Nanoparticles as Drug Delivery Systems for the Targeted Treatment of Atherosclerosis.

Atherosclerosis continues to be a leading cause of morbidity and mortality globally. The precise evaluation of the extent of an atherosclerotic plaque is essential for forecasting its likelihood of causing health concerns and tracking treatment outcomes. When compared to conventional methods used, nanoparticles offer clear benefits and excellent development opportunities for the detection and characterisation of susceptible atherosclerotic plaques. In this review, we analyse the recent advancements of nanoparticles as theranostics in the management of atherosclerosis, with an emphasis on applications in drug delivery. Furthermore, the main issues that must be resolved in order to advance clinical utility and future developments of NP research are discussed. It is anticipated that medical NPs will develop into complex and advanced next-generation nanobotics that can carry out a variety of functions in the bloodstream.

Surgical Anatomy and Exercises Using the Chicken Thigh Sciatic Nerve for Microsurgery Training.

Introduction Vessel repair in a chicken thigh is commonly used in microsurgery training model. The sciatic nerve is closely associated with the vessels and has been used for training nerve coaptation, which has different technical considerations from vessel anastomosis. We describe in detail the relevant surgical anatomy and training exercises that can be used with this model. Methods With 32 fresh store-bought chicken thighs, 16 were used to analyze the gross and histological features of the sciatic nerve, and 16 were intended to create and perform training models. Results The average visible length of the nerve in the thigh was 51 mm (standard deviation [SD] 2.57 mm). The average diameter of the nerve was 2 mm (SD 0.33 mm) and was largest at its proximal end (3.21 mm, SD 0.27 mm). The nerve consistently branched into two along the chicken thigh, with more branching subsequently. This simulation model is appropriate not only for the classical end-to-end epineural suture, but also for advanced exercises, in terms of longitudinal fasciculus dissection, mismatched size nerve transfer, injured nerve preparation, and vein conduit technique. Dyeing of nerve fascicles enhanced the visibility of nerve surface quality. Conclusion The sciatic nerve in the chicken thigh is a suitable and accessible model for microsurgery training. The branching and fascicular patterns of the nerve lends itself well to both novice training and advanced simulation. We have incorporated this model into our training curricula.

Achieving Molecular Recognition of Structural Analogues in Surface-Enhanced Raman Spectroscopy: Inducing Charge and Geometry Complementarity to Mimic Molecular Docking.

Molecular recognition of complex isomeric biomolecules remains challenging in surface-enhanced Raman scattering (SERS) spectroscopy due to their small Raman cross-sections and/or poor surface affinities. To date, the use of molecular probes has achieved excellent molecular sensitivities but still suffers from poor spectral specificity. Here, we induce "charge and geometry complementarity" between probe and analyte as a key strategy to achieve high spectral specificity for effective SERS molecular recognition of structural analogues. We employ 4-mercaptopyridine (MPY) as the probe, and chondroitin sulfate (CS) disaccharides with isomeric sulfation patterns as our proof-of-concept study. Our experimental and in silico studies reveal that "charge and geometry complementarity" between MPY's binding pocket and the CS sulfation patterns drives the formation of site-specific, multidentate interactions at the respective CS isomerism sites, which "locks" each CS in its analogue-specific complex geometry, akin to molecular docking events. Leveraging the resultant spectral fingerprints, we achieve > 97 % classification accuracy for 4 CSs and 5 potential structural interferences, as well as attain multiplex CS quantification with < 3 % prediction error. These insights could enable practical SERS differentiation of biologically important isomers to meet the burgeoning demand for fast-responding applications across various fields such as biodiagnostics, food and environmental surveillance.

SIRIUS, Ultra-Scintillating Upconversion Breast Implant for Remote Orthotopic Photodynamic Therapy.

Present day strategies for delivery of wireless photodynamic therapy (PDT) to deep-seated targets are limited by the inadequacy of irradiance and insufficient therapeutic depth. Here we report the design and preclinical validation of a flexible wireless upconversion nanoparticle (UCNP) implant (SIRIUS) that is capable of large field, high intensity illumination for PDT of deep-seated tumors. The implant achieves this by incorporating submicrometer core-shell-shell NaYF4 UCNPs into its design, which significantly enhances upconversion efficiency and mitigates light loss from surface quenching. We demonstrate the efficacy of SIRIUS UCNP implant mediated PDT in preclinical breast cancer disease models. In our in vitro experiments, SIRIUS directed 5-Aminolevulinic Acid (5-ALA) based wireless PDT leads to significant reactive oxygen species (ROS) generation and tumor apoptosis in hormonal receptor+/HER2+ (MCF7) and triple-negative (MDA-MB-231) breast cancer cell lines. In our in vivo rodent model, SIRIUS-driven PDT is shown to be significant in regressing tumors when applied to orthotopically inoculated breast tumors. Following successful preclinical validation, we also describe a clinical prototype of UCNP breast implant with potential dual cosmetic and onco-therapeutic functions. SIRIUS is an upconversion breast implant for wireless PDT that fulfils all the design prerequisites necessary for seamless clinical translation.

