Molecular machines open cell membranes.

Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

Induction of the antioxidant defense system using long-chain carotenoids extracted from extreme halophilic archaeon, Halovenus aranensis.

The field of microbial pigments is an emerging area in natural products science. Carotenoids form a major class of such pigments and are found to be diversely synthesized by microorganisms that reside in hypersaline ecosystems to provide resistance against oxidative stress. Human cells can benefit from compounds such as carotenoids as antioxidant agents through either their capability to quench free radicals or their effect on promoting the antioxidant defense pathway. In this study, the antioxidant effectiveness of carotenoid extract from an extremely halophilic archaeon Halovenus aranensis strain EB27T has been evaluated using different approaches. Finally, the ability of the extracted pigment to induce the antioxidant defense pathway of human primary skin fibroblast cells was studied. Hvn. aranensis carotenoid extract exhibited strong effectiveness such that at 2 µg/ml, the carotenoid extract fully neutralized the oxidative stress of hydrogen peroxide at its EC50 based on MTT assay. Results from real-time PCR of relevant genes, luciferase bioreporter of oxidative stress, and the western blot analysis further confirmed the antioxidant capability of the carotenoids. It was also shown the carotenoid extract had more antioxidant activity compared to ß-carotene the same concentration. Results suggest the carotenoid extract from this archaeon to have high potential for clinical and industrial applications.

Interrogating Amyloid Aggregates using Fluorescent Probes.

Amyloids are a broad class of proteins and peptides that can misfold and assemble into long unbranched fibrils with a cross-ß conformation. These misfolding and aggregation events are associated with the onset of a variety of human diseases, among them, Alzheimer's disease, Parkinson's disease, and Huntington disease. Our understanding of amyloids has been greatly supported by fluorescent molecular probes, such as thioflavin-T, which shows an increase in fluorescence emission upon binding to fibrillar aggregates. Since the first application of thioflavin-T in amyloid studies nearly 30 years ago, many probes have emerged exhibiting a variety of responses to amyloids, such as intensity changes, shifts in fluorescence maxima, and variations in lifetimes, among many others. These probes have shed light on a variety of topics including the kinetics of amyloid aggregation, the effectiveness of amyloid aggregation inhibitors, the elucidation of binding sites in amyloid structures, and the staining of amyloids aggregates in vitro, ex vivo, and in vivo. In this Review, we discuss the design, properties, and application of photoactive probes used to study amyloid aggregation, as well as the challenges faced by current probes and techniques, and the novel approaches that are emerging to address these challenges.

Monitoring the Formation of Amyloid Oligomers Using Photoluminescence Anisotropy.

The formation of oligomeric soluble aggregates is related to the toxicity of amyloid peptides and proteins. In this manuscript, we report the use of a ruthenium polypyridyl complex ([Ru(bpy)2(dpqp)]2+) to track the formation of amyloid oligomers at different times using photoluminescence anisotropy. This technique is sensitive to the rotational correlation time of the molecule under study, which is consequently related to the size of the molecule. [Ru(bpy)2(dpqp)]2+ presents anisotropy values of zero when free in solution (due to its rapid rotation and long lifetime) but larger values as the size and concentration of amyloid-ß (Aß) oligomers increase. Our assays show that Aß forms oligomers immediately after the assay is started, reaching a steady state at ∼48 h. SDS-PAGE, DLS, and TEM were used to confirm and characterize the formation of oligomers. Our experiments show that the rate of formation for Aß oligomers is temperature dependent, with faster rates as the temperature of the assay is increased. The probe was also effective in monitoring the formation of α-synuclein oligomers at different times.

Overexpression of miR-133 decrease primary endothelial cells proliferation and migration via FGFR1 targeting.

Angiogenesis is one of the essential hallmarks of cancer that is controlled by the balance between positive and negative regulators. FGFR1 signaling is crucial for the execution of bFGF-induced proliferation, migration, and tube formation of endothelial cells (ECs) and onset of angiogenesis on tumors. The purpose of this study is to identify whether or not miR-133 regulates FGFR1 expression and accordingly hypothesize if it plays a crucial role in modulating bFGF/FGFR1 activity in ECs and blocking tumor angiogenesis through targeting FGFR1. The influences of miR-133 overexpression on bFGF stimulated endothelial cells were assessed by cell growth curve, MTT assaying, tube formation, and migration assays. Forced expression of miR-133 caused significant reductions in bFGF-induced proliferation and migratory ability of ECs. MiR-133 Expression was negatively correlated with both mRNA and protein levels of FGFR1 in the transfected ECs isolated from peripheral blood. Moreover, overexpression of miR-133 drastically reduced the rate of cell division and disturbed capillary network formation of transfected ECs. These findings suggest that miR-133 plays an important function in bFGF-induced angiogenesis processes in ECs and provides a rationale for new therapeutic approaches to suppress tumor angiogenesis and cancer.

