GSK3ß phosphorylates Six1 transcription factor and regulates its APC/CCdh1 mediated proteosomal degradation.

Sine oculis homeobox homolog 1 (Six1) is a developmentally important transcription factor that regulates cellular proliferation, apoptosis, and dissemination during embryogenesis. Six1 overexpression as reported in multiple cancers modulates expression of a repertoire of its target genes causing an increase in proliferation, metastasis and survival of cancer cells. Six1 exists as a cell cycle regulated nuclear phosphoprotein and its cellular turnover is regulated by APC/C (Anaphase promoting complex / Cyclosome) complex mediated proteolysis. However, the kinases that regulate Six1 proteolysis have not been identified and the mechanistic details that cause its overproduction in various cancers are lacking. Here, we report that Six1 is a physiological GSK3ß substrate. GSK3ß interacts with Six1 and phosphorylates it at Ser221 within the conserved consensus sequence in its carboxy terminus. Using pharmacological inhibition, siRNA mediated knockdown and protein overexpression of GSK3ß; we show that GSK3ß regulates Six1 protein stability. Pulse chase analysis of Six1 revealed that GSK3ß regulates its ubiquitin proteolysis such that Six1 phosphomimicking mutant (Six1S221E) for Ser221 site had dramatically increased half-life than its phosphodeficient (Six1S221A) and wild type variants. Furthermore, we demonstrate that GSK3ß rescues Six1 from APC dependent proteolysis by regulating its binding with APC/C co-activator protein Cdh1. Importantly, strong positive correlation exists between GSK3ß and Six1 protein levels throughout the cell cycle and in multiple cancers indicating that GSK3ß activation may in part contribute to Six1 overproduction in a subset of human cancers.

Optimizing the fabrication of electrospun nanofibers of prochlorperazine for enhanced dissolution and permeation properties.

Despite being an approved antiemetic for more than five decades, the clinical usefulness of prochlorperazine is limited by its low solubility and inconsistent absorption in the gastrointestinal tract, which presents challenges for nanotherapeutic interventions. Here, we report the preparation of a highly soluble and permeable nanofiber formulation of prochlorperazine using the Quality-by-Design approach. The final nanofiber formulation with drug entrapment of 88.02 ± 1.14 % was obtained at 20.0 kV, with a flow rate of 0.5 ml/h and tip-to-collector distance of 19.9 cm. Physio-mechanical properties, such as thickness (0.42 ± 0.02 mm), pH resistance (7.04 ± 0.08), folding endurance (54 ± 5), and tensile strength (0.244 ± 0.02 N.mm-2), were appropriate for packaging and application to oromucosal surfaces. The content uniformity (93.48-106.63 %) and weight variation (<1.8 mg) of the optimal nanofiber formulation were within the permissible limits prescribed for orodispersible films. Microscopical investigations confirm a randomly deposited and dense network of woven nanofibers with an average diameter of 363 ± 5.66 nm. The drug particles were embedded homogeneously on the fiber in the nanoform (4.27 ± 1.34 nm). The spectral analysis using TEM-EDS shows diffraction peaks of sulfur and chlorine, the elemental constituents of prochlorperazine. The drug was amorphized in the nanofiber formulation, as led by the decline of the crystallinity index from 87.25 % to 7.93 % due to electrostatic destabilization and flash evaporation of the solvent. The enthalpy of fusion values of the drug in the nanofiber mat decreased significantly to 23.6 J/g compared to its pristine form, which exhibits a value of 260.7 J/g. The nanofibers were biocompatible with oral mucosal cells, and there were no signs of mucosal irritation compared to 1 % sodium lauryl sulfate. The fiber mats rapidly disintegrated within <1 s and released ≈91.49 ± 2.1 % of the drug within 2 min, almost 2-fold compared to the commercial Stemetil MD® tablets. Similarly, the cumulative amount of the drug permeated across the unit area of the oromucosal membrane was remarkably high (31.28 ± 1.30 µg) compared to 10.17 ± 1.11 µg and 13.10 ± 1.79 µg from the cast film and drug suspension. Our results revealed these nanofiber formulations have the potential to be fast-dissolving oromucosal delivery systems, which can result in enhanced bioavailability with an early onset of action due to rapid disintegration, dissolution, and permeation.