C1QBP Mediates Breast Cancer Cell Proliferation and Growth via Multiple Potential Signalling Pathways.

Breast carcinoma is the most prevalent cancer in women globally, with complex genetic and molecular mechanisms that underlie its development and progression. Several challenges such as metastasis and drug resistance limit the prognosis of breast cancer, and hence a constant search for better treatment regimes, including novel molecular therapeutic targets is necessary. Complement component 1, q subcomponent binding protein (C1QBP), a promising molecular target, has been implicated in breast carcinogenesis. In this study, the role of C1QBP in breast cancer progression, in particular cancer cell growth, was determined in triple negative MDA-MB-231 breast cancer cells. Depletion of C1QBP decreased cell proliferation, whereas the opposite effect was observed when C1QBP was overexpressed in MDA-MB-231 cells. Furthermore, gene expression profiling and pathway analysis in C1QBP depleted cells revealed that C1QBP regulates several signaling pathways crucial for cell growth and survival. Taken together, these findings provide a deeper comprehension of the role of C1QBP in triple negative breast cancer, and could possibly pave the way for future advancement of C1QBP-targeted breast cancer therapy.

The natural antisense transcript HAS2-AS1 regulates breast cancer cells aggressiveness independently from hyaluronan metabolism.

Hyaluronan (HA) is a ubiquitous extracellular matrix component playing a crucial role in the regulation of cell behaviors, including cancer. Aggressive breast cancer cells tend to proliferate, migrate and metastatize. Notably, triple-negative breast cancer cells lacking the expression of estrogen receptor (ER) as well as progesterone receptor and HER2 are more aggressive than ER-positive ones. As currently no targeted therapy is available for triple-negative breast cancer, the identification of novel therapeutic targets has a high clinical priority. In ER-negative cells, tumoral behavior can be reduced by inhibiting HA synthesis or silencing the enzymes involved in its metabolism, such as HA synthase 2 (HAS2). HAS2-AS1 is a long non-coding RNA belonging to the natural antisense transcript family which is known to favor HAS2 gene expression and HA synthesis, thus bolstering malignant progression in brain, ovary, and lung tumors. As the role of HAS2-AS1 has not yet been investigated in breast cancer, in this work we report that ER-positive breast cancers had lower HAS2-AS1 expression compared to ER-negative tumors. Moreover, the survival of patients with ER-negative tumors was higher when the expression of HAS2-AS1 was elevated. Experiments with ER-negative cell lines as MDA-MB-231 and Hs 578T revealed that the overexpression of either the full-length HAS2-AS1 or its exon 2 long or short isoforms alone, strongly reduced cell viability, migration, and invasion, whereas HAS2-AS1 silencing increased cell aggressiveness. Unexpectedly, in these ER-negative cell lines, HAS2-AS1 is involved neither in the regulation of HAS2 nor in HA deposition. Finally, transcriptome analysis revealed that HAS2-AS1 modulation affected several pathways, including apoptosis, proliferation, motility, adhesion, epithelial to mesenchymal transition, and signaling, describing this long non-coding RNA as an important regulator of breast cancer cells aggressiveness.

Theranostic Applications of Glycosaminoglycans in Metastatic Renal Cell Carcinoma.

Renal cell carcinoma (RCC) makes up the majority of kidney cancers, with a poor prognosis for metastatic RCC (mRCC). Challenges faced in the management of mRCC, include a lack of reliable prognostic markers and biomarkers for precise monitoring of disease treatment, together with the potential risk of toxicity associated with more recent therapeutic options. Glycosaminoglycans (GAGs) are a class of carbohydrates that can be categorized into four main subclasses, viz., chondroitin sulfate, hyaluronic acid, heparan sulfate and keratan sulfate. GAGs are known to be closely associated with cancer progression and modulation of metastasis by modification of the tumor microenvironment. Alterations of expression, composition and spatiotemporal distribution of GAGs in the extracellular matrix (ECM), dysregulate ECM functions and drive cancer invasion. In this review, we focus on the clinical utility of GAGs as biomarkers for mRCC (which is important for risk stratification and strategizing effective treatment protocols), as well as potential therapeutic targets that could benefit patients afflicted with advanced RCC. Besides GAG-targeted therapies that holds promise in mRCC, other potential strategies include utilizing GAGs as drug carriers and their mimetics to counter cancer progression, and enhance immunotherapy through binding and transducing signals for immune mediators.