Unprecedented Dual Light-Switching Response of a Metal Dipyridophenazine Complex toward Amyloid-ß Aggregation.

Probes for monitoring protein aggregation with a variety of photophysical properties are of importance for the fundamental understanding of the aggregation process as well as for drug discovery. In this manuscript we report the photoluminescence response of the metal dipyridophenazine complex [Re(CO)3(dppz)(Py)](+) in the presence of aggregated Aß. [Re(CO)3(dppz)(Py)](+) shows an instantaneous increase in photoluminescence with fibrillar Aß (primary light-switching), and an unprecedented further increase in photoluminescence upon light irradiation at 362 nm (secondary light switching). The total increase in photoluminescence amounts to 105-fold, which we show can be used to monitor Aß aggregation in real time.

Conjugated linoleic acid improves meiotic spindle morphology and developmental competence of heat-stressed bovine oocyte.

This study was conducted to elucidate the effects of introducing conjugated linoleic acid (CLA) on meiotic spindle organization of heat stressed (HS) matured oocytes and the resulting blastocysts DNA methylation as well as the expression of the genes involved in DNA methylation (DNMT3a, DNMT3b and DNMT1). Immature bovine oocytes were cultured at 41 °C for the first 12 h and 38.5 °C for the second 12 h of maturation time in the presence of 0 and 50 µM of CLA (HS and HS + CLA groups, respectively). A group of oocytes cultured in medium with no CLA supplementation at normal temperature (38.5 °C for 24 h) was considered as negative control (C). Percentage of normal spindle, and cleavage and blastocyst rates were significantly decreased in the HS group compared to the C group (P < 0.05). The global DNA methylation and expression level of DNMT3a gene were increased in HS group compared to the C groups (P < 0.05), while the expression level of DNMT3b was decreased. The CLA supplementation improved the percentage of normal microtubules shape in MII oocytes as well as the developmental competence in the HS + CLA group compared to the HS group (P < 0.05). However, global DNA methylation and expression level of DNMT3a/b were not ameliorated by CLA supplementation (P > 0.05). Based on the obtained results, CLA proved to be capable of improving the oocyte developmental competence as well as decreased the aberrant spindle organization of heat-stressed oocytes and it would not cause epigenetic alteration in the obtained blastocysts.

Near-Infrared Light Activates Molecular Nanomachines to Drill into and Kill Cells.

Using two-photon excitation (2PE), molecular nanomachines (MNMs) are able to drill through cell membranes and kill the cells. This avoids the use of the more damaging ultraviolet light that has been used formerly to induce this nanomechanical cell-killing effect. Since 2PE is inherently confocal, enormous precision can be realized. The MNMs can be targeted to specific cell surfaces through peptide addends. Further, the efficacy was verified through a controlled opening of synthetic bilayer vesicles using the 2PE excitation of MNM that had been trapped within the vesicles.

Microfluidic system for synthesis of nanofibrous conductive hydrogel and muscle differentiation.

Microscale hydrogels can be synthesized within microfluidic systems and subsequently assembled to make tissues composed of units such as myofibers in muscle tissue. Importantly, the nanofibrous surface of hydrogels is essential for tissue engineering aims due to inducing beneficial cell-surface interactions. In this study, a new microfluidic platform, embedded with a hydrogel, was introduced that allowed for performing multiple non-parallel steps for the synthetic approaches. Satellite cells, isolated from skeletal tissues of 10-day Naval Medical Research Institute-murine were cultured on the prepared hydrogel within the microfluidic system. The normal proliferation of satellite cells occurred after the employment of continuous perfusion cell culture. Interestingly, the positive results of the immuno-staining assay along with the cellular bridge formation between hydrogel fragments confirmed the muscle differentiation of seeded satellite cells. Further on, COMSOl simulations anticipated that the thermodynamic conditions of the microfluidic system during hydrogel synthesis had to be kept steady while a shear stress value of 15 × 10-6 Pa was calculated, exhibiting a cell culture condition free of environmental stress.

