Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma.

Our previous study of DNA methylation in the pediatric soft tissue tumor rhabdomyosarcoma (RMS) demonstrated that fusion-positive (FP) and fusion-negative (FN) RMS tumors exhibit distinct DNA methylation patterns. To further examine the significance of DNA methylation differences in RMS, we investigated genome-wide DNA methylation profiles in discovery and validation cohorts. Unsupervised analysis of DNA methylation data identified novel distinct subsets associated with the specific fusion subtype in FP RMS and with RAS mutation status in FN RMS. Furthermore, the methylation pattern in normal muscle is most similar to the FN subset with wild-type RAS mutation status. Several biologically relevant genes were identified with methylation and expression differences between the two fusion subtypes of FP RMS or between the RAS wild-type and mutant subsets of FN RMS. Genomic localization studies showed that promoter and intergenic regions were hypomethylated and the 3' untranslated regions were hypermethylated in FP compared to FN tumors. There was also a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Moreover, genes with PAX3-FOXO1 binding sites and promoter hypomethylation exhibited the highest frequency of overexpression in FP tumors. Finally, a comparison of RMS model systems revealed that patient-derived xenografts most closely recapitulate the DNA methylation patterns found in human RMS tumors compared to cell lines and cell line-derived xenografts. In conclusion, these findings highlight the interaction of epigenetic changes with mutational alterations and transcriptional organization in RMS tumors, and contribute to improved molecular categorization of these tumors.

A mutational comparison of adult and adolescent and young adult (AYA) colon cancer.

BACKGROUND: It is possible that the relative lack of progress in treatment outcomes among adolescent and young adult (AYA) patients with cancer is caused by a difference in disease biology compared with the corresponding diseases in younger and older individuals. There is evidence that colon cancer is more aggressive and has a poorer prognosis in AYA patients than in older adult patients. METHODS: To further understand the molecular basis for this difference, whole-exome sequencing was conducted on a cohort of 30 adult, 30 AYA, and 2 pediatric colon cancers. RESULTS: A statistically significant difference in mutational frequency was observed between AYA and adult samples in 43 genes, including ROBO1, MYC binding protein 2 (MYCBP2), breast cancer 2 (early onset) (BRCA2), MAP3K3, MCPH1, RASGRP3, PTCH1, RAD9B, CTNND1, ATM, NF1; KIT, PTEN, and FBXW7. Many of these mutations were nonsynonymous, missense, stop-gain, or frameshift mutations that were damaging. Next, RNA sequencing was performed on a subset of the samples to confirm the mutations identified by exome sequencing. This confirmation study verified the presence of a significantly greater frequency of damaging mutations in AYA compared with adult colon cancers for 5 of the 43 genes (MYCBP2, BRCA2, PHLPP1, TOPORS, and ATR). CONCLUSIONS: The current results provide the rationale for a more comprehensive study with a larger sample set and experimental validation of the functional impact of the identified variants along with their contribution to the biologic and clinical characteristics of AYA colon cancer. Cancer 2018;124:1070-82. © 2017 American Cancer Society.

Role of prephase treatment prior to definitive chemotherapy in patients with diffuse large B-cell lymphoma.

BACKGROUND: During the treatment of diffuse large B-cell lymphoma (DLBCL) patients, treatment-related toxicities are higher in the initial phase of treatment (First cycle effect). Toxicities can be tumor lysis syndrome, deterioration in performance status, febrile neutropenia, and rarely mortality. Prephase treatment before definitive chemotherapy is used in European countries to alleviate these toxicities. METHODS: This was a non-randomized study carried out with the aim to evaluate the role of prephase treatment given prior to definitive chemotherapy in newly diagnosed DLBCL patients. Patients were divided into 2 cohorts "prephase cohort" and "non-prephase cohort." Prephase cohort received prephase treatment consisting of vincristine (1 mg) on -6th day and prednisolone 100 mg daily for 7 days (-6th day to day 0). Prephase treatment was followed by CHOP/R-CHOP chemotherapy on day 1. Non-prephase cohort received chemotherapy without prephase. Both groups were followed up for 30 days post-first cycle chemotherapy. RESULTS: A total of 100 patients with DLBCL (50 in each cohort) were enrolled. There was a significant improvement in performance status of the patients who received prephase. A majority of 92% patients attained ECOG performance status of either 0 or 1 before starting chemotherapy in the prephase cohort. Febrile neutropenia was lower (16%) in the prephase cohort as compared with the non-prephase cohort (34%; P = .037). CONCLUSION: Prephase treatment prior to definitive chemotherapy (CHOP ± Rituximab) improves the performance status and decreases first cycle effect in DLBCL patients.

