Comprehensive analysis of structural variants in breast cancer genomes using single-molecule sequencing.

Improved identification of structural variants (SVs) in cancer can lead to more targeted and effective treatment options as well as advance our basic understanding of the disease and its progression. We performed whole-genome sequencing of the SKBR3 breast cancer cell line and patient-derived tumor and normal organoids from two breast cancer patients using Illumina/10x Genomics, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT) sequencing. We then inferred SVs and large-scale allele-specific copy number variants (CNVs) using an ensemble of methods. Our findings show that long-read sequencing allows for substantially more accurate and sensitive SV detection, with between 90% and 95% of variants supported by each long-read technology also supported by the other. We also report high accuracy for long reads even at relatively low coverage (25×-30×). Furthermore, we integrated SV and CNV data into a unifying karyotype-graph structure to present a more accurate representation of the mutated cancer genomes. We find hundreds of variants within known cancer-related genes detectable only through long-read sequencing. These findings highlight the need for long-read sequencing of cancer genomes for the precise analysis of their genetic instability.

Design, Synthesis, and Structural Analysis of Divalent N(I) Compounds and Identification of a New Electron-Donating Ligand.

The dative-bond representation (L→E) in compounds with main group elements (E) has triggered extensive debate in the recent past. The scope and limits of this nonclassical coordination bond warrant comprehensive exploration. Particularly compounds with (L→N←L')(+) arrangement are of special interest because of their therapeutic importance. This work reports the design and synthesis of novel chemical species with the general structural formula (L→N←L')(+) carrying the unusual ligand cyclohexa-2,5-diene-4-(diaminomethynyl)-1-ylidene. Four species belonging to the (L→N←L')(+) class carrying this unconventional ligand were synthesized. Quantum chemical and X-ray diffraction analyses showed that the electronic and geometric parameters are consistent with those of already reported divalent N(I) compounds. The molecular orbital analysis, geometric parameters, and spectral data clearly support the L→N and N←L' interactions in these species. The newly identified ligand has the properties of a reactive carbene and high nucleophilicity.

Possibility of the existence of donor-acceptor interactions in bis(azole)amines: an electronic structure analysis.

Donor-stabilized divalent N(I) systems have recently gained attention in the field of organic chemistry. Existence of low-valent nitrogen(I) species with moderate nucleophilicities in several pharmacophoric functionalities is prompting extensive exploration in this field. Quantum chemical analysis on the imidazole, oxazole, and thiazole derivatives of thiazole-2-amine indicated that these species preferably exist in the iminic state. Electronic structure analysis of these systems suggested the existence of hidden divalent N(I) character in a neutral state (L → N-R) and the explicit divalent N(I) character (L → N ← L)(+) in the protonated state. The strength of L → N interaction in these systems was analyzed, and the variations in the nucleophilicity trend at the coordinating nitrogen center were rationalized by estimating the electronic (TEP (Tolman electronic parameter) and MESP minimum (V(min))) as well as steric parameters (r-repulsiveness and ΔH elimination of CO group, in L → Ni(CO)3) of the coordinating ligands L. The importance of energetically preferred ionic and tautomeric representations of thiazol-2-amine derivatives in iminic and aminic forms was also demonstrated by carrying out comparative docking analysis with the enzyme lymphocyte-specific kinase (Lck).

Design and synthesis of guanylthiourea derivatives as potential inhibitors of Plasmodium falciparum dihydrofolate reductase enzyme.

A new class of compounds based on S-benzylated guanylthiourea has been designed as potential PfDHFR inhibitors using computer aided methods (molecular electrostatic potential, molecular docking). Several compounds in this class have been synthesized starting from guanylthiourea and alkyl bromides. In vitro studies showed that two compounds from this class are active with the IC50 value of 100 µM and 400 nM.

Existence of dynamic tautomerism and divalent N(I) character in N-(pyridin-2-yl)thiazol-2-amine.

N-(pyridin-2-yl)thiazol-2-amine is a versatile chemical functional unit present in many therapeutically important species. Quantum chemical analysis shows that there are six competitive isomeric structures possible for this class of compounds within a relative energy difference of ∼4 kcal/mol. Some of the isomeric structures possess divalent N(I) character. There appears to be a competition between the thiazole and pyridine groups to accommodate the tautomeric hydrogen, and consequently show electron donating property in the structure with R-N←L representation. Details of electron distribution, tautomeric preferences, protonation energy, and divalent N(I) character, and so on, of this class of compounds are presented in this article. Subsequently, upon protonation, (L→N←L)(⊕) character is clearly evident in these moieties as molecular orbital analysis clearly shows two lone pairs of electrons on the central nitrogen, in this system.