Overview on immobilization of enzymes on synthetic polymeric nanofibers fabricated by electrospinning.

The arrangement and type of support has a significant impact on the efficiency of immobilized enzymes. 1-dimensional fibrous materials can be one of the most desirable supports for enzyme immobilization. This is due to their high surface area to volume ratio, internal porosity, ease of handling, and high mechanical stability, all of which allow a higher enzyme loading, release and finally lead to better catalytic efficiency. Fortunately, the enzymes can reside inside individual nanofibers to remain encapsulated and retain their three-dimensional structure. These properties can protect the enzyme's tolerance against harsh conditions such as pH variations and high temperature, and this can probably enhance the enzyme's stability. This review article will discuss the immobilization of enzymes on synthetic polymers, which are fabricated into nanofibers by electrospinning. This technique is rapidly gaining popularity as one of the most practical ways to fibricate polymer, metal oxide, and composite micro or nanofibers. As a result, there is interest in using nanofibers to immobilize enzymes. Furthermore, present research on electrospun nanofibers for enzyme immobilization is primarily limited to the lab scale and industrial scale is still challanging. The primary future research objectives of this paper is to investigate the use of electrospun nanofibers for enzyme immobilization, which includes increasing yield to transfer biological products into commercial applications.

SIX1 transcription factor: A review of cellular functions and regulatory dynamics.

Sine Oculis Homeobox 1 (SIX1) is a member of homeobox transcription factor family having pivotal roles in organismal development and differentiation. This protein functionally acts to regulate the expression of different proteins that are involved in organ development during embryogenesis and in disorders like cancer. Aberrant expression of this homeoprotein has therefore been reported in multiple pathological complexities like hearing impairment and renal anomalies during development and tumorigenesis in adult life. Most of the cellular effects mediated by it are mostly due to its role as a transcription factor. This review presents a concise narrative of its structure, interaction partners and cellular functions vis a vis its role in cancer. We thoroughly discuss the reported molecular mechanisms that govern its function in cellular milieu. Its post-translational regulation by phosphorylation and ubiquitination are also discussed with an emphasis on yet to be explored mechanistic insights regulating its molecular dynamics to fully comprehend its role in development and disease.

Local dual delivery therapeutic strategies: Using biomaterials for advanced bone tissue regeneration.

Bone development is a complex process involving a vast number of growth factors and chemical substances. These factors include transforming growth factor-beta, platelet-derived growth factor, insulin-like growth factor, and most importantly, the bone morphogenetic protein, which exhibits excellent therapeutic value in bone repair. However, the spatial-temporal relationship in the expression of these factors during bone formation makes the bone repair a more complicated process to address. Thus, using a single therapeutic agent to address bone formation does not seem to provide a clinically effective option. Conversely, a dual delivery approach facilitating the co-delivery of agents has proved to be a dynamic alternative since such a strategy can provide more efficient spatial-temporal action. Such delivery systems can smartly target more than one pathway or differentiation lineage and thus offer more efficient bone regeneration. This review discusses various dual delivery strategies reported in the literature employed to achieve improved bone regeneration. These include concurrent use of different therapeutic agents (including growth factors and drugs), enhancing bone formation and cell recruitment, and improving the efficiency of bone healing.

Recent trends in peripheral nervous regeneration using 3D biomaterials.