Frequent inactivating germline mutations in DNA repair genes in patients with Ewing sarcoma.

PURPOSE: Ewing sarcoma is a small round blue cell tumor that is highly malignant and predominantly affects the adolescent and young adult population. It has long been suspected that a genetic predisposition exists for this cancer, but the germ-line genetic underpinnings of this disease have not been well established. METHODS: We performed germline variant analysis of whole-genome or whole-exome sequencing of samples from 175 patients affected by Ewing sarcoma. RESULTS: We discovered pathogenic or likely pathogenic germline mutations in 13.1% of our cohort. Pathogenic mutations were highly enriched for genes involved with DNA damage repair and for genes associated with cancer predisposition syndromes. CONCLUSION: Our findings reported here have important clinical implications for patients and families affected by Ewing sarcoma. Genetic counseling should be considered for patients and families affected by this disease to take advantage of existing risk management strategies. Our study also highlights the importance of germline sequencing for patients enrolled in precision-medicine protocols.Genet Med advance online publication 26 January 2017.

The genomic landscape of the Ewing Sarcoma family of tumors reveals recurrent STAG2 mutation.

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

Fluorescent carbon nanoparticles obtained from charcoal via green methods and their application for sensing Fe3+ in an aqueous medium.

Two green methods (microwave and hydrothermal) were employed for the preparation of water dispersible fluorescent carbon nanoparticles (CNPs) from activated charcoal. Microwave and hydrothermally synthesized carbon nanoparticles, (MW-CNPs) and (HT-CNPs), respectively were characterized by microscopic and spectroscopic techniques. A detailed study of their fluorescence characteristics was made. MW-CNPs and HT-CNPs were tested for metal ion selectivity in aqueous medium. MW-CNPs showed selectivity for Fe3+ among the tested metal ions and important studies such as for interference, linear range and limit of detection were carried out. The application of MW-CNPs for detection of Fe3+ in water was demonstrated.

Colorimetric and fluorogenic recognition of Hg2+ and Cr3+ in acetonitrile and their test paper recognition in aqueous media with the aid of rhodamine based sensors.

Two new rhodamine derivatives (L1 and L2) were synthesized, characterized and their ion recognition property has been investigated. Both of the ionophores exhibit colorimetric and fluorogenic response for Hg(2+) and Cr(3+) ions among large number of alkali, alkaline earth and transition metal ions tested in acetonitrile. Detail studies on determination of binding constant, binding mode, reversibility of binding, lower detection limit have been carried out. Detection of metal ions in aqueous media has also been demonstrated by preparation of simple, convenient and disposable test paper sensors with two approaches viz. filter paper and membrane filter loaded with these ionophores. Both of these methods responded sharply to both the metal ions (Hg(2+) and Cr(3+)) in aqueous solution, detectable by bared-eye. For better sensing at low concentration of metal ions, reprecipitation followed by filtration enrichment of ligands on membrane filter was employed.

A novel processing approach for free-standing porous non-oxide ceramic supports from polycarbosilane and polysilazane precursors.

In this contribution, a low-pressure/low-temperature casting technique for the preparation of novel free-standing macrocellular polymer-derived ceramic support structures is presented. Preceramic polymers (polycarbosilane and poly(vinyl)silazane) are combined with sacrificial porogens (ultra-high molecular weight polyethylene microbeads) to yield porous ceramic materials in the Si-C or Si-C-N systems, exhibiting well-defined pore structures after thermal conversion. The planar-disc-type specimens were found to exhibit biaxial flexural strengths of up to 60 MPa. In combination with their observed permeability characteristics, the prepared structures were found to be suitable for potential applications in filtration, catalysis, or membrane science.

Simple and sensitive technique for alignment of the pinhole of a spatial filter of a high-energy, high-power laser system.

In this paper, a new method for alignment of the pinhole of a spatial filter (SF) has been proposed and demonstrated experimentally. The effect of the misalignment of the pinhole on the laser beam profiles has been calculated for circular and elliptical Gaussian laser beams. Theoretical computation has been carried out to illustrate the effect of an intensity mask, placed before the focusing lens of the SF, on the spatial beam profile after the pinhole of the SF. It is shown, both theoretically and experimentally, that a simple intensity mask, consisting of a black dot, can be used to visually align the pinhole with a high accuracy of 5% of the pinhole diameter. The accuracy may be further improved using a computer-based image processing algorithm. Finally, the proposed technique has been demonstrated to align a vacuum SF of a compact 40 J Nd:phosphate glass laser system.

