Supercharged coiled-coil protein with N-terminal decahistidine tag boosts siRNA complexation and delivery efficiency of a lipoproteoplex.

Short interfering RNA (siRNA) therapeutics have soared in popularity due to their highly selective and potent targeting of faulty genes, providing a non-palliative approach to address diseases. Despite their potential, effective transfection of siRNA into cells requires the assistance of an accompanying vector. Vectors constructed from non-viral materials, while offering safer and non-cytotoxic profiles, often grapple with lackluster loading and delivery efficiencies, necessitating substantial milligram quantities of expensive siRNA to confer the desired downstream effects. We detail the recombinant synthesis of a diverse series of coiled-coil supercharged protein (CSP) biomaterials systematically designed to investigate the impact of two arginine point mutations (Q39R and N61R) and decahistidine tags on liposomal siRNA delivery. The most efficacious variant, N8, exhibits a twofold increase in its affinity to siRNA and achieves a twofold enhancement in transfection activity with minimal cytotoxicity in vitro. Subsequent analysis unveils the destabilizing effect of the Q39R and N61R supercharging mutations and the incorporation of C-terminal decahistidine tags on α-helical secondary structure. Cross-correlational regression analyses reveal that the amount of helical character in these mutants is key in N8's enhanced siRNA complexation and downstream delivery efficiency.

Effect of H-Bond Donor Lipids on Phosphatidylinositol-3,4,5-Trisphosphate Ionization and Clustering.

The phosphoinositide, phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), is a key signaling lipid in the inner leaflet of the cell plasma membrane, regulating diverse signaling pathways including cell growth and migration. In this study we investigate the impact of the hydrogen-bond donor lipids phosphatidylethanolamine (PE) and phosphatidylinositol (PI) on the charge and phase behavior of PI(3,4,5)P3. PE and PI can interact with PI(3,4,5)P3 through hydrogen-bond formation, leading to altered ionization behavior and charge distribution within the PI(3,4,5)P3 headgroup. We quantify the altered PI(3,4,5)P3 ionization behavior using a multistate ionization model to obtain micro-pKa values for the ionization of each phosphate group. The presence of PE leads to a decrease in the pKa values for the initial deprotonation of PI(3,4,5)P3, which describes the removal of the first proton of the three protons remaining at the phosphomonoester groups at pH 4.0. The decrease in these micro-pKa values thus leads to a higher charge at low pH. Additionally, the charge distribution changes lead to increased charge on the 3- and 5-phosphates. In the presence of PI, the final deprotonation of PI(3,4,5)P3 is delayed, leading to a lower charge at high pH. This is due to a combination of hydrogen-bond formation between PI and PI(3,4,5)P3, and increased surface charge due to the addition of the negatively charged PI. The interaction between PI and PI(3,4,5)P3 leads to the formation of PI and PI(3,4,5)P3-enriched domains within the membrane. These domains may have a critical impact on PI(3,4,5)P3-signaling. We also reevaluate results for all phosphatidylinositol bisphosphates as well as for PI(4,5)P2 in complex lipid mixtures with the multistate ionization model.

In situ hybridization of superparamagnetic iron-biomolecule nanoparticles.

The increase in interest in the integration of organic-inorganic nanostructures in recent years has promoted the use of hybrid nanoparticles (HNPs) in medicine, energy conversion, and other applications. Conventional hybridization methods are, however, often long, complicated, and multistepped, and they involve biomolecules and discrete nanostructures as separate entities, all of which hinder the practical use of the resulting HNPs. Here, we present a novel, in situ approach to synthesizing size-specific HNPs using Fe-biomolecule complexes as the building blocks. We choose an anticancer peptide (p53p, MW 1.8 kDa) and an enzyme (GOx, MW 160 kDa) as model molecules to demonstrate the versatility of the method toward different types of molecules over a large size range. We show that electrostatic interaction for complex formation of metal hydroxide ion with the partially charged side of biomolecule in the solution is the key to hybridization of metal-biomolecule materials. Electrochemical deposition is then used to produce hybrid NPs from these complexes. These HNPs with controllable sizes ranging from 30 nm to 3.5 µm are found to exhibit superparamagnetic behavior, which is a big challenge for particles in this size regime. As an example of greatly improved properties and functionality of the new hybrid material, in vitro toxicity assessment of Fe-GOx HNPs shows no adverse effect, and the Fe-p53p HNPs are found to selectively bind to cancer cells. The superparamagnetic nature of these HNPs (superparamagnetic even above the size regime of 15-20 nm!), their biocompatibility, and the direct integration approach are fundamentally important to biomineralization and general synthesis strategy for bioinspired functional materials.

Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia.

The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C6-ceramide (C6) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C6-ceramide may be a promising therapeutic approach for a fatal leukemia.

Occlusal accommodation and mouthguards for prevention of orofacial trauma.

PURPOSE: The aim of this study was to investigate the effect of two types of occlusal accommodation on the arch separation in centric and eccentric arch positions and to assess the opposing tooth contacts in professionally made, thermoformed sports mouthguards. MATERIALS AND METHODS: Maxillary and mandibular alginate impressions, a wax interocclusal record of centric occlusion together with maxillary/condylar face-bow registrations, were recorded clinically for 10 undergraduate dental students who are sports activist volunteers of the School of Medicine and Dentistry, Queen's University Belfast. Two ethylene vinyl acetate thermoformed maxillary mouthguards were made for each player (N = 20) using a standardised procedure. Ten mouthguards served both as the control (i.e. the non-accommodated) group and also the accommodated, occlusally 'imprinted' group. The other 10 mouthguards served as the accommodated, occlusally 'ground' group. Casts were articulated, each non-accommodated and accommodated mouthguard was seated and the extent of the interocclusal opening was recorded in all three arch relationships. The number of mouthguard and mandibular tooth contacts were also recorded in each position. RESULTS: The increased vertical occlusal dimension that was found in the presence of non-accommodated mouthguards equated to the full-sheet thickness of the material that was used to form the mouthguards. Only mouthguards accommodated by grinding retained high levels of occlusal contact in all arch relationships that were tested. CONCLUSIONS: Within the limitations of this study, the modification of the occlusal surface made by flat grinding reduced the arch separation in eccentric movements and increased the opposing tooth contacts in custom-made mouthguards. This may contribute to increased comfort, compliance and the protective effect of these appliances thus resulting in a reduction of injuries to the teeth, arches and soft tissues.

Identification of Specific Anaerobic Bacteria in Endodontic Infections of Primary Teeth-A PCR Study.

INTRODUCTION: Invasion of microorganisms and their multiplication in root canals (RCs) results in endodontic infections of primary teeth. Acute and chronic inflammation may be present in the periapical area and are based on the amount and virulence of microorganisms, especially anaerobic bacteria present in the RC. To identify microorganisms very precisely in endodontic infections, polymerase chain reaction (PCR) is used. AIM: The aim of the present study is to identify the specific anaerobic bacteria like Porphyromonas gingivalis, Prevotella intermedia, and Actinomyces naeslundii in the RCs of primary teeth using real-time PCR. METHODOLOGY: Fifteen subjects aged 3-8 years who had endodontic infections in primary molars were selected. The cases who had been selected did not receive any endodontic treatment and antibiotics within 3 months, and children with systemic diseases were not included. SAMPLE COLLECTION: Samples were taken by placing absorbent paper points into the largest canals of maxillary and mandibular molars for 60 seconds and are then transferred to a sterile Eppendorf tube with tris-hydochloride EDTA (TE) buffer. The samples were stored at -80°C. All samples were subjected to PCR analysis. RESULT: The specific anaerobes detected in the samples were A. naeslundii (93.3%), Prevotella intermedia (53.3%), and Porphyromonas gingivalis (13.3%). CONCLUSION: The results suggested a high bacterial diversity in the RCs of infected primary teeth. HOW TO CITE THIS ARTICLE: Thimmegowda U, Thomas J, et al. Identification of Specific Anaerobic Bacteria in Endodontic Infections of Primary Teeth-A PCR Study. Int J Clin Pediatr Dent 2019;12(1):1-4.

Analysis of a rapid, simple, and inexpensive technique used to obtain platelet-rich plasma for use in clinical practice.

