Modified graphene oxide nanoplates reinforced 3D printed multifunctional scaffold for bone tissue engineering.

Successful regeneration of load-bearing bone defects remains a major challenge in clinical orthopaedics. Designing biologically active 3-dimensional scaffolds possessing physiological responsiveness can potentially overcome current limitations. Here, we have described a novel approach to fabricate scaffolds with modified nanosheets reinforced on mechanically customized thermoplastic polymer-based 3D printed constructs. In this article, we have developed polydopamine-reduced graphene oxide (PD-RGO) reinforced 3D printed PLA scaffold for bone tissue construction. RGO was synthesized by reduction of GO under alkaline conditions using dopamine. 3D printed polylactic acid (PLA) scaffold with defined porosity was doped with PD-RGO. The designed scaffold was studied for its physiochemical properties and human umbilical cord-derived mesenchymal stem cell (hMSC) behaviour within the scaffold. In vitro hMSC studies revealed the influence of fibre direction and nanocoating on directional cell growth and proliferation. The fabricated scaffold showed antioxidant property along with pro-angiogenic and osteoinductive potential. The designed scaffold also successfully prevented the formation of biofilm. In vivo heterotopic implantation of the differentiated hMSC loaded scaffold confirmed the biocompatibility and bio functionality of the scaffold. In summary, the designed nanoplates doped 3D printed scaffold displays stem cell responsive, integrative and pro-regenerative multi functionalities, thereby, exhibiting potential application as bone tissue regeneration treatment alternative.

Cardiac Differentiation of Mesenchymal Stem Cells: Impact of Biological and Chemical Inducers.

Cardiovascular disorders (CVDs) are the leading cause of global death, widely occurs due to irreparable loss of the functional cardiomyocytes. Stem cell-based therapeutic approaches, particularly the use of Mesenchymal Stem Cells (MSCs) is an emerging strategy to regenerate myocardium and thereby improving the cardiac function after myocardial infarction (MI). Most of the current approaches often employ the use of various biological and chemical factors as cues to trigger and modulate the differentiation of MSCs into the cardiac lineage. However, the recent advanced methods of using specific epigenetic modifiers and exosomes to manipulate the epigenome and molecular pathways of MSCs to modify the cardiac gene expression yield better profiled cardiomyocyte like cells in vitro. Hitherto, the role of cardiac specific inducers triggering cardiac differentiation at the cellular and molecular level is not well understood. Therefore, the current review highlights the impact and recent trends in employing biological and chemical inducers on cardiac differentiation of MSCs. Thereby, deciphering the interactions between the cellular microenvironment and the cardiac inducers will help us to understand cardiomyogenesis of MSCs. Additionally, the review also provides an insight on skeptical roles of the cell free biological factors and extracellular scaffold assisted mode for manipulation of native and transplanted stem cells towards translational cardiac research.

5FU encapsulated polyglycerol sebacate nanoparticles as anti-cancer drug carriers.

The majority of anti-cancer drugs fail to reach clinical trials due to their low water solubility. A biocompatible drug delivery system that encapsulates and efficiently delivers hydrophobic drugs to the target site is the need of the hour. This study addresses the issue by focusing on a polymeric polyglycerol sebacate (PGS) nanoparticles loaded with 5-fluorouracil (5FU), a primary line chemotherapy drug for many types of cancers. The generated nanoparticle (PGS-NP) was biocompatible and had minimal cytotoxicity against the MDA-MB-231 and A549 cell lines, even at a high concentration of 100 µg mL-1. The cell viability post treatment with PGS nanoparticles encapsulated with 5FU (PGS-5FU) decreased to as low as around 40% whereas, in the case of treatment with 5FU, the viability percentage increased. The nanoparticles also showed controlled drug release when encapsulated with 5FU. This striking observation suggested that these nanoparticles can improve the efficacy of drug delivery to tumor sites. Apoptosis assay and caspase-3 activity quantification supported these data wherein PGS-5FU treatment showed almost three times caspase-3 activity as compared to control cells. Additionally, throughout all the experiments, MDA-MB-231 cells were more sensitive to PGS-5FU than A549 cells, indicating that these nanoparticles are ideal for breast cancer treatment. In summary, 5FU encapsulated PGS nanoparticles are a potential drug carrier to deliver 5FU efficiently to cancer cells.