KIF21A regulates breast cancer aggressiveness and is prognostic of patient survival and tumor recurrence.

PURPOSE: Invasion of carcinoma cells into surrounding tissue affects breast cancer staging, influences choice of treatment, and impacts on patient outcome. KIF21A is a member of the kinesin superfamily that has been well-studied in congenital extraocular muscle fibrosis. However, its biological relevance in breast cancer is unknown. This study investigated the functional roles of KIF21A in this malignancy and examined its expression pattern in breast cancer tissue. METHODS: The function of KIF21A in breast carcinoma was studied in vitro by silencing its expression in breast cancer cells and examining the changes in cellular activities. Immunohistochemical staining of breast cancer tissue microarrays was performed to determine the expression patterns of KIF21A. RESULTS: Knocking down the expression of KIF21A using siRNA in MDA-MB-231 and MCF7 human breast cancer cells resulted in significant decreases in tumor cell migration and invasiveness. This was associated with reduced Patched 1 expression and F-actin microfilaments. Additionally, the number of focal adhesion kinase- and paxillin-associated focal adhesions was increased. Immunohistochemical staining of breast cancer tissue microarrays showed that KIF21A was expressed in both the cytoplasmic and nuclear compartments of carcinoma cells. Predominance of cytoplasmic KIF21A was significantly associated with larger tumors and high grade cancer, and prognostic of cause-specific overall patient survival and breast cancer recurrence. CONCLUSION: The data demonstrates that KIF21A is an important regulator of breast cancer aggressiveness and may be useful in refining prognostication of this malignant disease.

Epigenetic derepression converts PPARγ into a druggable target in triple-negative and endocrine-resistant breast cancers.

Clinical trials repurposing peroxisome proliferator-activated receptor-gamma (PPARγ) agonists as anticancer agents have exhibited lackluster efficacy across a variety of tumor types. Here, we report that increased PPARG expression is associated with a better prognosis but is anticorrelated with histone deacetylase (HDAC) 1 and 2 expressions. We show that HDAC overexpression blunts anti-proliferative and anti-angiogenic responses to PPARγ agonists via transcriptional and post-translational mechanisms, however, these can be neutralized with clinically approved and experimental HDAC inhibitors. Supporting this notion, concomitant treatment with HDAC inhibitors was required to license the tumor-suppressive effects of PPARγ agonists in triple-negative and endocrine-refractory breast cancer cells, and combination therapy also restrained angiogenesis in a tube formation assay. This combination was also synergistic in estrogen receptor-alpha (ERα)-positive cells because HDAC blockade abrogated ERα interference with PPARγ-regulated transcription. Following a pharmacokinetics optimization study, the combination of rosiglitazone and a potent pan-HDAC inhibitor, LBH589, stalled disease progression in a mouse model of triple-negative breast cancer greater than either of the monotherapies, while exhibiting a favorable safety profile. Our findings account for historical observations of de-novo resistance to PPARγ agonist monotherapy and propound a therapeutically cogent intervention against two aggressive breast cancer subtypes.

Knockdown of the prognostic cancer stem cell marker Musashi-1 decreases radio-resistance while enhancing apoptosis in hormone receptor-positive breast cancer cells via p21WAF1/CIP1.

PURPOSE: While the stem cell marker Musashi-1 (MSI-1) has been identified as a key player in a wide array of malignancies, few findings exist on its prognostic relevance and relevance for cancer cell death and therapy resistance in breast cancer. METHODS: First, we determined prognostic relevance of MSI-1 in database analyses regarding multiple survival outcomes. To substantiate findings, MSI-1 was artificially downregulated in MCF-7 breast cancer cells and implications for cancer stem cell markers, cell apoptosis and apoptosis regulator p21, proliferation and radiation response were analyzed via flow cytometry and colony formation. Radiation-induced p21 expression changes were investigated using a dataset containing patient samples obtained before and after irradiation and own in vitro experiments. RESULTS: MSI-1 is a negative prognostic marker for disease-free and distant metastasis-free survival in breast cancer and tends to negatively influence overall survival. MSI-1 knockdown downregulated stem cell gene expression and proliferation, but increased p21 levels and apoptosis. Similar to the MSI-1 knockdown effect, p21 expression was strongly increased after irradiation and was expressed at even higher levels in MSI-1 knockdown cells after irradiation. Finally, combined use of MSI-1 silencing and irradiation reduced cancer cell survival. CONCLUSION: MSI-1 is a prognostic marker in breast cancer. MSI-1 silencing downregulates proliferation while increasing apoptosis. The anti-proliferation mediator p21 was upregulated independently after both MSI-1 knockdown and irradiation and even more after both treatments combined, suggesting synergistic potential. Radio-sensitization effects after combining radiation and MSI-1 knockdown underline the potential of MSI-1 as a therapeutic target.