Mechanism of invasion of lung epithelial cells by filamentous Legionella pneumophila.

Legionella, the aetiological agent responsible for Legionellosis, is an opportunistic pathogen that infects humans upon the inhalation of contaminated aerosolized water droplets. Legionella is pleomorphic and its different morphotypes exhibit varying degrees of virulence. While the filamentous forms of Legionella pneumophila (Lp) have been reported in patient samples since the first description of legionellosis, their role in disease has not been studied. Our results show that both E-cadherin and ß1 integrin receptors mediate filamentous Lp (FLp) attachment to lung epithelial cells (LECs). The activation of these receptors induces the formation of actin enriched membrane surface structures that we designated 'hooks' and 'membrane wraps'. These structures entrap the filaments on the cell surface leading to their gradual internalization through a zipper mechanism of phagocytosis dependent on actomyosin activity. The supply of E-cadherin receptors from the recycling pathway and ß1 integrins released from focal adhesion turnover are required to sustain this process. Intracellular FLp inhabits a vacuolar compartment where filaments differentiate into short rods and replicate to produce infective progeny. Here we are reporting a first description of the invasion mechanism used by FLp to invade LECs. Therefore, filamentous morphotype of Lp can induce its own uptake by LECs and has the potential ability to cause disease.

Establishment and Culture of Patient-Derived Breast Organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

Divalent N(I) character in 2-(thiazol-2-yl)guanidine: an electronic structure analysis.

Several medicinally important compounds carry a 2-(thiazol-2-yl)guanidine unit. These species are generally (erroneously) represented as 1-(thiazol-2-yl)guanidine species. Quantum chemical studies were performed to identify the appropriate tautomeric state of this class of compounds. B3LYP/6-31+G(d) calculations indicate the preferred tautomeric state of these species is associated with the 2-(thiazol-2-yl)guanidine structure rather than the 1-(thiazol-2-yl)guanidine structure. G2MP2 calculations on the model system were carried out to study the electronic structure, electron delocalization, and protonation energy; MESP, ELF, HOMA, AIM, and NBO analyses were also carried out. The results indicate that this class of compounds may be treated as species with hidden ::N(←L)R character. Upon protonation of the thiazole ring nitrogen, these systems show the electronic structure as in ::N(←L)2(⊕) systems with divalent N(I) oxidation state.

Thiazole derivative based topical nanoemulgel for inhibition of bacterial virulence in surface infections.

Objectives: Antimicrobial resistance emerged as a global challenge owing to limited therapeutic options to control infections. Pseudomonas aeruginosa, an MDR pathogen already developed resistance against many conventional antibiotics. An "anti-virulence strategy" that targets bacterial virulence rather than growth proves effective against drug-resistant pathogens. Materials and Methods: Here, we used a structure-based drug design approach to identify lead molecules using the LasR receptor protein of P. aeruginosa as a target responsible for virulence production in this bacterium. From the identified hits, we developed lead-based nanoformulation and investigated its effectiveness for treating the P. aeruginosa associated surface-infection in-vivo. First, TC-based nanoemulsions were fabricated by high-pressure homogenization and evaluated for various in vitro parameters. The optimized nanoemulsions were thereby utilized to prepare NEG. Results: The nanoemulsion (F3) exhibited low droplet size (51.04±1.88 nm), PDI (0.065±1.14), and negative zeta potential (-33.65±0.82 mV). In animals, topical application of NEG-3 demonstrated significant improvement on skin permeability (459±10.17 µg), drug influx (18.99±0.76 µg/cm2 hr), and repressed the CFU of P. aeruginosa induced-surface infection (P≤ 0.001). The histology of rat skin demonstrated a significant effect for groups treated with TC-based NEGs as compared with a negative control group, whereas no significant effect was seen on rat liver indicating low systemic exposure to the drug. Also, NEG3 showed no significant changes under different stability conditions after 3 months. Conclusion: TC-based NEGs open up the possibility of a more effective way to combat serious surface infections caused by P. aeruginosa.

Potential Assessment of Topical Felbinac-Loaded Cubosomal Gel in Soft Tissue Injury in Albino Rats.

