Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity.

In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated to be neither too high nor too low. Here, we show that in Caenorhabditis elegans, meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4PPH-4.1 phosphatase and ATRATL-1 kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants and in the wild-type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4PPH-4.1, ATRATL-1, and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan.

Cellulose nanocrystal (CNC)-inorganic hybrid systems: synthesis, properties and applications.

Cellulose nanocrystal (CNC), a class of sustainable nanomaterial derived from forest and agro-biomass can serve as nature's storage for carbon dioxide. It has many attractive features, such as large specific surface area, high tensile strength and stiffness, abundance of surface hydroxyl groups, and they are also biocompatible, biodegradable and renewable. When dispersed in a polar solvent, they assemble to form multiphase or higher order structures yielding desirable optical and structural properties. They are being explored as templates for the design of a wide range of new functional nanomaterials. CNCs are excellent support for the loading of inorganic nanoparticles (e.g. Ag, Au, Pt, Pd etc.) yielding stable nano-hybrids in aqueous media. Additional surface functionalization of CNCs impart new and attractive physicochemical properties that are being exploited for application in sensors, catalysts, drug delivery vehicles, anti-microbial agents, scaffold for tissue engineering, biomarkers etc. This review provides an overview and future perspective on recent advances in the development on functional CNC-inorganic hybrids with potential applications in biomedical and chemical systems.

Use of CdS quantum dot-functionalized cellulose nanocrystal films for anti-counterfeiting applications.

Structural colors and photoluminescence have been widely used for anti-counterfeiting and security applications. We report for the first time the use of CdS quantum dot (QD)-functionalized cellulose nanocrystals (CNCs) as building blocks to fabricate nanothin films via layer-by-layer (LBL) self-assembly for anti-counterfeiting applications. Both negatively- and positively-charged CNC/QD nanohybrids with a high colloidal stability and a narrow particle size distribution were prepared. The controllable LBL coating process was characterized by scanning electron microscopy and ellipsometry. The rigid structure of CNCs leads to nanoporous structured films on poly(ethylene terephthalate) (PET) substrates with high transmittance (above 70%) over the entire range of visible light and also resulted in increased hydrophilicity (contact angles of ∼40 degrees). Nanothin films on PET substrates showed good flexibility and enhanced stability in both water and ethanol. The modified PET films with structural colors from thin-film interference and photoluminescence from QDs can be used in anti-counterfeiting applications.

Synthesis and characterization of pH-responsive and fluorescent poly (amidoamine) dendrimer-grafted cellulose nanocrystals.

Poly (amidoamine) (PAMAM) dendrimers have found promising applications in biomedicine and in the encapsulation of inorganic nanoparticles. G6 PAMAM dendrimer-grafted cellulose nanocrystals (CNC-PAMAM) were prepared via a simple carbodiimide-mediated amidation process and they displayed pH-responsive and fluorescent characteristics as confirmed by zeta potential, transmittance, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Stable aqueous dispersions of CNC-PAMAM were obtained at pH⩽4 and pH⩾10, driven by electrostatic repulsion from positive charge and negative charge respectively. However, large aggregates were formed at pH values from 5 to 9 due to electrostatic attraction. In addition, strong blue fluorescent emission was observed, and the fluorescent behaviour of CNC-PAMAM was influenced by the formation of aggregates. The pH-responsive and fluorescent properties of CNC-PAMAM may be suitable for their applications in pH-responsive nanodevices, fluorescent-based pH sensors, optical markers, and nanoreactors for the encapsulation of inorganic nanoparticles.

Synthesis and self-assembly of stimuli-responsive poly(2-(dimethylamino) ethyl methacrylate)-block-fullerene (PDMAEMA-b-C60) and the demicellization induced by free PDMAEMA chains.

Well-defined poly(2-(dimethylamino) ethyl methacrylate)-block-fullerene [60] ((PDMAEMA)-b-C(60)) with a galactose targeting moiety was prepared by atom-transfer radical polymerization (ATRP). This copolymer was designed for possible use as a targeted drug carrier. The chemical composition and the self-assembly behavior were characterized using different techniques, including GPC, NMR, UV, and DLS. The self-assembly of the galactose-functionalized PDMAEMA-b-C(60) structure in aqueous solutions was investigated using dynamic light scattering (DLS) under different pH conditions. At pH 3 and 10, the DLS results showed the presence of both polymeric micelles and unimers. However, a smaller R(h) was observed at pH 10 than at pH 3 because of electrostatic repulsion at low pH values. In addition, free PDMAEMA chains induced the demicellization of self-assembled nanostructures caused by the formation of a charge-transfer complex between PDMAEMA and C(60.) This phenomenon offers possible applications for free-polymer-triggered drug release.