Prognostic significance of phosphoglycerate dehydrogenase in breast cancer.

PURPOSE: Breast cancer is the most common type of cancer affecting women worldwide. Phosphoglycerate dehydrogenase (PHGDH) is an oxidoreductase in the serine biosynthesis pathway. Although it has been reported to affect growth of various tumors, its role in breast cancer is largely unknown. This study aimed to analyze the expression of PHGDH in breast cancer tissue samples and to determine if PHGDH regulates breast cancer cell proliferation. METHODS: Tissue microarrays consisting of 305 cases of breast invasive ductal carcinoma were used for immunohistochemical evaluation of PHGDH expression. The role of PHGDH in breast cancer was investigated in vitro by knocking down its expression and determining the effect on cell proliferation and cell cycling, and in ovo by using a chorioallantoic membrane (CAM) assay. RESULTS: Immunohistochemical examination showed that PHGDH is mainly localized in the cytoplasm of breast cancer cells and significantly associated with higher cancer grade, larger tumor size, increased PCNA expression, and lymph node positivity. Analysis of the GOBO dataset of 737 patients demonstrated that increased PHGDH expression was associated with poorer overall survival. Knockdown of PHGDH expression in breast cancer cells in vitro resulted in a decrease in cell proliferation, reduction in cells entering the S phase of the cell cycle, and downregulation of various cell cycle regulatory genes. The volume of breast tumor in an in ovo CAM assay was found to be smaller when PHGDH was silenced. CONCLUSION: The findings suggest that PHGDH has a regulatory role in breast cancer cell proliferation and may be a potential prognostic marker and therapeutic target in breast cancer.

The heparan sulfate proteoglycan syndecan-1 regulates colon cancer stem cell function via a focal adhesion kinase-Wnt signaling axis.

In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of ß1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751.

The Role of Hydroxychloroquine in COVID-19: A Systematic Review and Meta-Analysis.

OBJECTIVE: A systematic review and meta-analysis was carried out to examine the role of hydroxychloroquine (HCQ) in the treatment of COVID-19. METHODS: We performed a systematic search in PubMed, Scopus, Embase, CochraneLibrary, Web of Science, Google Scholar, and medRxiv pre-print databases using available MeSH terms for COVID-19 and hydroxychloroquine. Data from all studies that focused on the effectiveness of HCQ with or without the addition of azithromycin (AZM) in confirmed COVID-19 patients, which were published up to 12 September 2020, were collated for analysis using CMA v.2.2.064. RESULTS: Our systematic review retrieved 41 studies. Among these, 37 studies including 45,913 participants fulfilled the criteria for subsequent meta-analysis. The data showed no significant difference in treatment efficacy between the HCQ and control groups (RR: 1.02, 95% CI, 0.81-1.27). Combination of HCQ with AZM also did not lead to improved treatment outcomes (RR: 1.26, 95% CI, 0.91-1.74). Furthermore, the mortality difference was not significant, neither in HCQ treatment group (RR: 0.86, 95% CI, 0.71-1.03) nor in HCQ plus AZM treatment group (RR: 1.28, 95% CI, 0.76-2.14) in comparison to controls. Meta-regression analysis showed that age was the factor that significantly affected mortality (P<0.00001). CONCLUSION: The meta-analysis found that there was no clinical benefit of using either HCQ by itself or in combination with AZM for the treatment of COVID-19 patients. Hence, it may be prudent for clinicians and researchers to focus on other therapeutic options that may show greater promise in this disease.

Diagnostic value of serum HER2 levels in breast cancer: a systematic review and meta-analysis.