Computational insight towards the binding affinity and participation of aliphatic unsaturated sidearms of aza-18-crown-6 extractants for Sr2+ encapsulation in different solvent medium.

CONTEXT: Macrocyclic extractants capture cations in solution phase; therefore, their structures and energetics in different solvents are worth investigating. In this computational research work, the optimized geometry of three aza-18-crown-6 extractants (1-3) has been obtained in suitable solvent mediums to work out the influence of the solvation on their binding affinity with Sr2+. The designed macrocyclic extractants are remarkable as the N-substituted side chain bears the rarely examined simple aliphatic unsaturated double and triple bond. All significant structural perturbations of the macro ring wherein Sr2+ binds are examined. Prediction of the auxiliary effect of the simple aliphatic unsaturated side arm on the binding of Sr2+ through solvent competition or cation-pi interaction is undertaken. The binding affinity of the complex formed in the solvent and gas phases is calculated. Results obtained in this study favor the utilization of solvents of low dielectric constant (CHCl3 and DCM) for effective binding of Sr2+ ions by the extractants. METHOD: All DFT calculations were performed using the Gaussian 09 program. The optimized geometries and their structural features were visualized by GaussView. Density functional calculation involving B3LYP functional and LANL2DZ basis set was employed to obtain optimized geometry and energy of the extractants and their Sr2+ complex. The solvation effects were considered by employing the calculations with the polarized continuum model (PCM). The computational method's reliability was assured by comparing the optimized structure with that of X-ray reported structure of a similar type of complex.

Promoter G-Quadruplex Binding Styryl Benzothiazolium Derivative for Applications in Ligand Affinity Ranking and as Ethidium Bromide Substitute in Gel Staining.

Fluorescent compounds that can preferentially interact with certain nucleic acids are of great importance in new drug discovery in a multitude of functions including fluorescence-based displacement assays and gel staining. Here, we report the discovery of an orange emissive styryl-benzothiazolium derivative (compound 4) which interacts preferentially with Pu22 G-quadruplex DNA among a pool of nucleic acid structures containing G-quadruplex, duplex, and single-stranded DNA structures as well as RNA structures. Fluorescence-based binding analysis revealed that compound 4 interacts with Pu22 G-quadruplex DNA in a 1:1 DNA to ligand binding stoichiometry. The association constant (Ka) for this interaction was found to be 1.12 (±0.15) × 106 M-1. Circular dichroism studies showed that the binding of the probe does not cause changes in the overall parallel G-quadruplex conformation; however, signs of higher-order complex formation were seen in the form of exciton splitting in the chromophore absorption region. UV-visible spectroscopy studies confirmed the stacking nature of the interaction of the fluorescent probe with the G-quadruplex which was further complemented by heat capacity measurement studies. Finally, we have shown that this fluorescent probe can be used toward G-quadruplex-based fluorescence displacement assays for ligand affinity ranking and as a substitute for ethidium bromide in gel staining.

Metastatic Renal Cell Carcinoma: An Enigmatic Nasal Mass.

Metastatic Renal Cell Carcinoma rarely presents in head and neck and is even rarer in the sinonasal region. However, a sinonasal metastatic mass is usually of RCC origin. These metastases may present prior to the renal symptoms or may appear after primary treatment. Report a 60-year lady with epistaxis due to metastatic RCC. Calculate total published cases of sino-nasal metastasis of RCC. Classify according to sequence of primary and metastatic presentation. A computer aided search of PubMed and Google scholar databases was done using pertinent combinations of the keywords "renal cell carcinoma", "nose and paranasal sinus", "metastasis", "delayed metastasis" and "unusual presentation", revealing 1350 articles. 38 relevant articles were included in the review. Our case presented with epistaxis 3 years after primary RCC. She had a vascular left sided nasal mass which was excised enblock. Immunohistochemistry confirmed metastatic RCC. She is on oral chemotherapy and asymptomatic 1 year post excision. Literature search revealed 116 such cases. 19 patients presented within 10 years of RCC while 7 more were delayed metastasis. 17 cases presented primarily with nasal symptoms with subsequent incidental renal mass. Chronology of presentation was unavailable in the rest 73 cases. We recommend to consider the diagnosis of sinonasal metastatic RCC in a patient presenting with epistaxis or nasal mass, particularly with a past history of RCC. Also, any person with known diagnosis of RCC should undergo regular ENT examination for early diagnosis of sinonasal metastasis.

Nile Blue: A Red-Emissive Fluorescent Dye That Displays Differential Self-Assembly and Binding to G-Quadruplexes.