Binding and release studies of a cationic drug from a star-shaped four-arm poly(ethylene oxide)-b-poly(methacrylic acid).

Star-shape polymers possess higher densities of terminal functional groups and three-dimensional tetrahedron structure that induce significantly different association and interactions with drug compared to linear structure of identical molecular weights. Four-arm poly(ethylene oxide)-b-poly(methacrylic acid) block copolymer was synthesized by atom transfer radical polymerization technique, and it self-assembled into core-shell micelles and extended unimers at low and high pH respectively. The negatively charged carboxylate groups on the polymer chains interacted with a cationic drug through electrostatic interaction forming polymer/drug complexes stabilized by biocompatible hydrophilic PEO segments. The hydrodynamic radius (R(h)) of the polymeric aggregates and polymer/drug complexes ranged from 46 to 84 nm and 32 to 55 nm at pH of 4.6 and 8.0 respectively, making them suitable for drug delivery applications. The thermodynamic parameters and interactions between polymer and drug were determined by isothermal titration calorimetric technique. The electrostatic force, hydrogen bonding and hydrophobic interactions controlled the characteristics of polymer/drug formation and complexes when the molar ratios of drug and polymer were varied. Drug selective electrode system was used to measure the dynamic release of imipramine hydrochloride (IPH) from multi-arm PEO-b-PMAA star polymer. The release exponent n was greater than 0.5 indicating a non-Fickian type diffusion behavior, where the release behavior was dominated by chain relaxation induced by ion exchange that was dependent on pH.

Association behavior of star-shaped pH-responsive block copolymer: four-arm poly(ethylene oxide)-b-poly(methacrylic acid) in aqueous medium.

A four arm pH-responsive poly(ethylene oxide)-b-poly(methacrylic acid) block copolymer was synthesized by atom transfer radical polymerization technique. The conformation transition over the course of neutralization was investigated using a combination of potentiometric and conductometric titrations, dynamic and static light scattering, and transmission electron microscopy. The multiarm block copolymer existed as an extended unimer at high pH due to the negatively charged carboxylate groups and hydrophilic poly(ethylene oxide) segments. The block copolymers self-assembled into core-shell micelles and large spherical aggregates that flocculated at low degree of neutralization (alpha). Such behavior is controlled by the fine balance of electrostatic, hydrophobic, and hydrogen bond interactions. The hydrodynamic radius (R(h)) of the aggregates was approximately 84 nm at alpha of 0.3, and it decreased to 63 and 46 nm at alpha approximately 0.2 and 0.1, respectively, as a result of the reduced electrostatic interaction between ionized carboxylate groups. The thermodynamic parameters obtained from isothermal titration calorimetric technique in different salt concentrations indicated that the energy to extract a proton from a charged polyion was reduced by the addition of salt, which favors the neutralization process.

Polyplex formation between four-arm poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate) and plasmid DNA in gene delivery.

Amphiphilic polyelectrolytes comprising cationic and uncharged hydrophilic segments condensed negatively charged DNA to form a core-shell structure stabilized by a layer of hydrophilic corona chains. At physiological pH, four-arm star-shaped poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate) (four-arm PEO-b-PDEAEMA) block copolymer possessed positively charged amine groups that interacted with negatively charged plasmid DNA to form polymer/DNA complexes. The mechanism and physicochemical properties of the complex formation were investigated at varying molar ratio of amine groups on polymer chains and phosphate group on plasmid DNA segments (N/P ratio). The capability of the star block copolymer to condense DNA was demonstrated through gel electrophoresis and ethidium bromide exclusion assay. In the absence of salt, the hydrodynamic radius of polyplexes was about 94 nm at low polymer/DNA ratio, and it decreased to about 34 nm at large N/P ratios, forming a compact spherical structure with a weighted average molecular weight of 4.39 +/- 0.22 x 10(6) g/mol. Approximately 15 polymeric chains were required to condense a plasmid DNA. The addition of monovalent salt to the polyplexes significantly altered the size of the complexes, which would have an impact on cell transfection. Because of the electrostatic interaction induced by the diffusion of small ions, the polyplex increased in size to about 53 nm with a less compact structure. In vitro cytotoxicty of polymer and polymer/pDNA complexes were evaluated, and the polyplexes exhibited low toxicity at low N/P ratios. At N/P ratio of 4.5, the four-arm PEO-b-PDEAEMA showed the highest level of transfection in Neuro-2A cells. These observations showed that the star-shaped multi-arm polymers offers interesting properties in self-association and condensation ability for plasmid DNA and can serve as a nonviral DNA delivery system.

