Decoding the therapeutic landscape of alpha-linolenic acid: a network pharmacology and bioinformatics investigation against cancer-related epigenetic modifiers.

Omega-3 (n - 3) and omega-6 (n - 6) polyunsaturated fatty acids (PUFAs) are vital for human health, but an imbalance between these types is associated with chronic diseases, including cancer. Alpha-linolenic acid (ALA), a n - 3 PUFA, shows promise as an anticancer agent in both laboratory and animal studies. However, the precise molecular mechanisms underlying ALA's actions against cancer-related epigenetic modifiers (CaEpM) remain unclear. To understand this, we employed network pharmacology (NP) and molecular docking techniques. Our study identified 51 potential ALA targets and GO and KEGG pathway analysis revealed possible molecular targets and signaling pathways of ALA against CaEpM. From PPI analysis, EZH2, KAT2B, SIRT1, KAT2A, KDM6B, EHMT2, WDR5, SETD7, SIRT2, and HDAC3 emerged as the top 10 potential targets. Additionally, GeneMANIA functional association (GMFA) network analysis of these top 10 targets was performed to enhance NP insights and explore ALA's multi-target approach. After an exhaustive analysis of the core FGN subnetwork, it became evident that 9 out of the 15 targets-namely EZH2, SUZ12, EED, PARP1, HDAC3, DNMT1, NCOR2, KAT2B, and TRRAP-manifested evidently strong and abundant interconnections among each other. Molecular docking of both top 10 targets and core FGN targets confirmed strong binding affinity between ALA and SIRT2, WDR5, KDM6B, EHMT2, HDAC3, EZH2, PARP1, and KAT2B, underscoring their roles in ALA's anti-CaEpM mechanism. Our findings suggest that ALA may target key signaling pathways related to transcriptional regulation, microRNA involvement, stem cell pluripotency and cellular senescence in cancer epigenetics. These findings illuminate ALA's potential as a multi-target agent against CaEpM.Communicated by Ramaswamy H. Sarma.

Awareness about Tobacco Causing Head and Neck Cancers via Mass Media: A Case-Control Study from India.

OBJECTIVE: This study aimed to assess the awareness of people about the adverse effects of tobacco (smoking and chewing) consumption causing head and neck cancers (HNCs) via mass media channels like television, cinema, radio and newspapers or magazines, wall painting or billboards / hoardings, public transportation and packets of chewing tobacco, bidis or cigarettes. METHODS: Hospital-based case-control was conducted in Pune, Maharashtra, India. Face to face interviews were conducted for the purpose of data collection on 225 cases and 240 controls. The relationship between two categorical variables were estimated using chi-square test with a  2-tailed P value of <.05.  SPSS software was used for data analysis. RESULTS: Controls as compared to cases had good awareness scores for chewing (59.9%) and smoking tobacco (63.7%), P<0.001. The most common form of mass media was television where the cases (60.4%) and controls (77.9%) had heard messages about tobacco in chewing and smoking form causing HNCs. Level of awareness of tobacco causing HNCs amongst tobacco users, stratified by their status (cases versus controls) showed that cases were 1.68 times less likely than controls to have heard or seen messages about the association between chewing tobacco and HNCs via radio. Males (61.3% and 61.0%) had significantly (P<0.001) more awareness as compared to females (46.9% and 43.5%) about chewing and smoking tobacco as a causal factor for  HNCs. CONCLUSION: Mass media needs to create a social environment which discourages tobacco consumption and promotes oral health at the population level. Additionally, there should be easy access to the availability of support services like Quitline and other community support services.

cGAS-STING pathway targeted therapies and their applications in the treatment of high-grade glioma.

Median survival of patients with glioblastoma (GBM) treated with standard of care which consists of maximal safe resection of the contrast-enhancing portion of the tumor followed by radiation therapy with concomitant adjuvant temozolomide (TMZ) remains 15 months. The tumor microenvironment (TME) is known to contain immune suppressive myeloid cells with minimal effector T cell infiltration. Stimulator of interferon genes (STING) is an important activator of immune response and results in production of Type 1 interferon and antigen presentation by myeloid cells. This review will discuss important developments in STING agonists, potential biomarkers for STING response, and new combinatorial therapeutic approaches in gliomas.

Hypoxia-activated neuropeptide Y/Y5 receptor/RhoA pathway triggers chromosomal instability and bone metastasis in Ewing sarcoma.