Bacterial diversity and functional analysis of severe early childhood caries and recurrence in India.

Dental caries is the most prevalent oral disease affecting nearly 70% of children in India and elsewhere. Micro-ecological niche based acidification due to dysbiosis in oral microbiome are crucial for caries onset and progression. Here we report the tooth bacteriome diversity compared in Indian children with caries free (CF), severe early childhood caries (SC) and recurrent caries (RC). High quality V3-V4 amplicon sequencing revealed that SC exhibited high bacterial diversity with unique combination and interrelationship. Gracillibacteria_GN02 and TM7 were unique in CF and SC respectively, while Bacteroidetes, Fusobacteria were significantly high in RC. Interestingly, we found Streptococcus oralis subsp. tigurinus clade 071 in all groups with significant abundance in SC and RC. Positive correlation between low and high abundant bacteria as well as with TCS, PTS and ABC transporters were seen from co-occurrence network analysis. This could lead to persistence of SC niche resulting in RC. Comparative in vitro assessment of biofilm formation showed that the standard culture of S. oralis and its phylogenetically similar clinical isolates showed profound biofilm formation and augmented the growth and enhanced biofilm formation in S. mutans in both dual and multispecies cultures.

Fabrication of nanopatterned PLGA films of curcumin and TPGS for skin cancer.

Squamous cell carcinoma treatment has limited therapeutic options and the incidence rate is increasing recently. In the present investigation, we developed poly(lactic-co-glycolic acid) (PLGA) nanopatterned films (NPFs) through poly dimethyl siloxane (PDMS) cast molding technique and explored its therapeutic efficacy in combination with curcumin and tocopherol poly (ethylene glycol) 1000 succinate (TPGS). Herein, we demonstrate the preparation and characterization of curcumin loaded tocopherol polyethylene glycol 1000 succinate stabilized poly (lactic-co-glycolic) acid nanopatterned films (CTP-NPFs). CTP-NPFs showed good in vitro cytotoxicity towards human skin cancer cell line (A431) when compared to that of unpattern films. CTP-NPFs effectively inhibited the progression of 7, 12-Dimethylbenz[a]anthracene (DMBA)/croton oil induced skin cancer in Swiss albino mice. The nanopatterned films could be used as an alternate treatment for skin cancer.

Deacetylation of LAMP1 drives lipophagy-dependent generation of free fatty acids by Abrus agglutinin to promote senescence in prostate cancer.

Therapy-induced senescence in cancer cells is an irreversible antiproliferative state, which inhibits tumor growth and is therefore a potent anti-neoplastic mechanism. In this study, low doses of Abrus agglutinin (AGG)-induced senescence through autophagy in prostate carcinoma cells (PC3) and inhibited proliferation. The inhibition of autophagy with 3-methyl adenine reversed AGG-induced senescence, thus confirming that AGG-triggered senescence required autophagy. AGG treatment also led to lipophagy-mediated accumulation of free fatty acids (FFAs), with a concomitant decrease in the number of lipid droplets. Lalistat, a lysosomal acid lipase inhibitor, abrogated AGG-induced lipophagy and senescence in PC3 cells, indicating that lipophagy is essential for AGG-induced senescence. The accumulation of FFAs increased reactive oxygen species generation, a known facilitator of senescence, which was also reduced in the presence of lalistat. Furthermore, AGG upregulated silent mating type information regulator 2 homolog 1 (SIRT1), while the presence of sirtinol reduced autophagy flux and the senescent phenotype in the AGG-treated cells. Mechanistically, AGG-induced cytoplasmic SIRT1 deacetylated a Lys residue on the cytoplasmic domain of lysosome-associated membrane protein 1 (LAMP1), an autolysosomal protein, resulting in lipophagy and senescence. Taken together, our findings demonstrate a novel SIRT1/LAMP1/lipophagy axis mediating AGG-induced senescence in prostate cancer cells.

Microwave-Assisted Synthesis of Quasi-Pyramidal CuInS2 -ZnS Nanocrystals for Enhanced Near-Infrared Targeted Fluorescent Imaging of Subcutaneous Melanoma.