Thermodynamics of micellization of beta-sheet forming poly(acrylic acid)-block-poly(l-valine) hybrids.

The phase behavior and thermodynamic of micellization of three hybrid poly(acrylic acid)- block-poly( l-valine), namely PAA 40-b-PLVAL 100, PAA 80-b-PLVAL 100, and PAA 80-b-PLVAL 80, were investigated. beta-sheet formation in these polymeric systems resulted in a dominant enthalpic micellization process that exhibited an upper critical solution temperature (UCST). Micelle dissociation at higher temperatures is attributed to the disruption of favorable hydrogen bonds in the micellar core. Separation of hydrogen bond contributions to the micellization thermodynamics through the addition of urea as an external denaturing agent, revealed a shift from a dominant enthalpic contribution of PLVAL segments at low degree of deprotonation (alpha), where significant beta-sheet is formed, to a balanced enthalpy and entropy contributions at high alpha. At high alpha, an enhanced "water cage" hydration of unimers was observed due to the formation of water-PLVAL hydrogen bonds. Hydrophobic forces played an indirect role in enhancing the compactness of the hydrophobic core, which enhanced the strength of hydrogen bonds in the beta-sheet structures.

Self-assembly of poly(ethylene oxide)-block-poly(acrylic acid) induced by CaCl2: mechanistic study.

The interaction between CaCl 2 and double hydrophilic block copolymer, poly(ethylene oxide) 45- block-poly(acrylic acid) 70, PEO 45- b-PAA 70, was investigated. At a stoichiometric ratio of Ca2+:COO (-) = 0.5, Ca2+ ions were bound to COO (-) groups on PAA segments via electrostatic interaction. Small particles of 4-8 nm in diameter were observed, suggesting the formation of coil-like polymeric globule induced by charge neutralization. At Ca2+:COO (-) >or= 2.5, monodispersed aggregates of average hydrodynamic diameter of 52.0 +/- 7.4 nm were produced. The ISE, ITC, surface tension and fluorescence spectroscopic data confirmed that the formation of these aggregates is not the result of interaction between excess Ca2+ ions and the polymer, but rather it is due to changes in the water activity that triggers the structural rearrangement of Ca2+/PEO 45- b-PAA 70 complex.

Poly(acrylic acid)-block-poly(L-valine): evaluation of beta-sheet formation and its stability using circular dichroism technique.

Secondary structure formation in four novel hybrid poly(acrylic acid)-b-poly(L-valine) (PAA-b-PLVAL) block copolymers, that is, PAA(40)-PLVAL(100), PAA(80)-PLVAL(100), PAA(80)-PLVAL(80), and PAA(80)-PLVAL(60), was investigated by circular dichroism. The formation of stable and well-defined beta-sheet structure in the PLVAL hydrophobic domains was observed for all the copolymers. At pH 5, PAA(80)-PLVAL(60) with the lowest PLVAL/PAA molar ratio possessed the lowest beta-sheet content of 12%, and it increased to 62% for PAA(40)-PLVAL(100) system. The beta-sheet formation in the block copolymers was controlled by both random PAA-PLVAL hydrogen bonds at low pH and electrostatic repulsive forces on the PAA segment at high pH; hence, the beta-sheet structure was most stable at intermediate pH. The length of PAA segments was critical in the beta-sheet solubilization and in providing sufficient shielding of the hydrophobic core from denaturing agents such as urea.

Correlating transfection barriers and biophysical properties of cationic polymethacrylates.

Transfection efficiencies of several polymeric gene carriers were compared and correlated quantitatively to the amounts of cellular accumulation of plasmid DNA and to the expression of mRNA by quantitative real-time polymerase chain reaction (real-time PCR). Three polycations polymers with similar chemical structure were used in this study: poly(dimethylamino)ethyl methacrylate (PDMA) homopolymer, PEO-b-PDMA copolymer, and PEO-b-poly(diethylamino)ethyl methacrylate (PEO-b-PDEA) copolymer. Despite their similar chemical structures, the transfection efficiencies were significantly different. PEO-b-PDEA copolymer was significantly less efficient as gene carrier as compared to both PDMA and PEO-b-PDMA. Correlations between cytotoxicity, cellular uptake of plasmid DNA, expression levels of transgene and protein, and the physical properties of the polymers were observed. With the PEO-b-PDEA studies, cytotoxicity was due primarily to the excess of polymers that did not participate in the DNA binding. In addition, the inability of the polymer/DNA polyplexes to interact with cell effectively was identified as a critical barrier for high efficiency of transfection. This study demonstrated that the use of quantitative real-time PCR in combination with physical characterization techniques could provide useful insights into the transfection barrier at different cellular levels.