Adverse prognosis in Ewing sarcoma (ES) is associated with the presence of metastases, particularly in bone, tumor hypoxia and chromosomal instability (CIN). Yet, a mechanistic link between these factors remains unknown. We demonstrate that in ES, tumor hypoxia selectively exacerbates bone metastasis. This process is triggered by hypoxia-induced stimulation of the neuropeptide Y (NPY)/Y5 receptor (Y5R) pathway, which leads to RhoA over-activation and cytokinesis failure. These mitotic defects result in the formation of polyploid ES cells, the progeny of which exhibit high CIN, an ability to invade and colonize bone, and a resistance to chemotherapy. Blocking Y5R in hypoxic ES tumors prevents polyploidization and bone metastasis. Our findings provide evidence for the role of the hypoxia-inducible NPY/Y5R/RhoA axis in promoting genomic changes and subsequent osseous dissemination in ES, and suggest that targeting this pathway may prevent CIN and disease progression in ES and other cancers rich in NPY and Y5R.

Spherical Nucleic Acids as Precision Therapeutics for the Treatment of Cancer-From Bench to Bedside.

Spherical Nucleic Acids (SNAs) emerged as a new class of nanotherapeutics consisting of a nanoparticle core densely functionalized with a shell of radially oriented synthetic oligonucleotides. The unique three-dimensional architecture of SNAs protects the oligonucleotides from nuclease-mediated degradation, increases oligonucleotide bioavailability, and in the absence of auxiliary transfection agents, enables robust uptake into tumor and immune cells through polyvalent association with cell surface pattern recognition receptors. When composed of gene-regulatory small interfering (si)RNA or immunostimulatory DNA or RNA oligonucleotides, SNAs silence gene expression and induce immune responses superior to those raised by the oligonucleotides in their "free" form. Early phase clinical trials of gene-regulatory siRNA-based SNAs in glioblastoma (NCT03020017) and immunostimulatory Toll-like receptor 9 (TLR9)-agonistic SNAs carrying unmethylated CpG-rich oligonucleotides in solid tumors (NCT03086278) have shown that SNAs represent a safe, brain-penetrant therapy for inhibiting oncogene expression and stimulating immune responses against tumors. This review focuses on the application of SNAs as precision cancer therapeutics, summarizes the findings from first-in-human clinical trials of SNAs in solid tumors, describes the most recent preclinical efforts to rationally design next-generation multimodal SNA architectures, and provides an outlook on future efforts to maximize the anti-neoplastic activity of the SNA platform.

Delayed Tibial Shaft Fracture Healing Associated with Smoking: A Systematic Review and Meta-Analysis of Observational Studies Conducted Worldwide.

Tibial fractures represent a great burden of disease globally, being the most common long-bone fracture; smoking is a known risk factor for delayed skeletal healing and post-fracture complications. This systematic review and meta-analysis aims to analyse the effect of smoking on healing of tibial shaft fractures. PubMed, CINAHL, EMBASE, and Cochrane Library databases were searched from inception to March 2021, with no limitation on language, to find relevant research. All observational studies that assessed the association between cigarette smoking and tibial shaft fracture healing in adults (≥18 years) were included. The quality of studies was evaluated using the Newcastle Ottawa Quality Assessment Scale. A random effects model was used to conduct meta-analysis. Tobacco smoking was associated with an increased rate of non-union and delayed union as well as an increase in time to union in fractures of the tibial shaft. Among the 12 included studies, eight reported an increased rate of non-union, three reported delayed union, and five reported an increase in time to union. However, the results were statistically significant in only three studies for non-union, one for delayed union, and two studies for increased time to union. This review confirms the detrimental impact of smoking on tibial shaft fracture healing and highlights the importance of patient education regarding smoking cessation.

Impact of COVID-19 pandemic on orthopaedic surgeons in terms of anxiety, sleep outcomes and change in management practices: A cross-sectional study from India.

PURPOSE: The aim of this study was to assess the effects of the COVID-19 pandemic on anxiety, sleep outcomes and change in clinical management practices among orthopaedic surgeons following a nation-wide lockdown. METHODS: We conducted an online cross-sectional study using piloted structured questionnaires with self-reported responses from Indian orthopaedic surgeons. Study participants were identified through social networking sites: Facebook and WhatsApp. The extent of anxiety and sleep quality was assessed by the standardised seven-item Generalised Anxiety Disorder (GAD-7) scale, single-item sleep quality scale, questions on unavailability of personal protective equipment, training module on COVID-19 and change in orthopaedic patient management. RESULTS: One hundred male orthopaedic surgeons responded to the survey with majority (79%) in 30-44 years age group. Severe anxiety scores were observed in 8%; moderate, mild and minimal anxiety was observed in 12%, 27% and 53% surgeons respectively. Changes in management practice due to the pandemic was admitted by 65% respondents. We also observed an association between higher anxiety among surgeons and primary or secondary level of healthcare facility: (p = 0.04). Sleep disturbance was significantly associated with change in management practice to non-operative procedures (p = 0.03). CONCLUSION: Anxiety among orthopaedic surgeons during the COVID-19 pandemic is related to factors like younger age group, working in a primary or secondary healthcare facility. Early recognition of anxiety is essential to prevent serious psychological sequelae.