Muscle strain is one of the most common injuries with high intermittence rate. Due to diverseness of strain injuries, different experimental animal models are employed to investigate such injuries with reproducible results. Cubosomes, an emerging nano drug delivery tool, are considered ideal carriers for the topical delivery of lipophilic drugs to treat local inflammations with reduced frequency of application for prolonged periods. This work describes the development of Felbinac-loaded cubosomal gel and investigated the treatment of inflammation and tissue injury in vivo. Sciatic Function Index (SFI) is a simple clinical method to observe hind limb recovery in rats after induced injuries. First, cubosomes were fabricated by high-pressure homogenization process and evaluated for in vitro parameters. The optimized cubosome formulation was chosen to develop cubosomal gel and evaluated for in vitro parameters and also investigated time to recovery of SFI after strain induction in tibialis anterior muscles in rats. The cubosome formulation (F4) exhibited low droplet size (51.04 ± 1.37 nm)and polydispersity index (0.085 ± 1.13), and negative zeta potential (-32.8 ± 0.67 mV). In rats, topical application of cubosomal gel formulation (CGF) exhibited significant improvement in skin permeation (402 ± 6.08 µg) and drug flux (15.71 ± 0.82 µg/cm2 h) compared to plain gel. Also, CGF demonstrated significant difference in SFI from first to seventh day. The histology of rat skin showed significant effect for groups treated with Felbinac-loaded CGF compared to a negative control group.

Patient-Derived Triple-Negative Breast Cancer Organoids Provide Robust Model Systems That Recapitulate Tumor Intrinsic Characteristics.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with poor patient outcomes, highlighting the unmet clinical need for targeted therapies and better model systems. Here, we developed and comprehensively characterized a diverse biobank of normal and breast cancer patient-derived organoids (PDO) with a focus on TNBCs. PDOs recapitulated patient tumor intrinsic properties and a subset of PDOs can be propagated for long-term culture (LT-TNBC). Single cell profiling of PDOs identified cell types and gene candidates affiliated with different aspects of cancer progression. The LT-TNBC organoids exhibit signatures of aggressive MYC-driven, basal-like breast cancers and are largely comprised of luminal progenitor (LP)-like cells. The TNBC LP-like cells are distinct from normal LPs and exhibit hyperactivation of NOTCH and MYC signaling. Overall, this study validates TNBC PDOs as robust models for understanding breast cancer biology and progression, paving the way for personalized medicine and tailored treatment options. SIGNIFICANCE: A comprehensive analysis of patient-derived organoids of TNBC provides insights into cellular heterogeneity and mechanisms of tumorigenesis at the single-cell level.

Quorum Sensing Inhibitors: Curbing Pathogenic Infections through Inhibition of Bacterial Communication.

Currently, most of the developed and developing countries are facing the problem of infectious diseases. The genius way of an exaggerated application of antibiotics led the infectious agents to respond by bringing a regime of persisters to resist antibiotics attacks prolonging their survival. Persisters have the dexterity to communicate among themself using signal molecules via the process of Quorum Sensing (QS), which regulates virulence gene expression and biofilms formation, making them more vulnerable to antibiotic attack. Our review aims at the different approaches applied in the ordeal to solve the riddle for QS inhibitors. QS inhibitors, their origin, structures and key interactions for QS inhibitory activity have been summarized. Solicitation of a potent QS inhibitor molecule would be beneficial, giving new life to the simplest antibiotics in adjuvant therapy.

Growing emergence of drug-resistant Pseudomonas aeruginosa and attenuation of its virulence using quorum sensing inhibitors: A critical review.

A perilous increase in the number of bacterial infections has led to developing throngs of antibiotics for increasing the quality and expectancy of life. Pseudomonas aeruginosa is becoming resistant to all known conventional antimicrobial agents thereby posing a deadly threat to the human population. Nowadays, targeting virulence traits of infectious agents is an alternative approach to antimicrobials that is gaining much popularity to fight antimicrobial resistance. Quorum sensing (QS) involves interspecies communication via a chemical signaling pathway. Under this mechanism, cells work in a concerted manner, communicate with each other with the help of signaling molecules called auto-inducers (AI). The virulence of these strains is driven by genes, whose expression is regulated by AI, which in turn acts as transcriptional activators. Moreover, the problem of antibiotic-resistance in case of infections caused by P. aeruginosa becomes more alarming among immune-compromised patients, where the infectious agents easily take over the cellular machinery of the host while hidden in the QS mediated biofilms. Inhibition of the QS circuit of P. aeruginosa by targeting various signaling pathways such as LasR, RhlR, Pqs, and QScR transcriptional proteins will help in blocking downstream signal transducers which could result in reducing the bacterial virulence. The anti-virulence agent does not pose an immediate selective pressure on growing bacterium and thus reduces the pathogenicity without harming the target species. Here, we review exclusively, the growing emergence of multi-drug resistant (MDR) P. aeruginosa and the critical literature survey of QS inhibitors with their potential application of blocking P. aeruginosa infections.