Synthesis and self-assembly behavior of four-arm poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate) star block copolymer in salt solutions.

A well-defined block copolymer consisting of four-arm poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate) (four-arm PEO56-b-PDEAEMA74) was synthesized by atom transfer radical polymerization. The pH-responsive self-assembly behavior was examined by potentiometric titration, surface tensiometry, laser light scattering, and transmission electron microscopy over the course of dissociation by the addition of a base or acid. The apparent hydrodynamic radius (Rh) of the micelle increased from 21 to 56 nm when the degree of protonation of the amine groups was increased from 0 to 0.5 in 0.01 M NaCl solution. In higher concentration NaCl solution, the micelle shrank due to the electrostatic charge screening of the protonated DEAEMA groups. At low pH, the micelles dissociated into unimers.

Polymeric nanostructures for drug delivery applications based on Pluronic copolymer systems.

The first part of this article reviewed the applications of nanostructures derived from Pluronic block copolymers that have potentials in the field of biomedical sciences. Pluronic block copolymers are used not only in drug delivery systems but also in gene and cancer therapies. In the second part, the chemical modifications of Pluronic copolymers and their applications in biomedical science were reviewed. Chemical modifications of Pluronic copolymers not only improve the properties of the polymers but they also impart more attractive properties to the block copolymers. The common systems used to modify Pluronic copolymers are polyacrylic acids, polybases, and biodegradable polyesters. Pluronics were also modified at both ends with functional groups to improve the mechanical properties of hydrogels.

Self-assembly of well-defined mono and dual end-capped C(60) containing polyacrylic acids in aqueous solution.

Well-defined stimuli-responsive mono and dual fullerene (C(60)) end-capped poly(acrylic acid)s (PAA-C(60) and C(60)-PAA-C(60)) were synthesized by reacting C(60) with well-defined mono and dual azide end-functionalized poly(tert-butyl acrylate)s, followed by hydrolysis. The aggregation behaviors of these C(60) end-capped polymers in aqueous solution were examined using potentiometric and conductometric titrations and static and dynamic light scattering as well as transmission electron microscopy (TEM). Both PAA-C(60) and C(60)-PAA-C(60) show pH-responsive and water-soluble properties at high pH. Both polymers self-assemble to form large compound micelles (LCMs) in aqueous solutions. The LCMs of PAA-C(60) exist as "compact aggregates", whereas the LCMs of C(60)-PAA-C(60) possess a "core-shell" structure with a larger size and aggregation number. The micelles for both polymers swell upon neutralization, where the R(h) of PAA-C(60) micelles increases from approximately 44 to approximately 102 nm and the R(h) of C(60)-PAA-C(60) aggregates varies from approximately 89 to approximately 128 nm with increasing degree of neutralization. The lower swelling of the dual end-capped C(60)-PAA-C(60) system is related to its higher C(60) content, which enhances the interpolymer chain hydrophobic association that restrains the swelling of micellar aggregates.

Aggregation behavior and thermodynamics of binding between poly(ethylene oxide)-block-poly(2-(diethylamino)ethyl methacrylate) and plasmid DNA.

The aggregation behavior and the thermodynamics of binding between poly(ethylene oxide)-block-poly(2-(diethylamino)ethyl methacrylate) (PEO-b-PDEAEMA) block copolymers and plasmid DNA were examined. Binding between the polymer and DNA were confirmed by gel electrophoresis. The high affinity between the polymer and DNA was demonstrated through the ethidium bromide (EtBr) displacement assay, and the binding was found to be related to the stoichiometric balance between the amine group of the polymer and the DNA nucleotide molar ratio (N/P molar ratio). The light scattering and TEM results showed that, at low polymer concentration, the hydrodynamic radii (R(h)) of the polymer/DNA complexes was around 90 nm; however, at sufficiently high polymer concentration, the complexes condensed to around 35 nm induced by a structural rearrangement of the amphiphilic nature of the block copolymer. The isothermal titration calorimetric results showed that the binding between the polymer and DNA is driven by a large favorable enthalpy.