Soft-Lithographic Patterning of Luminescent Carbon Nanodots Derived from Collagen Waste.

Luminescent carbon dots (Cdots) synthesized using inexpensive precursors have inspired tremendous research interest because of their superior properties and applicability in various fields. In this work, we report a simple, economical, green route for the synthesis of multifunctional fluorescent Cdots prepared from a natural, low-cost source: collagen extracted from animal skin wastes. The as-synthesized metal-free Cdots were found to be in the size range of ∼1.2-9 nm, emitting bright blue photoluminescence with a calculated Cdot yield of ∼63%. Importantly, the soft-lithographic method used was inexpensive and yielded a variety of Cdot patterns with different geometrical structures and significant cellular biocompatibility. This novel approach to Cdot production highlights innovative ways of transforming industrial biowastes into advanced multifunctional materials which offer exciting potential for applications in nanophotonics and nanobiotechnology using a simple and scalable technique.

Kaplan-Meier Meets Chemical Kinetics: Intrinsic Rate of SOD1 Amyloidogenesis Decreased by Subset of ALS Mutations and Cannot Fully Explain Age of Disease Onset.

Over 150 mutations in SOD1 (superoxide dismutase-1) cause amyotrophic lateral sclerosis (ALS), presumably by accelerating SOD1 amyloidogenesis. Like many nucleation processes, SOD1 fibrillization is stochastic (in vitro), which inhibits the determination of aggregation rates (and obscures whether rates correlate with patient phenotypes). Here, we diverged from classical chemical kinetics and used Kaplan-Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ∼103 replicate amyloid assays of wild-type (WT) SOD1 and nine ALS variants. The probability of apo-SOD1 fibrillization (expressed as a Hazard ratio) is increased by certain ALS-linked SOD1 mutations but is decreased or remains unchanged by other mutations. Despite this diversity, Hazard ratios of fibrillization correlated linearly with (and for three mutants, approximately equaled) Hazard ratios of patient survival (R2 = 0.67; Pearson's r = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (R2 = 0.09). Thus, Hazard ratios of fibrillization might explain rates of disease progression but not onset. Classical kinetic metrics of fibrillization, i.e., mean lag time and propagation rate, did not correlate as strongly with phenotype (and ALS mutations did not uniformly accelerate mean rate of nucleation or propagation). A strong correlation was found, however, between mean ThT fluorescence at lag time and patient survival (R2 = 0.93); oligomers of SOD1 with weaker fluorescence correlated with shorter survival. This study suggests that SOD1 mutations trigger ALS by altering a property of SOD1 or its oligomers other than the intrinsic rate of amyloid nucleation (e.g., oligomer stability; rates of intercellular propagation; affinity for membrane surfaces; and maturation rate).

Induction of the antioxidant defense system using long-chain carotenoids extracted from extreme halophilic archaeon, Halovenus aranensis / Inducción del sistema de defensa antioxidante utilizando carotenoides de cadena larga extraídos de arqueas halofílicas extremas, Halovenus aranensis

The field of microbial pigments is an emerging area in natural products science. Carotenoids form a major class of such pigments and are found to be diversely synthesized by microorganisms that reside in hypersaline ecosystems to provide resistance against oxidative stress. Human cells can benefit from compounds such as carotenoids as antioxidant agents through either their capability to quench free radicals or their effect on promoting the antioxidant defense pathway. In this study, the antioxidant effectiveness of carotenoid extract from an extremely halophilic archaeon Halovenus aranensis strain EB27T has been evaluated using different approaches. Finally, the ability of the extracted pigment to induce the antioxidant defense pathway of human primary skin fibroblast cells was studied. Hvn. aranensis carotenoid extract exhibited strong effectiveness such that at 2 µg/ml, the carotenoid extract fully neutralized the oxidative stress of hydrogen peroxide at its EC50 based on MTT assay. Results from real-time PCR of relevant genes, luciferase bioreporter of oxidative stress, and the western blot analysis further confirmed the antioxidant capability of the carotenoids. It was also shown the carotenoid extract had more antioxidant activity compared to β-carotene the same concentration. Results suggest the carotenoid extract from this archaeon to have high potential for clinical and industrial applications.(AU)