BACKGROUND: Measurement of serum human epidermal growth factor receptor-2 (HER-2/neu) levels might play an essential role as a diagnostic/screening marker for the early selection of therapeutic approaches and predict prognosis in breast cancer patients. We aimed to undertake a systematic review and meta-analysis focusing on the diagnostic/screening value of serum HER-2 levels in comparison to routine methods. METHODS: We performed a systematic search via PubMed, Scopus, Cochrane-Library, and Web of Science databases for human diagnostic studies reporting the levels of serum HER-2 in breast cancer patients, which was confirmed using the histopathological examination. Meta-analyses were carried out for sensitivity, specificity, accuracy, area under the ROC curve (AUC), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), and negative likelihood ratio (NLR). RESULTS: Fourteen studies entered into this investigation. The meta-analysis indicated the low sensitivity for serum HER2 levels (Sensitivity: 53.05, 95%CI 40.82-65.28), but reasonable specificity of 79.27 (95%CI 73.02-85.51), accuracy of 72.06 (95%CI 67.04-77.08) and AUC of 0.79 (95%CI 0.66-0.92). We also found a significant differences for PPV (PPV: 56.18, 95%CI 44.16-68.20), NPV (NPV: 76.93, 95%CI 69.56-84.31), PLR (PLR: 2.10, 95%CI 1.69-2.50) and NLR (NLR: 0.58, 95%CI 0.44-0.71). CONCLUSION: Our findings revealed that although serum HER-2 levels showed low se nsitivity for breast cancer diagnosis, its specificity, accuracy and AUC were reasonable. Hence, it seems that the measurement of serum HER-2 levels can play a significant role as a verification test for initial negative screening test results, especially in low-income regions due to its cost-effectiveness and ease of implementation.

Protecting-Group-Free Synthesis of Chondroitin 6-Sulfate Disaccharide and Tetrasaccharide.

Chondroitin 6-sulfate (CS-C) is an important glycosaminoglycan that regulates many physiological functions including the development, progression, and metastasis of cancer. To understand its mechanism of action at the molecular level, CS-C molecules of defined length are required. A protecting group-free synthesis of CS-C disaccharide and tetrasaccharide from the CS-A polymer that involves only three steps and furnishes CS-O disaccharide and tetrasaccharide as intermediates is reported.

In Vitro Evaluation of Candidate Gene Targets for Cancer Therapy.

Discovery and development of gene targets for cancer therapeutics are lengthy and highly costly processes. Identification and evaluation of candidate gene targets are of fundamental importance. RNA interference allows candidate genes to be specifically and effectively knocked down in cancer cells. This tool can be easily incorporated into a loss-of-function approach in the initial evaluation of candidate gene targets for cancer treatment prior to moving on to animal studies and clinical trials. This chapter describes a relatively simple and straightforward protocol that makes use of small interfering RNA to achieve knockdown of the candidate gene target and to evaluate the resultant effects on four aspects of cancer cell behavior: migration, invasion, proliferation, and adhesion.

Small Maf functions in the maintenance of germline stem cells in the Drosophila testis.

Reactive oxygen species (ROS) are byproducts generated during normal cellular metabolism, and redox states have been shown to influence stem cell self-renewal and lineage commitment across phyla. However, the downstream effectors of ROS signaling that control stem cell behavior remain largely unexplored. Here, we used the Drosophila testis as an in vivo model to identify ROS-induced effectors that are involved in the differentiation process of germline stem cells (GSCs). In the Affymetrix microarray analysis, 152 genes were either upregulated or downregulated during GSC differentiation induced by elevated levels of ROS, and a follow-up validation of the gene expression by qRT-PCR showed a Spearman's rho of 0.9173 (P<0.0001). Notably, 47 (31%) of the identified genes had no predicted molecular function or recognizable protein domain. These suggest the robustness of this microarray analysis, which identified many uncharacterized genes, possibly with an essential role in ROS-induced GSC differentiation. We also showed that maf-S is transcriptionally downregulated by oxidative stress, and that maf-S knockdown promotes GSC differentiation but Maf-S overexpression conversely results in an over-growth of GSC-like cells by promoting the mitotic activity of germ cell lineage. Together with the facts that Maf-S regulates ROS levels and genetically interacts with Keap1/Nrf2 in GSC maintenance, our study suggests that Maf-S plays an important role in the Drosophila testis GSC maintenance by participating in the regulation of redox homeostasis.

Divergent Synthesis of Chondroitin Sulfate Disaccharides and Identification of Sulfate Motifs that Inhibit Triple Negative Breast Cancer.

Glycosaminoglycans (GAGs) regulate many important physiological processes. A pertinent issue to address is whether GAGs encode important functional information via introduction of position specific sulfate groups in the GAG structure. However, procurement of pure, homogenous GAG motifs to probe the "sulfation code" is a challenging task due to isolation difficulty and structural complexity. To this end, we devised a versatile synthetic strategy to obtain all the 16 theoretically possible sulfation patterns in the chondroitin sulfate (CS) repeating unit; these include rare but potentially important sulfated motifs which have not been isolated earlier. Biological evaluation indicated that CS sulfation patterns had differing effects for different breast cancer cell types, and the greatest inhibitory effect was observed for the most aggressive, triple negative breast cancer cell line MDA-MB-231.