Effect of enzymatic degradation on the release kinetics of model drug from Pluronic F127/poly(lactic acid) nano-particles.

Poly(lactic acid) (PLA) was successfully grafted to both ends of Pluronic F127 block copolymer (PEO-PPO-PEO) to obtain amphiphilic PLA-F127-PLA block copolymers. The effect of enzymatic degradation on the release behaviors of hydrophobic model drug 9-(methylaminomethyl)anthracene (MAMA) from PLA-F127-PLA nano-particles with vesicular structure was studied by UV-Vis spectroscopy. It was observed that the release rate of MAMA from PLA-F127-PLA nano-particles with the enzymatic degradation varied with temperature due to the activity of the enzyme with temperature. However, the enzyme concentration has negligible effect on the release rates of MAMA.

Is symptomatology useful in distinguishing between carpal tunnel syndrome and cervical spondylosis?

Hand paraesthesia is a common symptom found in patients either with carpal tunnel syndrome or cervical spondylosis. To differentiate between the two conditions, it is important to identify additional diagnostic symptoms. Ninety-two patients with operated carpal tunnel syndrome and 138 patients with spinal surgery for cervical spondylosis were reviewed. After exclusion of cases co-morbid with both cervical spondylosis and carpal tunnel syndrome or other neurological disorders, 44 patients with carpal tunnel syndrome and 41 patients with cervical spondylosis were compared. There were significant differences in the symptomatology between the two groups. In carpal tunnel syndrome, 84% had nocturnal paraesthesia, 82% hand paraesthesia were aggravated by hand activity, and hand pain occurred in 64%. The incidences were only 10%, 7% and 10%, respectively in cervical spondylosis. Neck pain was present in 76% of cervical spondylosis but only in 14% of carpal tunnel syndrome, and lower limb symptoms were present in 44% of cervical spondylosis and only 9% in carpal tunnel syndrome.

Dissolution and swelling behaviors of random and cross-linked methacrylic acid-ethyl acrylate copolymers.

The neutralization behaviors of random and cross-linked methacrylic acid-ethyl acrylate (MAA-EA) copolymers were examined as a function of degree of neutralization (alpha) using potentiometric titration and laser light scattering techniques. The random MAA-EA copolymers exhibit a conformational transition from a compact latex particle to a swollen randomly coiled aggregate upon neutralization over a certain range of alpha. With further addition of NaOH, the swollen aggregates dissociate into several smaller clusters. This conformational change is controlled by the balance between electrostatic repulsion within ionized MAA groups and hydrophobic attraction of EA. The cross-linked MAA-EA copolymers do not undergo a drastic conformational change during neutralization. The polymer latex particles swell slightly upon neutralization, and the extent of chain expansion is proportional to MAA molar composition and inversely proportional to cross-linked density. The electrostatic Gibbs energy (DeltaG(el)) obtained from the potentiometric titration data indicates that a higher MAA portion is favorable for the deprotonization of both the random and cross-linked MAA-EA copolymers, suggesting that the dissociation is mainly dominated by polymer structure instead of the electrostatic attraction between H(+) and -COO(-). Moreover, static and dynamic light scattering results confirmed that the cross-linked latex particle exists as monodispersed hard sphere in the collapsed state, whereas in its swollen state the latex particle possesses a core-shell structure.

Release kinetics of hydrophobic and hydrophilic model drugs from pluronic F127/poly(lactic acid) nanoparticles.

Poly(lactic acid) (PLA) was successfully grafted to both ends of Pluronic F127 block copolymers (PEO-PPO-PEO) to obtain amphiphilic PLA-F127-PLA block copolymers. The block composition and structure of PLA-F127-PLA block copolymers were studied by nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), differential scanning calorimetric (DSC) and wide angle X-ray diffraction (WXRD) techniques. Data from DSC and WXRD measurements indicated that Tg and Tm of PLA blocks in PLA-F127-PLA block polymers are lower than those of PLA homopolymer. Furthermore, Tm and crystallinity of PLA blocks decrease with decreasing PLA block length in PLA-F127-PLA block copolymers. The release behaviors of both hydrophobic 9-(methylaminomethyl)anthracene (MAMA) and hydrophilic procaine hydrochloride (PrHy) model drugs from PLA-F127-PLA nanoparticles with vesicular structure in PBS solution at 37 degrees C were examined by UV spectroscopy. The release kinetics of both MAMA and PrHy model drugs from PLA-F127-PLA nanoparticles exhibit burst release characteristics, which are believed to be controlled by concentration gradient resulting from the slow hydrolytic degradation of PLA segments.