The use of platelet-rich plasma (PRP) has become more generally accepted, and implant dentists are using PRP more frequently to promote the healing of oral surgical and/or periodontal wounds. Critical elements of PRP are thought to be growth factors contained within the concentrated platelets. These growth factors are known to promote soft-tissue healing, angiogenesis and osteogenesis. We present a rapid, simple, and inexpensive methodology for preparing PRP using the Cliniseal centrifuge method. This study demonstrates that platelets are concentrated approximately 6-fold without altering platelet morphology. Further we demonstrate that key growth factors, platelet-derived growth factor BB (PDGF-BB), transforming growth factor B (TGF-B1), vasculature endothelial growth factor (VEGF), and epidermal growth factor (EGF) are present in comparable or higher concentrations than those reported with the use of other techniques. Prolonged bench set time (>3 hours) after centrifugation resulted in decreased concentration of TGF-B1 but not decreased concentration of PDGF-BB, VEGF, or EGF. This study confirms the molecular aspects of PRP obtained using this inexpensive and efficient methodology.

Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials.

The possible combination of specific physicochemical properties operating at unique sites of action within cells and tissues has led to considerable uncertainty surrounding nanomaterial toxic potential. We have investigated the importance of proteins adsorbed onto the surface of two distinct classes of nanomaterials (single-walled carbon nanotubes [SWCNTs]; 10-nm amorphous silica) in guiding nanomaterial uptake or toxicity in the RAW 264.7 macrophage-like model. Albumin was identified as the major fetal bovine or human serum/plasma protein adsorbed onto SWCNTs, while a distinct protein adsorption profile was observed when plasma from the Nagase analbuminemic rat was used. Damaged or structurally altered albumin is rapidly cleared from systemic circulation by scavenger receptors. We observed that SWCNTs inhibited the induction of cyclooxygenase-2 (Cox-2) by lipopolysaccharide (LPS; 1 ng/ml, 6 h) and this anti-inflammatory response was inhibited by fucoidan (scavenger receptor antagonist). Fucoidan also reduced the uptake of fluorescent SWCNTs (Alexa647). Precoating SWCNTs with a nonionic surfactant (Pluronic F127) inhibited albumin adsorption and anti-inflammatory properties. Albumin-coated SWCNTs reduced LPS-mediated Cox-2 induction under serum-free conditions. SWCNTs did not reduce binding of LPS(Alexa488) to RAW 264.7 cells. The profile of proteins adsorbed onto amorphous silica particles (50-1000 nm) was qualitatively different, relative to SWCNTs, and precoating amorphous silica with Pluronic F127 dramatically reduced the adsorption of serum proteins and toxicity. Collectively, these observations suggest an important role for adsorbed proteins in modulating the uptake and toxicity of SWCNTs and nano-sized amorphous silica.

Osteogenic differentiation of marrow stromal cells cultured on nanoporous alumina surfaces.

A major goal in orthopedic biomaterials research is to design implant surfaces, which will enhance osseointegration in vivo. Several microscale as well as nanoscale architectures have been shown to significantly affect the functionality of bone cells i.e., osteoblasts. In this work, nanoporous alumina surfaces fabricated by a two-step anodization process were used. The nanostructure of these surfaces can be controlled by varying the voltage used for anodization process. Marrow stromal cells were isolated from mice and seeded on nanoporous and amorphous (control) alumina surfaces. Cell adhesion, proliferation, and viability were investigated for up to 7 days of culture. Furthermore, the cell functionality was investigated by calcein staining. The cells were provided with differentiation media after 7 days of culture. The alkaline phosphatase (ALP) activity and matrix production were quantified using a colorimetric assay and X-ray photoelectron spectroscopy (XPS) for up to 3 weeks of culture (2 weeks after providing differentiation media). Further, scanning electron microscopy (SEM) was used to investigate osteoblast morphology on these nanoporous surfaces. Over the 3-week study, the nanoporous alumina surfaces demonstrated approximately 45% increase in cell adhesion, proliferation, and viability, 35% increase in ALP activity, and 50% increase in matrix production when compared with the control surfaces.

A comparison of the stress corrosion cracking susceptibility of commercially pure titanium grade 4 in Ringer's solution and in distilled water: a fracture mechanics approach.