Antiviral therapeutics directed against RNA dependent RNA polymerases from positive-sense viruses.

Viruses with positive-sense single stranded RNA (+ssRNA) genomes are responsible for different diseases and represent a global health problem. In addition to developing new vaccines that protect against severe illness on infection, it is imperative to identify new antiviral molecules to treat infected patients. The genome of these RNA viruses generally codes for an enzyme with RNA dependent RNA polymerase (RdRP) activity. This molecule is centrally involved in the duplication of the RNA genome. Inhibition of this enzyme by small molecules will prevent duplication of the RNA genome and thus reduce the viral titer. An overview of the different therapeutic strategies used to inhibit RdRPs from +ssRNA viruses is provided, along with an analysis of these enzymes to highlight new binding sites for inhibitors.

PHAROH lncRNA regulates Myc translation in hepatocellular carcinoma via sequestering TIAR.

Hepatocellular carcinoma, the most common type of liver malignancy, is one of the most lethal forms of cancer. We identified a long non-coding RNA, Gm19705, that is overexpressed in hepatocellular carcinoma and mouse embryonic stem cells. We named this RNA Pluripotency and Hepatocyte Associated RNA Overexpressed in HCC, or PHAROH. Depletion of PHAROH impacts cell proliferation and migration, which can be rescued by ectopic expression of PHAROH. RNA-seq analysis of PHAROH knockouts revealed that a large number of genes with decreased expression contain a Myc motif in their promoter. MYC is decreased in knockout cells at the protein level, but not the mRNA level. RNA-antisense pulldown identified nucleolysin TIAR, a translational repressor, to bind to a 71-nt hairpin within PHAROH, sequestration of which increases MYC translation. In summary, our data suggest that PHAROH regulates MYC translation by sequestering TIAR and as such represents a potentially exciting diagnostic or therapeutic target in hepatocellular carcinoma.

Cancer stem cells: advances in biology and clinical translation-a Keystone Symposia report.

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability. On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.

Development and Characterization of Niosomal Gel System using Lallementia royaleana Benth. mucilage for the treatment of Rheumatoid Arthritis.

Niosomes structural framework comprises of non-ionic surfactant-based microscopic lamellar structures which carries the potential to sustain the effect of drug from its delivery system. In present work, the attempt was made to identify the effect of different ingredients such as effect of Tweens and natural mucilage of Lallemantia royaleana Benth. on the performance of developed niosomal gel formulations in order to prolong the duration of action of drug and to minimize its side effects of topical conventional drug administration. All Ibuprofen loaded niosomes formulationswere prepared by ether injection method; using cetosteryl alcohol with different variants of Tweens and Spans. Various evaluation parameters were performed to confirm niosome formation. Further, the niosomes were incorporated into gel system and evaluated for in-vitro permeability study (ex-vivo) on excised rat skin by membrane diffusion method and in-vivo study by carrageenan induced rat paw edema model. The best selected niosome formulation F9 gave no sedimentation, layer separation and unchanged particle shapes and thus selected for gel preparation using Lallemantia royaleana Benth. mucilage and carbopol in different ratios. Ex-vivo and in-vivo studies indicated high skin retention and penetration rates within the skin for tests niosomal gel formulations (G1 & G2). The present study suggested that developed topical gel formulation provides enhance permeability and longer duration of drug action over conventional gels.

MaTAR25 lncRNA regulates the Tensin1 gene to impact breast cancer progression.