The orphan nuclear receptor estrogen-related receptor beta (ERRß) in triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC)/basal-like breast cancer (BLBC) is a highly aggressive form of breast cancer. We previously reported that a small molecule agonist ligand for the orphan nuclear receptor estrogen-related receptor beta (ERRß or ESRRB) has growth inhibitory and anti-mitotic activity in TNBC cell lines. In this study, we evaluate the association of ESRRB mRNA, copy number levels, and protein expression with demographic, clinicopathological, and gene expression features in breast tumor clinical specimens. METHODS: ESRRB mRNA-level expression and clinical associations were analyzed using RNAseq data. Array-based comparative genomic hybridization determined ESRRB copy number in African-American and Caucasian women. Transcription factor activity was measured using promoter-reporter luciferase assays in TNBC cell lines. Semi-automatic quantification of immunohistochemistry measured ERRß protein expression on a 150-patient tissue microarray series. RESULTS: ESRRB mRNA expression is significantly lower in TNBC/BLBC versus other breast cancer subtypes. There is no evidence of ESRRB copy number loss. ESRRB mRNA expression is correlated with the expression of genes associated with neuroactive ligand-receptor interaction, metabolic pathways, and deafness. These genes contain G/C-rich transcription factor binding motifs. The ESRRB message is alternatively spliced into three isoforms, which we show have different transcription factor activity in basal-like versus other TNBC cell lines. We further show that the ERRß2 and ERRßsf isoforms are broadly expressed in breast tumors at the protein level. CONCLUSIONS: Decreased ESRRB mRNA expression and distinct patterns of ERRß isoform subcellular localization and transcription factor activity are key features in TNBC/BLBC.

Implementation of Quality by Design (QbD) approach in development of silver sulphadiazine loaded egg oil organogel: An improved dermatokinetic profile and therapeutic efficacy in burn wounds.

Silver Sulphadiazine (SSD) is an effective antibacterial agent considered as the gold standard for burn wound treatment. The present study aimed to investigate EO-based organogel (SSD-EOOG) as an effective carrier system for SSD delivery in burn wound management employing Quality by Design (QbD) paradigm. The organogel-based formulations were prepared employing QbD-oriented approach and further evaluated for in vivo efficacy and stability. The developed formulations were characterized for particle size, drug content, morphology, in vitro drug release, skin safety studies, ex vivo permeation, skin retention, textural analysis and pharmacodynamic studies in murine burn wound model. I-optimal mixture design was employed for optimization and evaluating different critical quality attributes (CQAs). The optimized formulation exhibited particle size of 256.5 nm with enhanced permeation (72.33 ± 1.73%) and retention (541.20 ± 22.16 µg/cm2) across skin barrier as compared to SSD-MKT. The pharmacodynamic results proved superior therapeutic efficacy of SSD-EOOG in topical burn wounds inflicted with MRSA bacterium. The results indicated wound contraction rate (78.23 ± 5.65%) and faster re-epithelialization in SSD-EOOG treated group. The present study concluded that egg oil based organogel promoted therapeutic efficacy of SSD for burn wound treatment.

Integrated copy number and miRNA expression analysis in triple negative breast cancer of Latin American patients.

Triple negative breast cancer (TNBC), a clinically aggressive breast cancer subtype, affects 15-35% of women from Latin America. Using an approach of direct integration of copy number and global miRNA profiling data, performed simultaneously in the same tumor specimens, we identified a panel of 17 miRNAs specifically associated with TNBC of ancestrally characterized patients from Latin America, Brazil. This panel was differentially expressed between the TNBC and non-TNBC subtypes studied (p ≤ 0.05, FDR ≤ 0.25), with their expression levels concordant with the patterns of copy number alterations (CNAs), present mostly frequent at 8q21.3-q24.3, 3q24-29, 6p25.3-p12.2, 1q21.1-q44, 5q11.1-q22.1, 11p13-p11.2, 13q12.11-q14.3, 17q24.2-q25.3 and Xp22.33-p11.21. The combined 17 miRNAs presented a high power (AUC = 0.953 (0.78-0.99);95% CI) in discriminating between the TNBC and non-TNBC subtypes of the patients studied. In addition, the expression of 14 and 15 of the 17miRNAs was significantly associated with tumor subtype when adjusted for tumor stage and grade, respectively. In conclusion, the panel of miRNAs identified demonstrated the impact of CNAs in miRNA expression levels and identified miRNA target genes potentially affected by both CNAs and miRNA deregulation. These targets, involved in critical signaling pathways and biological functions associated specifically with the TNBC transcriptome of Latina patients, can provide biological insights into the observed differences in the TNBC clinical outcome among racial/ethnic groups, taking into consideration their genetic ancestry.