Nile Blue (NB) is a red-emissive dye that is well-known for imaging and staining applications. In this work, we describe the interaction of NB with various types of G-quadruplexes belonging to different topologies, molecularities, and conformations. Using spectroscopic techniques, we have determined the preferential binding of NB to c-Myc G-quadruplex and the other aspects of its binding. Concentration- and temperature-dependent studies showed that NB exists in a dynamic equilibrium between monomeric and H-aggregated states, which could be modulated by the addition of external agents such as anionic surfactants. NB displayed differential self-assembly with different types of G-quadruplex and duplex DNAs modulating its dynamic equilibrium between the monomeric and H-aggregated states. Fluorescence-based displacement studies revealed a 1:1 binding stoichiometry upon interaction with c-Myc G-quadruplex and an association constant of Kapp = 6.7 × 106 M-1. Circular dichroism studies indicated that NB does not cause changes in the overall conformation of either G-quadruplexes or duplexes; however, it does indicate nucleic acid-dependent self-assembly at higher concentrations. Heat capacity measurement showed a more negative change when compared to that in DNA duplex, indicating more burial of the polar surface area by NB to the G-quadruplex host.

Initial Experiences in Adolescents and Young Adults with T-Cell Acute Lymphoblastic Leukemia/Lymphoma Treated with the Modified BFM 2002 Protocol in a Resource-Constrained Setting.

Prutha Jinwala T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma (T-ALL/LBL) in adolescents and young adults (AYAs) is a clinically aggressive malignancy and life-threatening at diagnosis. Intensive chemotherapy protocols, inspired by the Berlin-Frankfurt-Münster (BFM) regimen, along with central nervous system (CNS) prophylaxis, have achieved a 75 to 85% 5-year disease-free survival rate. However, in cases of marrow and CNS relapses, second-line chemotherapy is usually ineffective. This study aimed to assess the safety and efficacy of the BFM 2002 protocol and to correlate clinical profiles and prognostic factors with survival outcomes in AYA T-ALL/LBL patients. We retrospectively analyzed data from T-ALL/LBL patients treated at the Department of Medical Oncology, Sri Aurobindo Institute of Medical Sciences (SAIMS), Indore, between 2018 and 2021. Twenty-one patients aged 15 to 29 years were studied for their clinical course and laboratory parameters over 36 months. Diagnosis and risk stratification were performed following the guidelines of the BFM 2002 protocol. All patients received treatment and monitoring according to this pediatric-inspired protocol. The median age of the patients was 17 years (range: 15-28 years). Eleven patients presented with mediastinal lymph node enlargement, 10% exhibited CNS involvement, and none had testicular involvement. Eleven patients had marrow blasts greater than 25%, indicative of acute lymphoblastic leukemia. All 21 patients were treated according to the intensive modified BFM 2002 protocol and achieved morphological remission after a median follow-up of 24 months (range: 18-36 months). Seventeen patients achieved minimal residual disease (MRD) negativity post-induction. MRD at day 33 showed a significant association with the probability of disease relapse ( p = 0.0015). There were five deaths (24%), one due to toxicity and four due to relapse. The study recorded an 18-month overall survival of 76%. These results were achieved despite financial constraints. Data were entered into a spreadsheet, and statistical analysis was performed using IBM SPSS version 23. Continuous data are presented as ranges and medians, while categorical variables are shown as percentages and numbers. A chi-squared test for association, with a significance level set at p < 0.05, was conducted as indicated. AYA T-ALL/LBL requires intensive treatment regimens. With biological characterization of LBL/ALL and close therapy monitoring, encouraging outcomes can be achieved even in resource-limited settings.

High-throughput molecular and histopathologic profiling of tumor tissue in a novel transplantable model of murine neuroblastoma: new tools for pediatric drug discovery.

Using two MYCN transgenic mouse strains, we established 10 transplantable neuroblastoma cell lines via serial orthotopic passage in the adrenal gland. Tissue arrays demonstrate that by histochemistry, vascularity, immunohistochemical staining for neuroblastoma markers, catecholamine analysis, and concurrent cDNA microarray analysis, there is a close correspondence between the transplantable lines and the spontaneous tumors. Several genes closely associated with the pathobiology and immune evasion of neuroblastoma, novel targets that warrant evaluation, and decreased expression of tumor suppressor genes are demonstrated. These studies describe a unique and generalizable approach to expand the utility of transgenic models of spontaneous tumor, providing new tools for preclinical investigation.

Profiling of trace elemental impurities in caustic soda matrix by inductively coupled plasma mass spectrometric technique.

