated from the leaves of Rhodomyrtus tomentosa. Compounds 1-3 represent the first examples of ß-calacorene-based meroterpenoids. Their structures and absolute configurations were determined by a combination of NMR and ECD spectroscopy and X-ray diffraction analysis. On the basis of ECD data analysis for isolated and synthesized compounds, an empirical rule was proposed to determine the absolute configuration at C-7' of syncarpic acid-conjugated terpenoids. Additionally, a study of the reversal effect of multidrug resistance in doxorubicin-resistant human breast cancer cells showed that the noncytotoxic (+)-4 exerted the strongest potentiation effect of doxorubicin susceptibility, with an enhancement of 16.5-fold at a concentration of 30 µM.

Three new alkaloids from the seeds of Nigella glandulifera.

Three new alkaloids namely 8-(4-hydroxyphenyl)-6-methoxy-3,4-dihydroisoquinolin-1(2H)-one (1), 4-aminonigellidine (2), and N-[(4-hydroxy-2-isopropyl-5-methyl)]phenylurea (3), along with six known ones (4-9), were isolated from the seeds of Nigella glandulifera. The structures of 1-3 were determined through spectroscopic analyses (HRESIMS, 1D/2D NMR). Compound 1 was a rare isoquinolinone alkaloid with phenyl substituted at C-8.

[Recurrent pulmonary infection and oral mucosal ulcer].

An 8-year-old girl who had experienced intermittent cough and fever over a 3 year period, was admitted after experiencing a recurrence for one month. One year ago the patient experienced a recurrent oral mucosal ulcer. Physical examination showed vitiligo in the skin of the upper right back. Routine blood tests and immune function tests performed in other hospitals had shown normal results. Multiple lung CT scans showed pulmonary infection. The patient had recurrent fever and cough and persistent presence of some lesions after anti-infective therapy. The antitubercular therapy was ineffective. Routine blood tests after admission showed agranulocytosis. Gene detection was performed and she was diagnosed with dyskeratosis congenita caused by homozygous mutation in RTEL1. Patients with dyskeratosis congenita with RTEL1 gene mutation tend to develop pulmonary complications. Since RTEL1 gene sequence is highly variable with many mutation sites and patterns and can be inherited via autosomal dominant or recessive inheritance, this disease often has various clinical manifestations, which may lead to missed diagnosis or misdiagnosis. For children with unexplained recurrent pulmonary infection, examinations of the oral cavity, skin, and nails and toes should be taken and routine blood tests should be performed to exclude dyskeratosis congenita. There are no specific therapies for dyskeratosis congenita at present, and when bone marrow failure and pulmonary failure occur, hematopoietic stem cell transplantation and lung transplantation are the only therapies. Androgen and its derivatives are effective in some patients. Drugs targeting the telomere may be promising for patients with dyskeratosis congenita.

(1) H†NMR-Guided Isolation of Formyl-Phloroglucinol Meroterpenoids from the Leaves of Eucalyptus robusta.

Nine formyl-phloroglucinolmeroterpenoids (FPMs), namely, eucalrobusones A-I (1-9), were isolated from the leaves of Eucalyptus robusta by tracking the phenolic hydroxyl (1) H NMR peaks. The Snatzke helicity rules for the Cotton effects of twisted benzene rings were applied to elucidate the absolute configurations of the FPMs. These findings, along with NMR spectroscopy, the circular dichroism (CD) exciton chirality method, and CD calculations, allowed complete structures for the FPMs to be assigned. Eucalrobusones A-F (1-6) are novel adducts formed between a formyl-derived carbon atom on the phloroglucinol ring and monoterpene and sesquiterpene components. Eucalrobusones G-I (7-9) are the first examples of FPMs with cubebane part structures connected by an unusual 1-oxaspiro[5.5]undecane subunit. Among these isolates, eucalrobusone C (3) showed significant cytotoxicity against HepG2, MCF-7, and U2OS cancer cell lines, with IC50 values less than 10 µm. Compound 3 significantly blocks cell proliferation in MCF-7 cells and induces MCF-7 cell death through apoptosis.

Trichiconlides A and B: two novel limonoids from the fruits of Trichilia connaroides.

Trichiconlides A (1) and B (2), two novel limonoids, were isolated from the fruits of Trichilia connaroides. Compound 1 is a novel limonoid with a highly rearranged A/B ring system and an intact D ring, which formed an unprecedented 5/6/5/6/5 carbon skeleton. Trichiconlide B (2) possesses a 10-oxatricyclo[3.3.1.(3,8)]decane core and formed a cage-like structure located between rings A and C. Their structures and absolute configurations were elucidated by spectroscopic methods, single crystal X-ray diffraction, the ECD exciton chirality method and TDDFT/ECD calculations. 1 showed a moderate inhibitory effect on nitric oxide (NO) production.