Mesenchymal stem cells (MSCs) owing their multipotency are known as progenitors for the regeneration of adult tissues including that of neuronal tissue. The repair and/or regeneration of traumatic nerves is still a challenging task for neurosurgeons. It is also a well-established fact that the microenvironment plays a primary role in determining the fate of stem cells to a specific lineage. In recent years, with the advent of nanotechnology and its positive influence on designing and fabrication of various 3D biomaterials have progressed to a greater extent. The production of 3D biomaterials such as nanofibers, conduits and hydrogels are providing a suitable environment for mimicking physiological niche of stem cells. These 3D biomaterials in combination with MSCs have been successfully analyzed for their potential in the regeneration of degenerative neurological disorders. This review primarily highlights the combinatorial effect of multipotent MSCs seeded on various 3D polymeric scaffolds in repair and regeneration of nervous tissue. The elaboration of MSCs from distinct sources reported so far in literature are summarized to understand their role in regeneration processes. Furthermore, we accentuate the application of 3D biomaterials especially the nanofibers, polymeric conduits, hydrogels infiltrated with MSCs harvested from distinct sources in the field of peripheral nerve regeneration studies.

Recent Trends in the Fabrication of Starch Nanofibers: Electrospinning and Non-electrospinning Routes and Their Applications in Biotechnology.

Electrospinning a versatile and the most preferred technique for the fabrication of nanofibers has revolutionized by opening unlimited avenues in biomedical fields. Presently, the simultaneous functionalization and/or post-modification of as-spun nanofibers with biomolecules has been explored, to serve the distinct goals in the aforementioned field. Starch is one of the most abundant biopolymers on the earth. Besides, being biocompatible and biodegradable in nature, it has unprecedented properties of gelatinization and retrogradation. Therefore, starch has been used in numerous ways for wide range of applications. Keeping these properties in consideration, the present article summarizes the recent expansion in the fabrication of the pristine/modified starch-based composite scaffolds by electrospinning along with their possible applications. Apart from electrospinning technique, this review will also provide the comprehensive information on various other techniques employed in the fabrication of the starch-based nanofibers. Furthermore, we conclude with the challenges to be overcome in the fabrication of nanofibers by the electrospinning technique and future prospects of starch-based fabricated scaffolds for exploration of its applications.

Impact of catechol-O-methyltransferase gene variants on methylation status of P16 and MGMT genes and their downregulation in colorectal cancer.

Globally, colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second most commonly diagnosed cancer in females, with 1.4 million new cases and almost 694 000 deaths estimated to have occurred in 2012. The development and progression of CRC is dictated by a series of alterations in diverse genes mostly proto-oncogenes and tumor suppressor genes. In this dreadful disease disturbances different from mutations called as epigenetic regulations are also taken into consideration and are thoroughly investigated. The present study was designed to analyze the promoter hypermethylation of CpG (cytosine, followed by guanine nucleotide) islands of cyclin-dependent kinase inhibitor 2A (P16) and O-methylguanine-DNA methyltransferase (MGMT) genes and its subsequent effect on the protein expression in CRC. The impact of the common functional polymorphism of the catechol-O-methyltransferase (COMT) gene, Val158Met, on promoter hypermethylation of P16 and MGMT genes in CRC was also investigated. The study included 200 CRC cases and equal numbers of normal samples. DNA was extracted using the kit method and methylation specific-PCR was performed for analysis of the promoter hypermethylation status. Total protein was isolated form all CRC cases and western blotting was performed for P16 and MGMT proteins. The COMT Val158Met polymorphism was analyzed by a PCR-restriction fragment length polymorphism assay. Epigenetic analysis showed that unlike other high-risk regions, the Kashmiri population has a different promoter hypermethylation profile of both P16 and MGMT genes, with frequent and significant promoter hypermethylation of both in CRC. The frequency of promoter hypermethylation of both genes was significantly higher in males and was insignificantly found to be higher in stage III/IV. The degree of P16 and MGMT promoter hypermethylation increased significantly with increasing severity of the lesion. We also found a significant correlation between P16 and MGMT promoter hypermethylation and loss of protein expression in CRC. A significant association was found between COMT polymorphism (homozygous variant) and P16 methylation status. Similar results were also found for MGMT hypermethylated cases.