An inductively coupled plasma mass spectrometric (ICP-MS) technique method is developed for simultaneous determination of Li, Cr, Mn, Fe, Ni, Co, Cu, Sr, Ag, Cd, Ba, and Pb at trace level in caustic soda. Operational parameters of the instrument were optimized and suitable accessory (argon gas dilution) was used in the method. Direct aspiration of high total dissolved solids (TDS) samples (beyond 0.2% TDS) and highly alkaline NaOH is not suitable for the instrument; therefore, strategy of neutralization of NaOH by HNO3 was adopted to handle its high alkalinity. Suitable internal standards of low, mid, and high atomic masses were used with external calibration. Features such as matrix matching, calibration verification, interference correction, etc. were undertaken in this work. Neutralized caustic soda samples were spiked with the analytes with lower, middle, and higher concentration. The results of spiking with 30, 70, 140, and 200 ppb were examined. The method exhibited excellent accuracy and precision.

Fragment-Based Design of Small Molecules to Study DNA Minor Groove Recognition.

DNA-protein interactions are ubiquitous in cellular processes. Impeding unwanted nucleic acid interactions and protein recognition have therapeutic implications. Therefore, new chemical scaffolds and studies related to their structural basis of nucleic acid recognition are essential for developing high-affinity DNA binders. In this study, we have employed a fragment-based strategy to design and synthesize benzimidazole-guanidinium hybrid compounds and study the individual fragment's role in imparting selectivity and specificity in DNA recognition. The fragments were extensively studied using thermal denaturation, circular dichroism, UV-vis absorption spectroscopy, and molecular docking techniques. The results indicate an interdependent role of the benzimidazole core, polar ends, and the DNA composition in imparting sequence-selective binding of the benzimidazole-guanidinium hybrid compounds in the DNA minor groove. Circular dichroism and molecular docking studies indicated minor groove binding analogous to classical minor groove binders such as DAPI and Hoechst 33258. Thermal denaturation studies indicated that the best binder (compound 8) gave similar thermal stabilization to B-DNA as given by DAPI.

Green-synthesized, pH-stable and biocompatible carbon nanosensor for Fe3+: An experimental and computational study.

Brightly fluorescent Carbon Dots (CDs) were synthesized by green hydrothermal method using commonly available biomass (Aloe vera) as carbon precursor. Their physiochemical and optical characterization was done by standard microscopic and spectroscopic techniques. Photophysical features of their aqueous dispersion were investigated in detail. The influence of wide pH range (2-12), high ionic load (2M) and temperature on their photoluminescence behavior was investigated. Their in-vitro cytotoxicity examination was conducted on Human Cervical Cancer Cells (HeLa) using MTT assay. Testing of their ion-recognition property for common metal ions was done in aqueous medium. These CDs exhibited preferential interaction with Fe3+ over other tested metal ions, without any functionalization. Interaction between CDs and Fe3+ was analyzed in the light of Density Functional Theory (DFT). The work demonstrates that these CDs are acting as nanoprobe for Fe3+ and sensing it at ultra-trace level (5 nM).

Recent Advances in the Discovery of Antiviral Metabolites from Fungi.

As the world manages the impact of a global pandemic caused by COVID-19, the discovery of new antiviral agents has become way more relevant and urgent. Viruses are submicroscopic infectious agents that replicate inside the living cells of different organisms. These viruses use nucleic acids (both DNA and RNA) for further replication and maturity inside the cells. Some of the viruses responsible for various human and plant diseases belong to the classes of Picornaviridae, Retroviridae, Orthomyxoviridae, Flaviviridae, Pneumoviridae, Virgaviridae, and Hepadnaviridae, and their treatment options are limited or non-existent. The consistent reemergence and resistance development in the viral strains demand the discovery and development of new antiviral drugs possessing better efficacy. Bio-active compounds isolated from fungi can be the source of new compounds with enhanced potency and new mechanisms of action. Fungi are known to produce a diverse lot of secondary metabolites due to their existence in harsh and testing climates which are often inhabitable for many organisms. Because of these unique environments, fungi produce a variety of secondary metabolites of different chemical classes like alkaloids, quinones, furanone, pyrones, benzopyranoids, xanthones, terpenes, steroids, peptides, and many acyclic compounds. Fungal metabolites are known to display a wide range of bioactive attributes, i.e., anticancer, antibacterial, antifungal, and anti-Alzheimer's, along with antiviral properties. In this review article, we report over 300 antiviral compounds from fungal sources during the period of 2009 to 2019. The source of these compounds is marine and endophytic fungi and they are arranged based on their antiviral action against different viral families. These compounds offer promise for their use and development as future antiviral drugs.