From the results of laboratory investigations reported in the literature, it has been suggested that stress corrosion cracking (SCC) mechanisms may contribute to early failures in titanium alloys that have elevated oxygen concentrations. However, the susceptibility of titanium alloys to SCC in physiological environments remains unclear. In this study, a fracture mechanics approach was used to examine the SCC susceptibility of CP titanium grade 4 in Ringer's solution and distilled de-ionized (DI) water, at 37°C. The study duration was 26 weeks, simulating the non-union declaration of a plated fracture. Four wedge loads were used corresponding to 86-95% of the alloy's ligament yield load. The longest cracks were measured to be 0.18 mm and 0.10 mm in Ringer's solution and DI water, respectively. SEM analysis revealed no evidence of extensive fluting and quasi-cleavage fracture features which, in literature reports, were attributed to SCC. We thus postulate that the Ringer's solution accelerated the wedge-loaded crack growth without producing the critical stresses needed to change the fracture mechanism. Regression analysis of the crack length results led to a significant best-fit relationship between crack growth velocity (independent variable) and test electrolyte, initial wedge load, and time of immersion of specimen in electrolyte (dependent variables).

Near-surface oxidized sulfur modifications and self-assembly of thiol-modified aptamer on Au thin film substrates influenced by piranha treatment.

Self-assembly of thiol-modified oligonucleotides on Au films has great importance for biosensor applications. Prior to the self-assembly, a piranha treatment (PT) is commonly used to clean the Au surface. Here we report that near-surface oxidized sulfur modifications on Au thin films by PT for longer than 60 s have serious effects on the self-assembled monolayer (SAM) formation of thiol-modified single-stranded thrombin binding aptamer (s-TBA), and a PT time of 10-30 s is optimal for s-TBA SAM formation. These results have important implication to SAM formation of biomolecules, especially for the thiol-modified ones where a careful consideration of this key step could significantly enhance the SAM formation and biosensor performance.

Induction and regulation of differentiation in neural stem cells on ultra-nanocrystalline diamond films.

The interaction of ultra-nanocrystalline diamond (UNCD) with neural stem cells (NSCs) has been studied in order to evaluate its potential as a biomaterial. Hydrogen-terminated UNCD (H-UNCD) films were compared with standard grade polystyrene in terms of their impact on the differentiation of NSCs. When NSCs were cultured on these substrates in medium supplemented with low concentration of serum and without any differentiating factors, H-UNCD films spontaneously induced neuronal differentiation on NSCs. By direct suppression of mitogen-activated protein kinase/extracellular signaling-regulated kinase1/2 (MAPK/Erk1/2) signaling pathway in NSCs using U0126, known to inhibit the activation of Erk1/2, we demonstrated that the enhancement of Erk1/2 pathway is one of the effects of H-UNCD-induced NSCs differentiation. Moreover, functional-blocking antibody directed against integrin beta1 subunit inhibited neuronal differentiation on H-UNCD films. This result demonstrated the involvement of integrin beta1 in H-UNCD-mediated neuronal differentiation. Mechanistic studies revealed the cell adhesion to H-UNCD films associated with focal adhesion kinase (Fak) and initiated MAPK/Erk1/2 signaling. Our study demonstrated that H-UNCD films-mediated NSCs differentiation involves fibronectin-integrin beta1 and Fak-MAPK/Erk signaling pathways in the absence of differentiation factors. These observations raise the potential for the use of UNCD as a biomaterial for central nervous system transplantation and tissue engineering.

The effect of ultra-nanocrystalline diamond films on the proliferation and differentiation of neural stem cells.

The interaction of ultra-nanocrystalline diamond (UNCD) with neural stem cells (NSCs) has been studied along with its surface modification in order to improve its function as a biomaterial. Hydrogen- and oxygen-terminated UNCD films were compared with standard grade polystyrene in terms of their impact on the growth, expansion and differentiation of NSCs. When NSCs were cultured on these substrates in low serum and without any differentiating factors, hydrogen-terminated UNCD films spontaneously induced cell proliferation and neuronal differentiation. Oxygen-terminated UNCD films were also shown to further improve neural differentiation, with a preference to differentiate into oligodendrocytes. Hence, controlling the surface properties of UNCD could manipulate the differentiation of NSCs for different biomedical applications. These observations raise the potential for the use of UNCD as a biomaterial for central nervous system transplantation and tissue engineering.