Reconstructing nanofibers from natural polymers using surface functionalization approaches for applications in tissue engineering, drug delivery and biosensing devices.

Previously, the nanofibers were predominantly fabricated from synthetic polymers due to their excellent mechanical properties. Understanding the different complex processes in fabrication and various process parameters involved have not only allowed the use of natural polymers for fabricating nanofibers but also broadened the scope of applications. To date, many of the natural polymeric composites have been fabricated by different functionalization techniques to increase their applicability. Nanofibers fabricated from natural polymers have been chemically functionalized by a variety of molecules like drugs, enzymes, metal ions etc. by techniques such as plasma treatment, wet chemical method, graft polymerization and co-electrospinning of surface-active molecules. Furthermore, the nanofibers derived from natural polymers have been surface-coated on the synthetic polymers to induce extracellular matrix mirroring properties like cell adhesion, migration, proliferation and differentiation. In this review, we have not only investigated the various novel and facile functionalization approaches but potential properties and applications are discussed as well. The various surface chemistry modifications of the natural polymeric nanofibers and their potential applications in drug delivery, enzymology, catalysis, filtration, biosensing and tissue engineering are discussed. In addition, a brief presentation of an overview of challenges and future scope with the aim of making them a clinical success has been presented.

Scaffolds Fabricated from Natural Polymers/Composites by Electrospinning for Bone Tissue Regeneration.

Naturally bone is a hierarchical and highly integrative dynamic tissue that is continuously remodeled by osteoblasts and osteoclasts. Deformities in bone due to trauma and/or disease are highly prevalent and mostly need surgical intervention. However, the methods of surgical treatments are associated with donor site morbidity, infection and/or complete rejection. Bone tissue-engineering provides a platform for growth of new bone tissue by fabricating scaffolds along with cells, growth factors and other dynamic forces. The polymeric materials especially natural polymers in their nanofibrous forms have been developed and introduced for bone tissue regeneration. At the nanoscale, natural polymers possess tunable properties and can be surface functionalized or blended with other polymers to provide juncture for cell-seeding, proliferation, differentiation and further resulting in regenerated tissue formation. These scaffolds fabricated from natural polymers and additives by electrospinning are bio-inspired to mimic the natural extracellular matrix resembling the native collagen of bone. This chapter focuses on the fabrication techniques as state of art nanofibrous scaffolds from natural polymers/additives during the recent years by the process of electrospinning for use in bone tissue regeneration. Further on, this chapter highlights the development in the scaffold fabrication from natural polymers like silk fibroin, chitosan, collagen, gelatin, cellulose, starch and, zein. The importance of add-on materials like stem cells, hydroxyapatite, apatite-wollanstonite, growth factors, osteogenic cells, bone morphogenic proteins and osteogenic drugs have been discussed and illustrated by various examples for enhancing the formation of new bone tissue. Furthermore, this chapter explains how these natural polymers influence the several signaling pathways to regulate bone regeneration.

Prospects of Natural Polymeric Scaffolds in Peripheral Nerve Tissue-Regeneration.

Tissue-engineering is emerging field and can be considered as a novel therapeutic intervention in nerve tissue-regeneration. The various pitfalls associated with the use of autografts in nerve-regeneration after injuries have inspired researchers to explore the possibilities using various natural polymers. In this context, the present chapter summarizes the advances of the various types of natural polymeric scaffolds such as fibrous scaffolds, porous scaffolds, and hydrogels in nerve-regeneration and repair process. The functionalization of the scaffolds with wide-range of biomolecules and their biocompatibility analysis by employing various cells (e.g., mesenchymal, neural progenitor stem cells) along with the in vivo regeneration outcomes achieved upon implantation are discussed here. Besides, the various avenues that have been explored so far in nerve tissue-engineering, the use of the extracellular matrix in enhancing the functional polymeric scaffolds and their corresponding outcomes of regeneration are mentioned. We conclude with the present challenges and prospects of efficient exploration of natural polymeric scaffolds in the future to overcome the problems of nerve-regeneration associated with various nerve injuries and neurodegenerative disorders.