Misregulation of long non-coding RNA (lncRNA) genes has been linked to a wide variety of cancer types. Here we report on Mammary Tumor Associated RNA 25 (MaTAR25), a nuclear enriched and chromatin associated lncRNA that plays a role in mammary tumor cell proliferation, migration, and invasion, both in vitro and in vivo. MaTAR25 functions by interacting with purine rich element binding protein B (PURB), and associating with a major downstream target gene Tensin1 (Tns1) to regulate its expression in trans. The Tns1 protein product is a critical component of focal adhesions linking signaling between the extracellular matrix and the actin cytoskeleton. Knockout of MaTAR25 results in down-regulation of Tns1 leading to a reorganization of the actin cytoskeleton, and a reduction of focal adhesions and microvilli. We identify LINC01271 as the human ortholog of MaTAR25, and importantly, increased expression of LINC01271 is associated with poor patient prognosis and metastasis. Our findings demonstrate that LINC01271 represents a potential therapeutic target to alter breast cancer progression.

Diminished AHR Signaling Drives Human Acute Myeloid Leukemia Stem Cell Maintenance.

Eliminating leukemic stem cells (LSC) is a sought after therapeutic paradigm for the treatment of acute myeloid leukemia (AML). While repression of aryl hydrocarbon receptor (AHR) signaling has been shown to promote short-term maintenance of primitive AML cells in culture, no work to date has examined whether altered AHR signaling plays a pathologic role in human AML or whether it contributes at all to endogenous LSC function. Here, we show AHR signaling is repressed in human AML blasts and preferentially downregulated in LSC-enriched populations within leukemias. A core set of AHR targets are uniquely repressed in LSCs across diverse genetic AML subtypes. In vitro and in vivo administration of the specific AHR agonist FICZ significantly impaired leukemic growth, promoted differentiation, and repressed self-renewal. Furthermore, LSCs suppressed a set of FICZ-responsive AHR target genes that function as tumor suppressors and promoters of differentiation. FICZ stimulation did not impair normal hematopoietic stem and progenitor (HSPC) function, and failed to upregulate a prominent LSC-specific AHR target in HSPCs, suggesting that differential mechanisms govern FICZ-induced AHR signaling manifestations in HSCs versus LSCs. Altogether, this work highlights AHR signaling suppression as a key LSC-regulating control mechanism and provides proof of concept in a preclinical model that FICZ-mediated AHR pathway activation enacts unique transcriptional programs in AML that identify it as a novel chemotherapeutic approach to selectively target human LSCs. SIGNIFICANCE: The AHR pathway is suppressed in leukemic stem cells (LSC), therefore activating AHR signaling is a potential therapeutic option to target LSCs and to treat acute myeloid leukemia.

In silico identification of albendazole as a quorum sensing inhibitor and its in vitro verification using CviR and LasB receptors based assay systems.

Introduction: Quorum sensing inhibition (QSI) is one of the vital tools to overcome emerging virulence of pathogenic bacteria which aims at curbing bacterial resistance. Targeting QS (quorum sensing) as chemotherapy is less likely to generate resistance among pathogens as it targets only the adaptation and not the survival mechanism of the pathogen. Several QS inhibitors were developed in the recent past but none of them managed to have clinical application due to known toxic effects for human consumption. A rapid development of QS inhibitor drugs could be achieved by verification of the QSI activity of drugs which are already in clinical use with known pharmacology. Recently, a known FDA approved clinical drug niclosamide belonging to an anthelmintic class is found to exhibit QSI activity. Methods: We have focused our study on Albendazole, another FDA approved clinical drug belonging to the same class for its potential to act as QSI. The structure-based molecular docking is used for finding putative interactions made by this drug with the CviR and LasB receptor protein of Chromobacterium violaceum and Pseudomonas aeruginosa , respectively. Further, the in vitro activity of this drug has been evaluated by employing CviR and LasB receptor-based bioassay. The efficacy of this drug alone and in combination with antibiotic Tobramycin to inhibit P. aeruginosa based biofilms was also analyzed by developing the biofilms on chambered glass slides and performing anti-biofilm assay. Results: Further, this drug found to inhibit purple pigment violacein production in C. violaceum , which is under the control of C6-AHL-CviR mediated QS in this human pathogen. The in vivo bioassays results suggested that albendazole has great potential to act as a QS inhibitor as found inhibiting violacein production in C. violaceum and biofilm formation in P. aeruginosa , respectively. Conclusion: It is that structure-based molecular docking guided bioassay evaluation is an efficient tool for finding the new therapeutic use of old drugs which could have more chances to come easily in clinical application for their newly identified therapeutic uses.