Near-infrared (NIR) fluorescent CuInS2 -ZnS nanocrystals (CIZS NCs) are synthesized via an ultra-fast, non-injection microwave (MW)-assisted nanoalloying process at 230 ºC within 5 min using 1-dodecanethiol (DDT) as both the sulfur source and solvent under solvothermal (ST) condition. The structural and surface analyses reveal that DDT-functionalized CIZS NCs exhibit quasi-pyramids of tetragonal-phase with well-defined facets. The DDT-functionalized CIZS NCs present a photoluminescence quantum yield (PLQY) of 76% and a long-lived fluorescence lifetime of ≈0.6 µs in organic-phase. Subsequently, DDT-functionalized CIZS NCs are phase-transferred via ligand-exchange using 11-mercaptoundecanoic acid (MUA) into water-soluble MUA-CIZS NCs that exhibit a substantial PLQY of 55%. In addition, the NIR-fluorescent MUA-functionalized CIZS NCs in conjugation with folic acid (FA), as a tumor-targeting ligand, demonstrates enhanced tumor-targeted imaging ability. The FA-MUA-CIZS NC conjugates exhibit a cell viability of ≈75% even at the highest concentration of 1 mg mL-1 and a labeling efficiency of 95.4%. The in vivo imaging results corroborate that FA-MUA-CIZS NCs conjugates are actively targeted to folate receptor-positive B16F10 tumor-bearing C57BL/6 mice in 2 h. The histopathological and hematological studies confirm no significant changes in tissue architecture and blood biochemical parameters. The confocal microscopy studies reveal deep penetration and uniform distribution of FA-MUA-CIZS NCs conjugates in subcutaneous melanoma.

Essential oils: from prevention to treatment of skin cancer.

The increasing incidence of cutaneous malignancies signifies the need for multiple treatment options. Several available reviews have emphasized the potential role of various botanical extracts and naturally occurring compounds as anti-skin-cancer agents. Few studies relate to the role of chemoprevention and therapeutic activity of essential oils (EOs) and EO components. The present review summarizes an overview of chemopreventive, anti-melanoma and anti-nonmelanoma activities of EOs from various plants and EO components in in vitro and in vivo models with special emphasis on skin cancer. Also, the mechanisms by which EOs and EO components exert their effects to induce cell death are presented.

Induction of apoptosis by essential oil from P. missionis in skin epidermoid cancer cells.

BACKGROUND: The genus Pamburus (Rutaceae) comprises the only species, Pamburus missionis (Wight) Swingle. Pamburus missionis is traditionally used in the treatment of swellings, chronic rheumatism, paralysis and puerperal diseases. PURPOSE: The present study investigates the cancer chemotherapeutic potential of essential oil (EO) from P. missionis. METHODS: EO was isolated by steam distillation and chemical composition was determined by GC-MS. Cell viability was used to detect cytotoxic activity. Mechanism of cell death was studied using Annexin V-FITC/PI binding, cell cycle analysis, measurement of MMP and ROS generation by flow cytometry. Expression of apoptosis related proteins was investigated by western blot. RESULTS: GC-MS analysis of the essential oil revealed the presence of 51 components. The major components were ß-Caryophyllene, 4(14),11-Eudesmadiene, Aromadendrene oxide-(2) and Phytol. EO inhibited the growth and colony formation ability of A431 and HaCaT cells. EO treatment induced nuclear condensation and loss of membrane integrity, DNA fragmentation, increase in sub-G1 DNA content and increase in intracellular ROS level. Inhibition of intracellular ROS by ascorbic acid and N-acetyl cysteine treatment blocked EO induced apoptosis, revealing that apoptotic activity was by ROS accumulation. EO induced apoptosis was found to be due to the loss of mitochondrial membrane potential (ΔΨm), increase in Bax/Bcl-2 ratio, release of cytochrome c and activation of caspases (cleaved form of caspase-3, caspase-8, caspase-9) and by PARP cleavage. CONCLUSION: The present study revealed cancer chemotherapeutic potential of EO from P. missionis. EO induces cell death through intrinsic (mitochondrial) and extrinsic apoptotic pathway in A431 and HaCaT cells. These results suggest that EO could be used as a potential therapeutic agent for the treatment of skin epidermoid cancer.

Synergistic interaction of ß-caryophyllene with aromadendrene oxide 2 and phytol induces apoptosis on skin epidermoid cancer cells.