Clinical Profile and Short Term Outcome of Children with Neutropenia.

OBJECTIVES: To study the clinical profile and short term outcome of neutropenia in children during hospital stay. METHODS: An observational study was carried out at a Children's Hospital. The study population comprised of 77 admitted children aged 1 mo to 18 y with a varied clinical profile and neutropenia; defined as absolute neutrophil count less than 1500/mm3. Patients known to have HIV, immunodeficiency, malignancy, aplastic anemia or chronic systemic illness were not enrolled. Necessary investigations were done to identify etiology as per clinical features. They were treated and followed up for a period of 4 wk or discharge; whichever was earlier. The primary outcome of duration of neutropenia and secondary outcomes of hospital stay duration, association with thrombocytopenia, incidence of complications and finally discharge/death were analyzed. RESULTS: Acute transient neutropenia was seen, the median duration being 3 d in younger patients. Dengue fever was the commonest etiology. The median duration of hospital stay was 8 d. Fifty three (68.8%) patients had associated thrombocytopenia. Three children developed complications like nosocomial sepsis and shock. Seventy two (93.5%) were discharged, 1 died, 3 left against medical advice and one patient was followed up for 4 wk. CONCLUSIONS: Acute febrile illnesses like dengue, enteric fever, malaria are the predominant causes of neutropenia. Mild neutropenia was seen in over 50% children requiring a short duration of admission (5-8 d); without any complications.

Cancer-associated mutation and beyond: The emerging biology of isocitrate dehydrogenases in human disease.

Isocitrate dehydrogenases (IDHs) are critical metabolic enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (αKG), NAD(P)H, and CO2. IDHs epigenetically control gene expression through effects on αKG-dependent dioxygenases, maintain redox balance and promote anaplerosis by providing cells with NADPH and precursor substrates for macromolecular synthesis, and regulate respiration and energy production through generation of NADH. Cancer-associated mutations in IDH1 and IDH2 represent one of the most comprehensively studied mechanisms of IDH pathogenic effect. Mutant enzymes produce (R)-2-hydroxyglutarate, which in turn inhibits αKG-dependent dioxygenase function, resulting in a global hypermethylation phenotype, increased tumor cell multipotency, and malignancy. Recent studies identified wild-type IDHs as critical regulators of normal organ physiology and, when transcriptionally induced or down-regulated, as contributing to cancer and neurodegeneration, respectively. We describe how mutant and wild-type enzymes contribute on molecular levels to disease pathogenesis, and discuss efforts to pharmacologically target IDH-controlled metabolic rewiring.

Integrated molecular analysis of Tamoxifen-resistant invasive lobular breast cancer cells identifies MAPK and GRM/mGluR signaling as therapeutic vulnerabilities.

Invasive lobular breast cancer (ILC) is an understudied malignancy with distinct clinical, pathological, and molecular features that distinguish it from the more common invasive ductal carcinoma (IDC). Mounting evidence suggests that estrogen receptor-alpha positive (ER+) ILC has a poor response to Tamoxifen (TAM), but the mechanistic drivers of this are undefined. In the current work, we comprehensively characterize the SUM44/LCCTam ILC cell model system through integrated analysis of gene expression, copy number, and mutation, with the goal of identifying actionable alterations relevant to clinical ILC that can be co-targeted along with ER to improve treatment outcomes. We show that TAM has several distinct effects on the transcriptome of LCCTam cells, that this resistant cell model has acquired copy number alterations and mutations that impinge on MAPK and metabotropic glutamate receptor (GRM/mGluR) signaling networks, and that pharmacological inhibition of either improves or restores the growth-inhibitory actions of endocrine therapy.

Genomic comparison of early-passage conditionally reprogrammed breast cancer cells to their corresponding primary tumors.