mTOR Signaling in Protein Translation Regulation: Implications in Cancer Genesis and Therapeutic Interventions.

mTOR is a central nutrient sensor that signals a cell to grow and proliferate. Through distinct protein complexes it regulates different levels of available cellular energy substrates required for cell growth. One of the important functions of the complex is to maintain available amino acid pool by regulating protein translation. Dysregulation of mTOR pathway leads to aberrant protein translation which manifests into various pathological states. Our review focuses on the role mTOR signaling plays in protein translation and its physiological role. It also throws some light on available data that show translation dysregulation as a cause of pathological complexities like cancer and the available drugs that target the pathway for cancer treatment.

Phosphorylation dynamics of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) is discordant with its potential to interact with eukaryotic initiation factor 4E (eIF4E).

Mammalian target of rapamycin (mTOR) controls cellular growth and proliferation by virtue of its ability to regulate protein translation. Eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) - a key mTOR substrate, binds and sequesters eIF4E to impede translation initiation that is supposedly overcome upon 4E-BP1 phosphorylation by mTOR. Ambiguity surrounding the precise identity of mTOR regulated sites in 4E-BP1 and their invariable resistance to mTOR inactivation raises concerns about phospho-regulated model proposed for 4E:4E-BP1 interaction. Our attempt to mimic dephosphorylation associated with rapamycin response by introducing phospho deficient mutants for sites implicated in regulating 4E:4E-BP1 interaction individually or globally highlighted no obvious difference in the quantum of their association with CAP bound 4E when compared with their phosphomimicked counterparts or the wild type 4E-BP1. TOS or RAIP motif deletion variants compromised for raptor binding and resultant phosphodeficiency did little to influence their association with CAP bound 4E. Interestingly ectopic expression of ribosomal protein S6 kinase 1 (S6K1) that restored 4E-BP1 sensitivity to rapamycin/Torin reflected by instant loss of 4E-BP1 phosphorylation, failed to bring about any obvious change in 4E:4E-BP1 stoichiometry. Our data clearly demonstrate a potential disconnect between rapamycin response of 4E-BP1 and its association with CAP bound 4E.

Rapamycin inhibition of baculovirus recombinant (BVr) ribosomal protein S6 kinase (S6K1) is mediated by an event other than phosphorylation.

BACKGROUND: Ribosomal protein S6 kinase 1(S6K1) is an evolutionary conserved kinase that is activated in response to growth factors and viral stimuli to influence cellular growth and proliferation. This downstream effector of target of rapamycin (TOR) signaling cascade is known to be directly activated by TOR- kinase mediated hydrophobic motif (HM) phosphorylation at Threonine 412 (T412). Selective loss of this phosphorylation by inactivation of TOR kinase or activation/recruitment of a phosphatase has accordingly been implicated in mediating inhibition by rapamycin. FINDINGS: We present evidence that baculovirus driven expression of S6K1 in insect cells (Sf9) fails to activate the enzyme and instead renders it modestly active representing 4-6 folds less activity than its fully active mammalian counterpart. Contrary to the contention that viral infection activates TOR signaling pathway, we report that BVr enzyme fails to exhibit putative TOR dependent phosphorylation at the HM and the resultant phosphorylation at the activation loop (AL) of the enzyme, correlating with the level of activity observed. Surprisingly, the BVr enzyme continued to exhibit sensitivity to rapamycin that remained unaffected by mutations compromised for TOR phosphorylation (T412A) or deletions compromised for TOR binding (ΔNH 2-46/ΔCT104). CONCLUSIONS: These data together with the ability of the BVr enzyme to resist inactivation by phosphatases indicate that inhibition by rapamycin is not mediated by any phosphorylation event in general and TOR dependent phosphorylation in particular.