BACKGROUND: Pamburus missionis (Wight) Swingle (Rutaceae) is traditionally used in the treatment of swellings, chronic rheumatism, paralysis and puerperal diseases. In a previous study the authors demonstrated apoptotic activity of Pamburus missionis essential oil (EO) on A431 and HaCaT cells. The major components of EO were ß-caryophyllene (25.40%), 4(14),11- eudesmadiene (7.17%), aromadendrene oxide 2 (14.01%) (AO-(2) and phytol (6.88%). PURPOSE OF STUDY: To investigate the role as well as the interactions among EO components inducing apoptosis in A431 and HaCaT cells. METHODS: Isobolographic analysis and combination index methods were used to detect the type of interactions among the essential oil (EO) components. Cell viability was used to detect cytotoxic activity. Mechanism of cell death was studied using Annexin V-FITC/PI binding assay, cell cycle analysis, measurement of MMP and ROS generation by flow cytometry. Expression of apoptosis associated proteins was investigated by western blot. RESULTS: Combination of P. missionis EO components: ß-caryophyllene/ aromadendrene oxide 2 (ß-C/AO-(2)), ß-caryophyllene/phytol (ß-C/P) and aromadendrene oxide 2 /phytol (AO-(2)/P) inhibited growth and colony formation ability of skin epidermoid A431 and precancerous HaCaT cells. Synergistic interaction was observed between ß-C/AO-(2) and ß-C/P combination while AO-(2)/P exhibited an additive effect. Combination of components induced chromatin condensation, phosphatidylserine externalisation, increase in sub-G1 DNA content, cell cycle arrest at G0/G1 phase and intracellular ROS accumulation. Inhibition of intracellular ROS by N-acetyl cysteine treatment blocked apoptosis induced by the combinations. The combinations induced apoptosis in A431 and HaCaT cells mediated by loss of mitochondrial membrane potential (ΔΨm), increase in Bax/Bcl-2 ratio, release of cytosolic cytochrome c and activation of caspases (cleaved form of caspase-3, caspase-8, caspase-9) and by PARP cleavage. CONCLUSION: The present study demonstrates interactions among ß-C, AO-(2) and P in the induction of apoptosis on A431 and HaCaT cells. These data suggest the combination of ß-caryophyllene with aromadendrene oxide 2 and phytol could be potential therapeutics for the treatment of skin epidermoid cancer and precancerous cells.

Expression Profiling of Differentially Regulated Genes in Fanconi Anemia.

Gene expression analysis mainly helps to study gene quantification methods by using various downstream detection approaches like imaging, amplification, probe hybridization, or sequencing. With respect to DNA, which is less static, mRNA levels vary over time, between cell types under divergent conditions. Gene expression analysis is principally focused on determination of mRNA levels transcribed from DNA. DNA microarrays are one of the robust and powerful tools to detect changes in multiple transcripts in larger cohorts in parallel. The basic principle of DNA microarray hybridization is complementary base pairing of single-stranded nucleic-acid sequences. On a microarray platform (also called a chip), known sequences called targets are attached at fixed locations (spots) to a solid surface such as glass using robotic spotting. Since a large number of samples (variables) are used in a typical hybridization experiment, which often leads to impreciseness for example, target mRNA transcribed from the same source should be identical every time. In such cases, developing an optimized protocol for microarray platform to study the expression profiling of differentially regulated genes is a challenging task. Thus genome-wide expression array analysis yields data about candidate genes that may be involved in disease acquisition progression, and helps in better understanding the pathophysiology of the disease. In this chapter we describe in detail the microarray technique, a well-accepted method for understanding the development and progression of Fanconi anemia (FA), a genetic disorder which is characterized by progressive bone marrow failure and a predisposition to cancer.

Aromadendrene oxide 2, induces apoptosis in skin epidermoid cancer cells through ROS mediated mitochondrial pathway.

AIMS: Aromadendrene oxide 2 (AO-(2)) is an oxygenated sesquiterpene naturally found as a chemical component of essential oils. In the present study anticancer activity of AO-(2) has been investigated on A431 human epidermoid cancer and precancerous HaCaT cells. MATERIAL AND METHODS: Cell viability was used to detect cytotoxic activity. Mechanism of cell death induced by AO-(2) treatments was studied using Annexin V-FITC/PI binding, cell cycle analysis, measurement of MMP and ROS generation by flow cytometry. Expression of apoptosis related proteins was investigated by western blot. KEY FINDINGS: AO-(2) inhibited the growth and colony formation ability of A431 and HaCaT cells in concentration dependent manner. It induced cell cycle arrest at G0/G1 phase and apoptosis through intracellular ROS accumulation. Inhibition of intracellular ROS by ascorbic acid and N-acetyl cysteine treatment completely blocked apoptotic effect. N-acetyl cysteine treatment significantly reversed G0/G1 arrest induced by AO-(2). AO-(2) treatment caused loss of mitochondrial membrane potential (ΔΨm), increase in Bax/Bcl-2 ratios, cytochrome c release, activation of caspases (cleaved caspase-3 and caspase-9) and PARP cleavage. AO-(2) also significantly inhibited the growth of multicellular tumor spheroids of A431 and HaCaT cells. SIGNIFICANCE: The results of the present study reveals that AO-(2) a chemical component of essential oils induces apoptosis in A431 and HaCaT cells.

Abrus agglutinin stimulates BMP-2-dependent differentiation through autophagic degradation of ß-catenin in colon cancer stem cells.

Eradicating cancer stem cells (CSCs) in colorectal cancer (CRC) through differentiation therapy is a promising approach for cancer treatment. Our retrospective tumor-specimen analysis elucidated alteration in the expression of bone morphogenetic protein 2 (BMP-2) and ß-catenin during the colon cancer progression, indicating that their possible intervention through "forced differentiation" in colon cancer remission. We reveal that Abrus agglutinin (AGG) induces the colon CSCs differentiation, and enhances sensitivity to the anticancer therapeutics. The low dose AGG (max. dose = 100 ng/mL) decreased the expression of stemness-associated molecules such as CD44 and ß-catenin in the HT-29 cell derived colonospheres. Further, AGG augmented colonosphere differentiation, as demonstrated by the enhanced CK20/CK7 expression ratio and induced alkaline phosphatase activity. Interestingly, the AGG-induced expression of BMP-2 and the AGG-induced differentiation were demonstrated to be critically dependent on BMP-2 in the colonospheres. Similarly, autophagy-induction by AGG was associated with colonosphere differentiation and the gene silencing of BMP-2 led to the reduced accumulation of LC3-II, suggesting that AGG-induced autophagy is dependent on BMP-2. Furthermore, hVps34 binds strongly to BMP-2, indicating a possible association of BMP-2 with the process of autophagy. Moreover, the reduction in the self-renewal capacity of the colonospheres was associated with AGG-augmented autophagic degradation of ß-catenin through an interaction with the autophagy adaptor protein p62. In the subcutaneous HT-29 xenograft model, AGG profoundly inhibited the growth of tumors through an increase in BMP-2 expression and LC3-II puncta, and a decrease in ß-catenin expression, confirming the antitumor potential of AGG through induction of differentiation in colorectal cancer.

Strategies for targeted drug delivery in treatment of colon cancer: current trends and future perspectives.

Despite advances in treatment modalities, colon cancer (CC) is the third most common cause of cancer-related death worldwide. Subsequent unfavorable effects owing to toxicity of conventional drugs are a challenging problem associated with chemotherapy. There is noticeable concern toward site-specific/targeted delivery of chemotherapeutic drugs specifically to the affected site of the colon in a predictable and reproducible manner. However, the biggest challenge in successful drug targeting for the colon is avoidance of drug absorption and/or degradation in the upper gastrointestinal tract before the drug reaches the colon. Nanoparticles endowed with targeting abilities offer a novel approach for site-specific delivery of chemotherapeutic agents. The present review focuses on recent approaches for colon-specific drug delivery (CDDS) and aims to unveil the emerging possibilities and advances in the treatment of CC with CDDS.

SNAP-Tag Technology: A Promising Tool for Ex Vivo Immunophenotyping.

BACKGROUND: SNAP-tag, a self-labeling protein tag, is commonly used for in vitro and in vivo analysis of bound target proteins. We report the first evidence that SNAP-tag could be used for ex vivo detection of enriched biological markers. METHODS: Proof of concept was established for target c-kit receptor, a pathological and diagnostic marker for a variety of cancers. SNAP-tag conjugates with stem-cell factor (SCF) fusion proteins were designed and their binding and specificity was validated in vitro using flow cytometry and immunostaining. RESULTS: Ex vivo diagnostic application of the fusion protein was demonstrated in comparison with anti-c-kit antibody for peripheral blood samples from leukemia patients and colorectal tissue specimens.

Impaired mitophagy in Fanconi anemia is dependent on mitochondrial fission.

Fanconi anemia (FA) is a rare genetic disorder associated with bone-marrow failure, genome instability and cancer predisposition. Recently, we and others have demonstrated dysfunctional mitochondria with morphological alterations in FA cells accompanied by high reactive oxygen species (ROS) levels. Mitochondrial morphology is regulated by continuous fusion and fission events and the misbalance between these two is often accompanied by autophagy. Here, we provide evidence of impaired autophagy in FA. We demonstrate that FA cells have increased number of autophagic (presumably mitophagic) events and accumulate dysfunctional mitochondria due to an impaired ability to degrade them. Moreover, mitochondrial fission accompanied by oxidative stress (OS) is a prerequisite condition for mitophagy in FA and blocking this pathway may release autophagic machinery to clear dysfunctional mitochondria.

A Patient-Inspired Ex Vivo Liver Tissue Engineering Approach with Autologous Mesenchymal Stem Cells and Hepatogenic Serum.

Design and development of ex vivo bioengineered liver tissue substitutes intended for subsequent in vivo implantation has been considered therapeutically relevant to treat many liver diseases that require whole-organ replacement on a long-term basis. The present study focus on patient-inspired ex vivo liver tissue engineering strategy to generate hepatocyte-scaffold composite by combining bone marrow mesenchymal stem cells (BMSCs) derived from cardiac failure patients with secondary hyperbilirubinemia as primers of hepatic differentiation and hepatocyte growth factor (HGF)-enriched sera from same individuals as hepatic inducer. A biodegradable and implantable electrospun fibrous mesh of poly-l-lactic acid (PLLA) and gelatin is used as supporting matrix (average fiber diameter = 285 ± 64 nm, porosity = 81 ± 4%, and average pore size = 1.65 ± 0.77 µm). The fibrous mesh supports adhesion, proliferation, and hepatic commitment of patient-derived BMSCs of adequate stemness using HGF-enriched sera generating metabolically competent hepatocyte-like cells, which is comparable to the hepatic induction with defined recombinant growth factor cocktail. The observed results confirm the combinatorial effects of nanofiber topography and biochemical cues in guiding hepatic specification of BMSCs. The fibrous mesh-hepatocyte construct developed in this study using natural growth factors and BMSCs of same individual is promising for future therapeutic applications in treating damaged livers.

Targeting c-kit receptor in neuroblastomas and colorectal cancers using stem cell factor (SCF)-based recombinant bacterial toxins.

Autocrine activation of c-kit (KIT receptor tyrosine kinase) has been postulated to be a potent oncogenic driver in small cell lung cancer, neuroblastoma (NB), and poorly differentiated colorectal carcinoma (CRC). Although targeted therapy involving tyrosine kinase inhibitors (TKIs) such as imatinib mesylate is highly effective for gastrointestinal stromal tumor carrying V560G c-kit mutation, it does not show much potential for targeting wild-type KIT (WT-KIT). Our study demonstrates the role of stem cell factor (SCF)-based toxin conjugates for targeting WT-KIT-overexpressing malignancies such as NBs and CRCs. We constructed SCF-based recombinant bacterial toxins by genetically fusing mutated form of natural ligand SCF to receptor binding deficient forms of Diphtheria toxin (DT) or Pseudomonas exotoxin A (ETA') and evaluated their efficacy in vitro. Efficient targeting was achieved in all receptor-positive neuroblastoma (IMR-32 and SHSY5Y) and colon cancer cell lines (COLO 320DM, HCT 116, and DLD-1) but not in receptor-negative breast carcinoma cell line (MCF-7) thereby proving specificity. While dose- and time-dependent cytotoxicity was observed in both neuroblastoma cell lines, COLO 320DM and HCT 116 cells, only an anti-proliferative effect was observed in DLD-1 cells. We prove that these novel targeting agents have promising potential as KIT receptor tyrosine kinase targeting system.