Conditionally reprogrammed cells (CRCs) are epithelial cells that are directly isolated from patients' specimens and propagated in vitro with feeder cells and a Rho kinase inhibitor. A number of these cells have been generated from biopsies of breast cancer patients, including ductal carcinoma in situ and invasive carcinomas. The characterization of their genomic signatures is essential to determine their ability to reflect the natural biology of their tumors of origin. In this study, we performed the genomic characterization of six newly established invasive breast cancer CRC cultures in comparison to the original patients' primary breast tumors (PBT) from which they derived. The CRCs and corresponding PBTs were simultaneously profiled by genome-wide array-CGH, targeted next generation sequencing and global miRNA expression to determine their molecular similarities in the patterns of copy number alterations (CNAs), gene mutations and miRNA expression levels, respectively. The CRCs' epithelial cells content and ploidy levels were also evaluated by flow cytometry. A similar level of CNAs was observed in the pairs of CRCs/PBTs analyzed by array-CGH, with >95% of overlap for the most frequently affected cytobands. Consistently, targeted next generation sequencing analysis showed the retention of specific somatic variants in the CRCs as present in their original PBTs. Global miRNA profiling closely clustered the CRCs with their PBTs (Pearson Correlation, ANOVA paired test, P<0.05), indicating also similarity at the miRNA expression level; the retention of tumor-specific alterations in a subset of miRNAs in the CRCs was further confirmed by qRT-PCR. These data demonstrated that the human breast cancer CRCs of this study maintained at early passages the overall copy number, gene mutations and miRNA expression patterns of their original tumors. The further characterization of these cells by other molecular and cellular phenotypes at late cell passages, are required to further expand their use as a unique and representative ex-vivo tumor model for basic science and translational breast cancer studies.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

High neuropeptide Y release associates with Ewing sarcoma bone dissemination - in vivo model of site-specific metastases.

Ewing sarcoma (ES) develops in bones or soft tissues of children and adolescents. The presence of bone metastases is one of the most adverse prognostic factors, yet the mechanisms governing their formation remain unclear. As a transcriptional target of EWS-FLI1, the fusion protein driving ES transformation, neuropeptide Y (NPY) is highly expressed and released from ES tumors. Hypoxia up-regulates NPY and activates its pro-metastatic functions. To test the impact of NPY on ES metastatic pattern, ES cell lines, SK-ES1 and TC71, with high and low peptide release, respectively, were used in an orthotopic xenograft model. ES cells were injected into gastrocnemius muscles of SCID/beige mice, the primary tumors excised, and mice monitored for the presence of metastases. SK-ES1 xenografts resulted in thoracic extra-osseous metastases (67%) and dissemination to bone (50%) and brain (25%), while TC71 tumors metastasized to the lungs (70%). Bone dissemination in SK-ES1 xenografts associated with increased NPY expression in bone metastases and its accumulation in bone invasion areas. The genetic silencing of NPY in SK-ES1 cells reduced bone degradation. Our study supports the role for NPY in ES bone invasion and provides new models for identifying pathways driving ES metastases to specific niches and testing anti-metastatic therapeutics.

Hypoxia shifts activity of neuropeptide Y in Ewing sarcoma from growth-inhibitory to growth-promoting effects.

Ewing sarcoma (ES) is an aggressive malignancy driven by an oncogenic fusion protein, EWS-FLI1. Neuropeptide Y (NPY), and two of its receptors, Y1R and Y5R are up-regulated by EWS-FLI1 and abundantly expressed in ES cells. Paradoxically, NPY acting via Y1R and Y5R stimulates ES cell death. Here, we demonstrate that these growth-inhibitory actions of NPY are counteracted by hypoxia, which converts the peptide to a growth-promoting factor. In ES cells, hypoxia induces another NPY receptor, Y2R, and increases expression of dipeptidyl peptidase IV (DPPIV), an enzyme that cleaves NPY to a shorter form, NPY3-36. This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death. Instead, NPY3-36 acts as a selective Y2R/Y5R agonist. The hypoxia-induced increase in DPPIV activity is most evident in a population of ES cells with high aldehyde dehydrogenase (ALDH) activity, rich in cancer stem cells (CSCs). Consequently, NPY, acting via Y2R/Y5Rs, preferentially stimulates proliferation and migration of hypoxic ALDHhigh cells. Hypoxia also enhances the angiogenic potential of ES by inducing Y2Rs in endothelial cells and increasing the release of its ligand, NPY3-36, from ES cells. In summary, hypoxia acts as a molecular switch shifting NPY activity away from Y1R/Y5R-mediated cell death and activating the Y2R/Y5R/DPPIV/NPY3-36 axis, which stimulates ES CSCs and promotes angiogenesis. Hypoxia-driven actions of the peptide such as these may contribute to ES progression. Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES.