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UO22+ was shown to form an interstrand crosslink between two different strands of a single DNA molecule. This crosslink hardly affected the hydrogen bonds between nucleobase pairs but destabilized the π–π stacking between the two nucleobases in the vicinity of UO22+-bound phosphate. Thereby, the fragility of the DNA backbone increased upon UO22+ binding. [ABSTRACT FROM AUTHOR]

UO22+ was shown to form an interstrand crosslink between two different strands of a single DNA molecule. This crosslink hardly affected the hydrogen bonds between nucleobase pairs but destabilized the π–π stacking between the two nucleobases in the vicinity of UO22+-bound phosphate. Thereby, the fragility of the DNA backbone increased upon UO22+ binding. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed at assessing the shortterm effect of corneal collagen crosslinking on higher order aberrations of cornea in patients with keratoconus using corneal topography. Patients and Methods: The study was a prospective cohort study that was conducted in a private specialized eye hospital on 40 eyes of 28 keratoconus patients. Each patient was fully assessed preoperatively including doing corneal topography using Pentacam® HR. Transepithelial accelerated CXL was done to all patients. Postoperative corneal topography was done at six months and data was retrieved and analyzed. Results: RMS HOA recorded a higher mean value preoperatively, with a high statistically significant difference (p=0.00). All elements of HOAs showed lower postoperative values except for trefoil 30°. The difference was statistically significant in comma 0°, comma 90°, spherical aberrations and fifth order comma 90° (p=0.026, p= 0.003, p=0.005, and p=0.001 respectively). Conclusion: Transepithelial corneal collagen cross-linking improves corneal higher order aberrations. The maximum effect of the procedure is on comma 0°, comma 90°, spherical aberrations and fifth order comma 90° elements. [ABSTRACT FROM AUTHOR]

With the goal of expanding the very limited toolkit of cross-linking agents available for nucleic acids and their protein complexes, we evaluated the merits of a wide range of bifunctional agents that may be capable of reacting with the functional groups characteristic of these types of biopolymers. The survey specifically focused on the ability of test reagents to produce desirable inter-molecular conjugates, which could reveal the identity of interacting components and the position of mutual contacts, while also considering a series of practical criteria for their utilization as viable nucleic acid probes. The survey employed models consisting of DNA, RNA, and corresponding protein complexes to mimic as close as possible typical applications. Denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometric (MS) analyses were implemented in concert to monitor the formation of the desired conjugates. In particular, the former was used as a rapid and inexpensive tool for the efficient evaluation of cross-linker activity under a broad range of experimental conditions. The latter was applied after preliminary rounds of reaction optimization to enable full-fledged product characterization and, more significantly, differentiation between mono-functional and intra- versus inter-molecular conjugates. This information provided the feedback necessary to further optimize reaction conditions and explain possible outcomes. Among the reagents tested in the study, platinum complexes and nitrogen mustards manifested the most favorable characteristics for practical cross-linking applications, whereas other compounds provided inferior yields, or produced rather unstable conjugates that did not survive the selected analytical conditions. The observed outcomes will help guide the selection of the most appropriate cross-linking reagent for a specific task, whereas the experimental conditions described here will provide an excellent starting point for approaching these types of applications. As a whole, the results of the survey clearly emphasize that finding a universal reagent, which may afford excellent performance with all types of nucleic acid substrates, will require extending the exploration beyond the traditional chemistries employed to modify the constitutive functional groups of these vital biopolymers. [ABSTRACT FROM AUTHOR]

Having previously highlighted the gelation of pectin with chlorhexidine (CX), pectinate microparticles were prepared here by vibrational prilling using CX, not only as an active ingredient encapsulated but also as a cross-linking agent. CX amount required for pectin gelation was smaller than usual dications (Ca 2+ , Zn 2+ ) used as cross-linking agent for pectin ionotropic gelation: CX seemed to bind more easily to pectin chains that could be explained by its large molecular size. Three batches of CX microparticles with different mean size were prepared. Whatever the droplet mean diameter, similar particle characteristics in terms of encapsulation efficiency, CX encapsulation yield and drug release were observed. The encapsulation efficiency was about 5.5%, the CX encapsulation yield was approximately 44% and the maximal amount of CX released after 6 h was about 7%. Finally, zinc diacetate was added to the formulation as a competitive pectin cross-linking agent in order to limit CX binding to pectin and to improve CX release. The influence of CX and Zn 2+ concentrations on the particles properties was studied by the means of a Doehlert design. Results showed the interest of such a mixture since the competition between both cations led to more or less structured and large microparticles, some of them having promoted the quantity of CX released. [ABSTRACT FROM AUTHOR]

In eukaryotes, cap-dependent translation initiation is a sophisticated process that requires numerous trans -acting factors, the eukaryotic Initiation Factors (eIFs). Their main function is to assist the ribosome for accurate AUG start codon recognition. The whole process requires a 5′-3′ scanning step and is therefore highly dynamic. Therefore translation requires a complex interplay between eIFs through assembly/release cycles. Here, we describe an original approach to assess the dynamic features of translation initiation. The principle is to use the m 7 Gcap located at the 5′ extremity of mRNAs as a tracker to monitor RNA and protein components that are in its vicinity. Cap-binding molecules are trapped by chemical and UV crosslinking. The combination of cap crosslinking methods in cell-free translation systems with the use of specific translation inhibitors for different steps such as edeine, GMP-PNP or cycloheximide allowed assessing the cap fate during eukaryotic translation. Here, we followed the position of the cap in the histone H4 mRNA and the cap binding proteins during H4 mRNA translation. [ABSTRACT FROM AUTHOR]

Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to investigate the dynamics of biomolecular events in real time. However, it requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules. Herein, we introduce a method for kinetic analysis based on control of fluorescence blinking (KACB), a general approach to investigate the dynamics of biomolecules by using a single fluorophore. By controlling the kinetics of the redox reaction the blinking kinetics or pattern can be controlled to be affected by microenvironmental changes around a fluorophore (rKACB), thereby enabling real-time single-molecule measurement of the structure-changing dynamics of nucleic acids. [ABSTRACT FROM AUTHOR]

We report novel capillary gel electrophoresis (CGE) with poly(ethylene glycol) (PEG)-based hydrogels for the effective separations of biomolecules containing sugars and DNAs based on a molecular size effect. The gel capillaries were prepared in a fused silica capillary modified with 3-(trimethoxysilyl)propylmethacrylate using a variety of the PEG-based hydrogels. After the fundamental evaluations in CGE regarding the separation based on the molecular size effect depending on the crosslinking density, the optimized capillary provided the efficient separation of glucose ladder (G1 to G20). In addition, another capillary showed the successful separation of DNA ladder in the range of 10–1100 base pair, which is superior to an authentic acrylamide-based gel capillary. For both glucose and DNA ladders, the separation ranges against the molecular size were simply controllable by alteration of the concentration and/or units of ethylene oxide in the PEG-based crosslinker. Finally, we demonstrated the separations of real samples, which included sugars carved out from monoclonal antibodies, mAbs, and then the efficient separations based on the molecular size effect were achieved. [ABSTRACT FROM AUTHOR]

Cross-linking of nucleic acids to proteins in combination with mass spectrometry permits the precise identification of interacting residues between nucleic acid--protein complexes. However, the mass spectrometric identification and characterization of cross-linked nucleic acid--protein heteroconjugates within a complex sample is challenging. Here we establish a novel enzymatic differential 16O/18O-labeling approach, which uniquely labels heteroconjugates. We have developed an automated data analysis workflow based on OpenMS for the identification of differentially isotopically labeled heteroconjugates against a complex background. We validated our method using synthetic model DNA oligonucleotide--peptide heteroconjugates, which were subjected to the labeling reaction and analyzed by high-resolution FTICR mass spectrometry. [ABSTRACT FROM AUTHOR]

Over the past decades, peptide nucleic acid/DNA (PNA:DNA) duplex stability has been improved via backbone modification, often achieved via introducing an amino acid side chain at the α- or γ-position in the PNA sequence. It was previously shown that interstrand cross-linking can further enhance the binding event. In this work, we combined both strategies to fine-tune PNA crosslinking towards single stranded DNA sequences using a furan oxidation-based crosslinking method; for this purpose, γ-L-lysine and γ-L-arginine furan-PNA monomers were synthesized and incorporated in PNA sequences via solid phase synthesis. It was shown that the L-lysine γ-modification had a beneficial effect on crosslink efficiency due to pre-organization of the PNA helix and a favorable electrostatic interaction between the positively-charged lysine and the negatively-charged DNA backbone. Moreover, the crosslink yield could be optimized by carefully choosing the type of furan PNA monomer. This work is the first to describe a selective and biocompatible furan crosslinking strategy for crosslinking of γ-modified PNA sequences towards single-stranded DNA. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the effects of ascorbic acid (vitamin C), the main antioxidant agent in the cornea on transepithelial corneal cross-linking (CXL) where the main mechanism is oxidation.Methods: Twenty eyes of 20 rabbits were divided into 3 groups: Group 1 (7 eyes) had transepithelial corneal CXL after being fed with normal diet; Group 2 (7 eyes) had corneal CXL after once-daily subcutaneous injections of 200 mg of ascorbic acid in addition to normal diet; and the control group (6 eyes) was fed with normal diet but did not have corneal CXL performed. Ascorbic acid levels were measured in aqueous humor and plasma, and biomechanical measurements were applied to the cornea.Results: There was a significant difference in ascorbic acid levels of plasma (P = 0.008) and aqueous humor (P = 0.006) between group 1 and 2. The Young's modulus values of group 1 and 2 were similar (P = 0.741) and were significantly higher than the control group (P = 0.02 and P = 0.01). The increase rate in Young's modulus values was 37.3% in group 1 and 43.9% in group 2 compared to control group. The ultimate strain values in group 1 and 2 were similar (P = 0.632) and were significantly higher than control group (P = 0.04, P = 0.03). The ultimate stress values in group 1 and 2 were similar (P = 0.836) and were significantly lower than control group (P = 0.001, P = 0.001).Conclusions: Systemic vitamin C does not appear to decrease effectiveness of transepithelial corneal CXL. Therefore, there is no reason to stop or reduce vitamin C supplementation before corneal CXL therapy. [ABSTRACT FROM AUTHOR]

Initiated chemical vapor deposition is used to integrate crosslinked poly(vinylpyrrolidone) (PVP) as a polymer electrolyte into the mesoporous TiO 2 photoanode of dye sensitized solar cells (DSSCs). By adjusting the fractional saturation ratio of ethylene glycol diacrylate (EGDA) crosslinker to vinylpyrrolidone (VP) monomer, the amount of crosslinker within the crosslinked polymer is controlled directly, and a minimum ratio of 0.2:1 (EGDA to VP) is found to form a robust insoluble crosslinked film. Fineman-Ross copolymer analysis indicates that both VP and EGDA prefer to polymerize with VP rather than with EGDA, likely due to reduced steric hindrance. DSSCs incorporating crosslinked PVP polymer electrolyte are found to have 51% higher power conversion efficiency (37% higher in open circuit voltage and 25% higher in fill factor) compared to DSSCs with liquid electrolytes. First-principles macroscopic mathematical modeling reveals that the polymer electrolyte shifts the conduction band of TiO 2 (vs. normal hydrogen electrode) negatively, increases shunt resistance, and affects interfacial processes like back-electron surface recombination in the cell. [ABSTRACT FROM AUTHOR]

Parkinson's disease has long been associated with redox imbalance and oxidative stress in dopaminergic neurons. The catecholaldehyde hypothesis proposes that 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate product of dopamine catabolism, is a central nexus in a network of pathways leading to disease-state neurodegeneration, owing to its toxicity and potent ability to oligomerize α-synuclein, the main component of protein aggregates in Lewy bodies. In this work we examine the connection between reactive oxygen species and DOPAL autoxidation. We show that superoxide propagates a chain reaction oxidation, and that this reaction is dramatically inhibited by superoxide dismutase. Moreover, superoxide dismutase prevents DOPAL from forming dicatechol pyrrole adducts with lysine and from covalently crosslinking α-synuclein. Given that superoxide is a major radical byproduct of impaired cellular respiration, our results provide a possible mechanistic link between mitochondrial dysfunction and synuclein aggregation in dopaminergic neurons. [ABSTRACT FROM AUTHOR]

Photographic methods of environmental monitoring have grown in popularity and now represent one of the main ways in which habitat and biodiversity are monitored for change through time. However, efficacy and efficiency of this technique compared with traditional approaches to environmental monitoring (direct count or observation) are lacking. This study compares the results and time-efficiency of manual versus photographic monitoring of floral abundance in low-growing flowering plants in a relatively open herbfield. Specifically, we compared 1) manual flower counting of individual plants for four species, followed by data entry in the laboratory, with 2) taking photographic images of each plant and quantifying flower counts in the laboratory. Photographic monitoring underestimated flower counts by an average of 7.5%. Manual counting was more time consuming in the field, but less time consuming in post-processing than photographic monitoring. Overall, photographic monitoring took almost twice as long as manual counting (81.5% longer in duration), which was attributed to the much longer post-processing associated with photographic monitoring. This suggests that perhaps the main benefit of photographic monitoring is a permanent record of the sampling frame rather than any cost savings or enhanced data accuracy, at least in the systems investigated in this study. [ABSTRACT FROM AUTHOR]

A novel series of bis(hydroxymethyl)indolizino[8,7- b ]indole hybrids composed of β-carboline (topoisomerase I/II inhibition) and bis(hydroxymethyl)pyrrole (DNA cross-linking) are synthesized for antitumor evaluation. Of tumor cell lines tested, small cell lung cancer (SCLC) cell lines are the most sensitive to the newly synthesized compounds. These hybrids induce cell cycle arrest at the G2/M phase, trigger tumor cell apoptotic death, and display diverse mechanisms of action involving topoisomerase II (Topo II) inhibition and induction of DNA cross-linking. Intriguingly, the substituent at N 11 (H or Me) plays a critical role in modulating Topo II inhibition and DNA cross-linking activities. N 11 -Me derivatives predispose to induce DNA crosslinks, whereas N 11 -H derivatives potently inhibit Topo II. Computational analysis implicates that N 11 -Me restrict the torsion angles of the two adjacent OH on pyrrole resulting in a favorable of DNA cross-linking. Among these hybrids, compound 17a with N 11 -H is more effective than cisplatin and etoposide, but as potent as irinotecan, against the growth of SCLC H526 cells in xenograft model. [ABSTRACT FROM AUTHOR]

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a family of sequence-selective DNA minor-groove binding agents that form a covalent aminal bond between their C11-position and the C2-NH2 groups of guanine bases. The first example of a PBD monomer, the natural product anthramycin, was discovered in the 1960s, and the best known PBD dimer, SJG-136 (also known as SG2000, NSC 694501 or BN2629), was synthesized in the 1990s and has recently completed Phase II clinical trials in patients with leukaemia and ovarian cancer. More recently, PBD dimer analogues are being attached to tumor-targeting antibodies to create antibody-drug conjugates (ADCs), a number of which are now in clinical trials, with many others in pre-clinical development. This Review maps the development from anthramycin to the first PBD dimers, and then to PBD-containing ADCs, and explores both structure-activity relationships (SARs) and the biology of PBDs, and the strategies for their use as payloads for ADCs. [ABSTRACT FROM AUTHOR]

Peptide nucleic acids (PNAs) are structural mimics of nucleic acids that form stable hybrids with DNA and RNA. Due to these characteristics, PNAs are widely used as biochemical tools, for example, in antisense/antigene therapy. In this study, we have synthesized PNAs incorporating 2-amino-6-vinylpurine (AVP) for the covalent targeting of single-stranded DNA and RNA, and evaluated their reactivities for these targets. PNA containing AVP at the N -terminal position showed a high reactivity to uracil in RNA and thymine in DNA at the complementary site with AVP. In addition, the crosslinking reactions to pre-miR122 with PNA containing AVP increased the inhibition effect for the Dicer processing of pre-miR122 in vitro. [ABSTRACT FROM AUTHOR]

Escherichia coli nitroreductase (NTR) may convert the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a bifunctional alkylating agent, which may lead to DNA crosslinks and the apoptosis of cancer cells. NTR/CB1954 has been demonstrated to be an effective gene therapy in cancer cells. The present study examined whether the NTR/CB1954 suicide gene system had cytotoxic effects on HeLa cells and may improve the radiosensitivity of HeLa cells to γ-rays. It was observed that the NTR/CB1954 suicide gene system exerted marked cytotoxic effects on HeLa cells. The combined therapeutic effects of NTR/CB1954 and γ-rays on HeLa cells demonstrated a synergistic effect. CB1954 at concentrations of 12.5 and 25 μmol/l increased the sensitization enhancement ratio of HeLa cells to 1.54 and 1.66, respectively. Therefore, when compared with monotherapy, the combined therapy of NTR/CB1954 and γ-rays may increase the apoptotic rate and enhance the radiosensitivity of HeLa cells. The combined therapy of γ-ray radiation and the NTR/CB1954 suicide gene system may be a novel and potent therapeutic method for the treatment of cervical carcinoma. [ABSTRACT FROM AUTHOR]

RNA functionalization is challenging due to the instability of RNA and the limited range of available enzymatic reactions. We developed a strategy based on solid phase synthesis and post-functionalization to introduce an electrophilic site at the 3′ end of tRNA analogues. The squarate diester used as an electrophile enabled sequential amidation and provided asymmetric squaramides with high selectivity. The squaramate-RNAs specifically reacted with the lysine of UDP-MurNAc-pentapeptide, a peptidoglycan precursor used by the aminoacyl-transferase FemXWv for synthesis of the bacterial cell wall. The peptidyl-RNA obtained with squaramate-RNA and unprotected UDP-MurNAc-pentapeptide efficiently inhibited FemXWv. The squaramate unit also promoted specific cross-linking of RNA to the catalytic Lys of FemXWv but not to related transferases recognizing different aminoacyl-tRNAs. Thus, squaramate-RNAs provide specificity for cross-linking with defined groups in complex biomolecules due to its unique reactivity. [ABSTRACT FROM AUTHOR]

Biodegradable starch nanocrystals prepared by an acid treatment process were modified through crosslinking modification using citric acid as reactant by a dry reaction method. The occurrence of crosslinking modification was evaluated by Fourier transform infrared spectroscopy and swelling degree. X-ray diffraction, wettability tests and contact angle measurements were used to characterize the modified starch nanocrystals. It was found that the crosslinked starch nanocrystals displayed a higher affinity for low polar solvents such as dichloromethane. The surface of starch nanocrystals became more roughness after crosslinking modification with citric acid and the size decreased as revealed by scanning electron microscopy and dynamic light scattering results. XRD analysis showed that the crystalline structure of starch nanocrystals was basically not changed after the crosslinking modification with shorter heating time. The resulting hydrophobic starch nanocrystals are versatile precursors to the development of nanocomposites. [ABSTRACT FROM AUTHOR]

Interstrand DNA crosslinks (ICLs) are the link between Watson-Crick strands of DNAs with the covalent bond and prevent separation of DNA strands. Since the ICL lesion affects both strands of the DNA, the ICL repair is not simple. So far, nucleotide excision repair (NER), structure-specific endonucleases, translesion DNA synthesis (TLS), homologous recombination (HR), and factors responsible for Fanconi anemia (FA) are identified to be involved in ICL repair. Since the presence of ICL lesions causes severe defects in transcription and DNA replication, mutations in these DNA repair pathways give rise to a various hereditary disorders. NER plays an important role for the ICL recognition and removal in quiescent cells, and defects of NER causes congential progeria syndrome, such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. On the other hand, the ICL repair in S phase requires more complicated orchestration of multiple factors, including structure-specific endonucleases, and TLS, and HR. Disturbed this ICL repair orchestration in S phase causes genome instability resulting a cancer prone disease, Fanconi anemia. So far more than 30 factors in ICL repair have already identified. Recently, a new factor, UHRF1, was discovered as a sensor of ICLs. In addition to this, numbers of nucleases that are involved in the first incision, also called unhooking, of ICL lesions have also been identified. Here we summarize the recent studies of ICL associated disorders and repair mechanism, with emphasis in the first incision of ICLs. [ABSTRACT FROM AUTHOR]

The synthesis of a shape-memory acrylamide-DNA hydrogel that includes two internal memories is introduced. The hydrogel is stabilized, at pH 7.0, by two different pH-responsive oligonucleotide crosslinking units. At pH 10.0, one of the T-A⋅T triplex DNA bridging units is dissociated, resulting in the dissociation of the hydrogel into a shapeless quasi-liquid state that includes the other oligonucleotide bridges as internal memory. Similarly, at pH 5.0, the second type of bridges is separated, through the formation of C-G⋅C+ triplex units to yield the shapeless quasi-liquid state that includes the other oligonucleotide bridges as internal memory. By reversible pH triggering of the hydrogel between the values 10.0⇔7.0⇔5.0, the two internal memories cycle the material across shaped hydrogel and shapeless quasi-liquid states. The two memories enable the pH-dictated formation of two different hydrogel structures. [ABSTRACT FROM AUTHOR]

In medical and pharmaceutical applications, chitosan is used as a component of hydrogels–macromolecular networks swollen in water. Chemical hydrogels are formed by covalent links between the crosslinking reagents and amino functionalities of chitosan. To date, the most commonly used chitosan crosslinkers are dialdehydes, such as glutaraldehyde (GA). We have developed novel GA like crosslinkers with additional functional groups–dialdehyde derivatives of uridine (oUrd) and nucleotides (oUMP and oAMP)–leading to chitosan-based biomaterials with new properties. The process of chitosan crosslinking was investigated in details and compared to crosslinking with GA. The rates of crosslinking with oUMP, oAMP, and GA were essentially the same, though much higher than in the case of oUrd. The remarkable difference in the crosslinking properties of nucleoside and nucleotide dialdehydes can be clearly attributed to the presence of the phosphate group in nucleotides that participates in the gelation process through ionic interactions with the amino groups of chitosan. Using NMR spectroscopy, we have not observed the formation of aldimine bonds. It can be concluded that the real number of crosslinks needed to cause gelation of chitosan chains may be less than 1%. [ABSTRACT FROM AUTHOR]

Purpose: Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer's disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. Methods: pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was transfected to insect cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE and western blot analysis. Functional analysis was evaluated by fluorometric assay and gel electrophoresis. Results: Recombinant bacmid was verified by amplification of a band near to 4500 bp. Expression analysis showed that the enzyme was expressed as a protein with a molecular weight near 80 kDa. Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional. The electrophoresis assay conformed that the expressed TG2 was the indeed capable of crosslinking in the presence of physiological concentration calcium ions. Conclusion: Human TG2 was expressed efficiently in the active biological form in the Bac- to-Bac baculovirus expression system. The expressed enzyme could be used for medical diagnostic, or studies which aim at finding novel inhibitors of the enzymes . To best of our knowledge, this is probably the first report of expression of full length human tissue transglutaminase (TG2) using the Bac-to-Bac expression system. [ABSTRACT FROM AUTHOR]

Although DNA interstrand crosslinking (ICL) agents such as mitomycin C, cisplatin and psoralen serve as potent anticancer drugs, these agents are known to have dose-limiting toxic effects on normal cells. Moreover, tumor resistance to these agents has been reported. Here, we show that trans-dichlorooxovanadium (IV) complex of pyrenyl terpyridine (VDC) is a novel photoinducible DNA crosslinking agent. By a combination of in vitro and ex vivo experiments including plasmid-based assays, we find that VDC forms monoadducts on the DNA and can be activated by UV-A and visible light to generate DNA interstrand crosslinks. VDC efficiently activates Fanconi anemia (FA) pathway of DNA interstrand crosslink repair. Strikingly, photoinduction of VDC induces prolonged activation of cell cycle checkpoint and a high degree of cell death in homologous recombination (HR)/ICL repair defective cells. Moreover, VDC specifically targets cells that express pathological RAD51C mutants. These data imply that VDC can be potentially used for cancer therapy and suggest that tumors arising in patients with gene mutations in FA and HR repair pathway can be specifically targeted by a photoactivatable VDC. [ABSTRACT FROM AUTHOR]

Initially developed as an elastomer with an excellent record of barrier and chemical resistance properties, poly(disulfide) has experienced a revival linked to the dynamic nature of the S-S covalent bond. A novel photobase-catalyzed oxidative polymerization of multifunctional thiols to poly(disulfide) network is reported. Based solely on air oxidation, the single-step process is triggered by the photodecarboxylation of a xanthone acetic acid liberating a strong bicyclic guanidine base. Starting with a 1 μm thick film based on trithiol poly(ethylene oxide) oligomer, the UV-mediated oxidation of thiols to disulfides occurs in a matter of minutes both selectively, i.e., without overoxidation, and quantitatively as assessed by a range of spectroscopic techniques. Thiolate formation and film thickness determine the reaction rates and yield. Spatial control of the photopolymerization serves to generate robust micropatterns, while the reductive cleavage of S-S bridges allows the recycling of 40% of the initial thiol groups. [ABSTRACT FROM AUTHOR]

Summary Microbial fatty acid biosynthetic enzymes are important targets for areas as diverse as antibiotic development to biofuel production. Elucidating the molecular basis of chain length control during fatty acid biosynthesis is crucial for the understanding of regulatory processes of this fundamental metabolic pathway. In Escherichia coli , the acyl carrier protein (AcpP) plays a central role by sequestering and shuttling the growing acyl chain between fatty acid biosynthetic enzymes. FabA, a β-hydroxyacyl-AcpP dehydratase, is an important enzyme in controlling fatty acid chain length and saturation levels. FabA-AcpP interactions are transient in nature and thus difficult to visualize. In this study, four mechanistic crosslinking probes mimicking varying acyl chain lengths were synthesized to systematically probe for modified chain length specificity of 14 FabA mutants. These studies provide evidence for the AcpP-interacting “positive patch,” FabA mutations that alter substrate specificity, and the roles that the FabA “gating residues” play in chain length control. [ABSTRACT FROM AUTHOR]

Poly(ethylene glycol) diglycidyl ether (PEG-DE) has been used as a crosslinker to construct artificial blood vessels of silk fibroin, endowing materials suitable compliance. As a biomaterial directly contacting with blood, it requires excellent anticoagulant property. In this article, hemolysis, platelet adhesion, platelet activity and plasma recalcification time of PEG-DE crosslinked silk fibroin were investigated using absorption spectrometry, scanning electron microscopy, methyl thiazolyl tetrazolium analysis and the time counting method. The hemolysis at ratios of PEG-DE crosslinked silk fibroin materials was < 0·5%, although it increased with increasing PEG-DE content. The platelet adhesion ratio and aggregation activity of PEG-DE crosslinked silk fibroin were higher than those of ethanol treated silk fibroin and increased with increasing PEG-DE content. However, the plasma recalcification time of PEG-DE crosslinked silk fibroin was longer than that of the ethanol treated silk fibroin and increased slightly with the increase of PEG-DE content. Based on these, PEG-DE used to crosslink silk fibroin showed different anticoagulant characteristics in different coagulation pathways. [ABSTRACT FROM AUTHOR]

Cells have evolved complex biochemical pathways for DNA interstrand crosslink (ICL) removal. Despite the chemotherapeutic importance of ICL repair, there have been few attempts to identify which mechanistic DNA repair inhibitor actually inhibits ICL repair. To identify such compounds, a new and robust ICL repair assay was developed using a novel plasmid that contains synthetic ICLs between a CMV promoter region that drives transcription and a luciferase reporter gene, and an SV40 origin of replication and the large T antigen ( LgT ) gene that enables self-replication in mammalian cells. In a screen against compounds that are classified as inhibitors of DNA repair or synthesis, the reporter generation was exquisitely sensitive to ribonucleotide reductase (RNR) inhibitors such as gemcitabine and clofarabine, but not to inhibitors of PARP, ATR, ATM, Chk1, and others. The effect was observed also by siRNA downregulation of RNR. Moreover, the reporter generation was also particularly sensitive to 3-AP, a non-nucleoside RNR inhibitor, but not significantly sensitive to DNA replication stressors, suggesting that the involvement of RNR in ICL repair is independent of incorporation of a nucleotide RNR inhibitor into DNA to induce replication stress. The reporter generation from a modified version of the plasmid that lacks the LgT-SV40ori motif was also adversely affected by RNR inhibitors, further indicating a role for RNR in ICL repair that is independent of DNA replication. Intriguingly, unhooking of cisplatin-ICL from nuclear DNA was significantly inhibited by low doses of gemcitabine, suggesting an unidentified functional role for RNR in the process of ICL unhooking. The assay approach could identify other molecules essential for ICLR in quantitative and flexible manner. [ABSTRACT FROM AUTHOR]

For the first time, mechanical properties of cross-linked protein products were quantitatively correlated with cross-linking degrees and molecular structures of polycarboxylic acids. Non-toxic polycarboxylic acids could effectively improve performance properties of multiple protein products via cross-linking. However, the reaction mechanism and relationship between reaction and performance properties remained unclear. In this study, the cross-linking reaction between gliadin, and polycarboxylic acids was verified. Natural logarithm relationship was found between the cross-linking degree and tensile strength of protein films. The polycarboxylic acids with more carboxyl groups and shorter backbones could more effectively increase the tensile strength of gliadin films. [ABSTRACT FROM AUTHOR]

Summary RNA polymerase binds tightly to DNA to recognize promoters with high specificity but then releases these contacts during the initial stage of transcription. We report a site-specific crosslinking approach to map the DNA path in bacterial transcription intermediates at amino acid and nucleotide resolution. After validating the approach by showing that the DNA path in open complexes (RP O ) is the same as in high-resolution X-ray structures, we define the path following substrate addition in “scrunched” complexes (RP ITC ). The DNA bulges that form within the transcription bubble in RP ITC are positioned differently on the two strands. Our data suggest that the non-template strand bulge is extruded into solvent in complexes containing a 5-mer RNA, whereas the template strand bulge remains within the template strand tunnel, exerting stress on interactions between the β flap, β′ clamp, and σ 3.2 . We propose that this stress contributes to σ 3.2 displacement from the RNA exit channel, facilitating promoter escape. [ABSTRACT FROM AUTHOR]

Summary Ionotropic glutamate receptors (iGluRs) are tetrameric ion channels that mediate excitatory neurotransmission. Recent structures of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and N -methyl-D-aspartate (NMDA) receptors permit a comparative analysis of whole-receptor dynamics for the first time. Despite substantial differences in the packing of their two-domain extracellular region, the two iGluRs share similar dynamics, elucidated by elastic network models. Motions accessible to either structure enable conformational interconversion, such as compression of the AMPA receptor toward the more tightly packed NMDA receptor conformation, which has been linked to allosteric regulation. Pivoting motions coupled to concerted rotations of the transmembrane ion channel are prominent between dimers of distal N-terminal domains in the loosely packed AMPA receptor. The occurrence and functional relevance of these motions is verified by cross-linking experiments designed to probe the computationally predicted distance changes. Together with the identification of hotspot residues acting as mediators of allosteric communication, our data provide a glimpse into the dynamic spectrum of iGluRs. [ABSTRACT FROM AUTHOR]

A novel approach is presented for the synthesis of thermoresponsive and pH-sensitive magnetic nanohydrogels by using the biodegradable starch-maleate as a crosslinker and magnetic nanoparticles stabilizer. Chemically modified starch-maleate produces highly stable Fe3O4magnetic nanopaticles (MNPs) aqueous dispersion. Then,N-isopropylacrylamide (NIPAAm) and itaconic acid (IA) are successfully polymerized from the vinyl double bonds of the starch-maleate-MNPs to prepare the thermo- and pH-responsive magnetic nanohydrogels. The obtained PNIPAAm-g-(starch-MNPs) and (PNIPAAm-co-IA)-g-(starch-MNPs) nanohydrogels are characterized by transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectrometer, X-ray diffraction, differential scanning calorimetry, thermal gravimetric analysis, and vibrating sample magnetometer. Also, pH- and temperature-responsive behaviors of the synthesized magnetic nanohydrogels are investigated and, finally, the cytotoxicity and drug loading are examined with mitoxantrone (MTX) as an anticancer drug model. The results confirm the low toxicity and enhanced anticancer effect of MTX-loaded magnetic nanohydrogels. [ABSTRACT FROM AUTHOR]

Conformational control of biomolecular activities can reveal functional insights and enable the engineering of novel activities. Here we show that conformational control through intramolecular cross-linking of a helicase monomer with undetectable unwinding activity converts it into a superhelicase that can unwind thousands of base pairs processively, even against a large opposing force. A natural partner that enhances the helicase activity is shown to achieve its stimulating role also by selectively stabilizing the active conformation. Our work provides insight into the regulation of nucleic acid unwinding activity and introduces a monomeric superhelicase without nuclease activities, which may be useful for biotechnological applications. [ABSTRACT FROM AUTHOR]

Several diagrams on various topics are presented including the peptide nucleic acid (PNA) crosslink yields, PNA molecular model, and the proton (HNMR) and carbon nuclear magnetic resonance spectra (CNMR).

Peptide nucleic acids (PNAs) are structural mimics of nucleic acids that form stable hybrids with DNA and RNA. In addition, PNAs can invade double-stranded DNA. Due to these characteristics, PNAs are widely used as biochemical tools, for example, in antisense/antigene therapy. Interstrand crosslink formation in nucleic acids is one of the strategies for preparing a stable duplex by covalent bond formation. In this study, we have synthesized PNAs incorporating 4-amino-6-oxo-2-vinylpyrimidine (AOVP) as a crosslinking agent and evaluated their reactivities for targeting DNA and RNA. [ABSTRACT FROM AUTHOR]

Chitosan film reinforced with nano-sized chitin whiskers and crosslinked using tannic acid was synthesized by the casting–vaporation method. The mechanical and physicochemical properties of several film samples (consisting of different ratio of chitin and tannic acid) were compared with neat chitosan. Tensile tests show that the addition of chitin improves the nanocomposite films mechanical properties up to 137% compared to neat chitosan, but this is slightly degraded when tannic acid is introduced. However, tannic acid and chitin whisker content greatly reduced moisture content by 294% and water solubility by 13%. Transmission electron microscopy (TEM) and Fourier-transform-infrared spectroscopy (FTIR) were used to investigate the morphology and molecular interaction of film. X-ray diffraction results indicated that the samples with chitin whiskers had a more rigid structure. The addition of tannic acid changed the structure into an anhydrous crystalline conformation when compared to neat chitosan film. [ABSTRACT FROM AUTHOR]

A kind of biocomposite was prepared by the intercalation of chitosan in bentonite and the crosslinking reaction of chitosan with glutaraldehyde, which was referred to as cross-linked chitosan/ bentonite (CCS/BT) composite. Adsorptive removal of methyl orange (MO) from aqueous solutions was investigated by batch method. The adsorption of MO onto CCS/BT composite was affected by the ratio of chitosan to BT and contact time. pH value had only a minor impact on MO adsorption in a wide pH range. Adsorption kinetics was mainly controlled by the pseudo-second-order kinetic model. The adsorption of MO onto CCS/BT composite followed the Langmuir isotherm model, and the maximum adsorption capacity of CCS/BT composite calculated by the Langmuir model was 224.8 mg/g. Experimental results indicated that this adsorbent had a potential for the removal of MO from aqueous solutions. [ABSTRACT FROM AUTHOR]

IgE-mediated, antigen-triggered activation of mast cells and basophils often results in bell-shaped dose–response curves for the release of various pro-inflammatory mediators. The degree of suppression of mediator release observed following supra-optimal stimulation varies widely for different allergens as well as for different experimental agents that cause crosslinking of high-affinity IgE receptors (FcɛRI) on these cells. While the reasons for these differences have not yet been resolved it has become increasingly apparent that supra-optimal stimulation in many cases causes a shift in the balance of stimulatory and inhibitory signal transduction mechanisms arising from FcɛRI triggering. In particular, the lipid phosphatase SHIP1 has been shown to be centrally involved in explaining the bell-shaped phenomena in both mast cells and basophils in different species and appears to play a fundamental role in limiting the IgE responsiveness of these allergic effector cells. Elucidating the nature of this inhibitory signaling pathway may provide crucial knowledge in order to optimize desensitization strategies in the treatment of allergic diseases. [ABSTRACT FROM AUTHOR]

Constructing safe and effective gene delivery carriers is becoming highly desirable for gene therapy. Herein, a series of supramolecular crosslinking system were prepared through host-guest binding of adamantyl-modified low molecular weight of polyethyleneimine with L-cystine-bridged bis(β-cyclodextrin)s and characterized by 1H NMR titration, electron microscopy, zeta potential, dynamic light-scattering, gel electrophoresis, flow cytometry and confocal fluorescence microscopy. The results showed that these nanometersized supramolecular crosslinking systems exhibited higher DNA transfection efficiencies and lower cytotoxicity than the commercial DNA carrier gold standard (25 kDa bPEI) for both normal cells and cancer cells, giving a very high DNA transfection efficiency up to 54% for 293T cells. Significantly, this type of supramolecular crosslinking system possesses a number of enzyme-responsive disulfide bonds, which can be cleaved by reductive enzyme to promote the DNA release but recovered by oxidative enzyme to make the carrier renewable. These results demonstrate that these supramolecular crosslinking systems can be used as promising gene carriers. [ABSTRACT FROM AUTHOR]

DNA interstrand cross-links (ICLs) are highly toxic lesions associated with cancer and degenerative diseases. ICLs can be repaired by the Fanconi anemia (FA) pathway and through FA-independent processes involving the FAN1 nuclease. In this work, FAN1-DNA crystal structures and biochemical data reveal that human FAN1 cleaves DNA successively at every third nucleotide. In vitro, this exonuclease mechanism allows FAN1 to excise an ICL from one strand through flanking incisions. DNA access requires a 5'-terminal phosphate anchor at a nick or a 1- or 2-nucleotide flap and is augmented by a 3' flap, suggesting that FAN1 action is coupled to DNA synthesis or recombination. FAN1's mechanism of ICL excision is well suited for processing other localized DNA adducts as well. [ABSTRACT FROM AUTHOR]

Summary Allergy is an immune response to complex mixtures of multiple allergens, yet current models use a single synthetic allergen. Multiple allergens were modeled using two well-defined tetravalent allergens, each specific for a distinct IgE, thus enabling a systematic approach to evaluate the effect of each allergen and percentage of allergen-specific IgE on mast cell degranulation. We found the overall degranulation response caused by two allergens is additive for low allergen concentrations or low percent specific IgE, does not change for moderate allergen concentrations with moderate to high percent specific IgE, and is reduced for high allergen concentrations with moderate to high percent specific IgE. These results provide further evidence that supraoptimal IgE crosslinking decreases the degranulation response and establishes the two-allergen model as a relevant experimental system to elucidate mast cell degranulation mechanisms. [ABSTRACT FROM AUTHOR]

All glassy epoxy polymers develop macroscopic properties at some degree of conversion. Our selected example, diglycidyl ether bisphenol-A epoxy pre-polymer, was cured with 3,3'-diaminodiphenylsulfone at stoichiometric equivalents using a series of cure profiles to produce distinct variation in the degree of epoxy conversion and result in varying network and network connectivity. Activation energy of epoxy-amine reaction in this selected system was ~61 kJ/mol. The calculated reaction energy barrier was found to vary with the extent of epoxy conversion and is attributed to multimechanistic reactions. Epoxy-amine conversion was tracked in situ via near infrared spectroscopic analysis. A single cure condition (90°C) was selected for experiments focused on preferential linear chain growth and minimal branching and/or crosslinking. The physical properties for matrix materials from samples prepared to varying degrees of conversion were characterized and tested for fracture toughness, tensile, flexural, compression properties, molecular weight between crosslinks/crosslink density, and glass transition temperature(s). An empirical equation was also designed to predict molecular weight between crosslinks based on chemical connectivity and extent of reaction. [ABSTRACT FROM AUTHOR]

Cholesteryl group-modified tilapia gelatins (Chol-T-Gltns) with various Chol contents from 3 to 69 mol % per amino group of Gltn were prepared for the assembly of liposomes and cells. Liposomes were physically crosslinked by anchoring Chol groups of Chol-T-Gltns into lipid membranes. The resulting liposome gels were enzymatically degraded by addition of collagenase. Liposome gels prepared using Chol-T-Gltn with high Chol content (69Chol-T-Gltn) showed slower enzymatic degradation when compared with gels prepared using Chol-T-Gltn with low Chol content (3Chol-T-Gltn). The hepatocyte cell line HepG2 showed good assembly properties and no cytotoxic effects after addition of 69Chol-T-Gltns. In addition, the number of HepG2 cells increased with concentration of 69Chol-T-Gltns. Therefore, Chol-T-Gltn, particularly, 69Chol-T-Gltn, can be used as an assembling material for liposomes and various cell types. The resulting organization can be applied to various biomedical fields, such as drug delivery systems, tissue engineering and regenerative medicine. [ABSTRACT FROM AUTHOR]

Highlights: [•] RIPiT-Seq is a novel RNP footprinting approach based on two sequential purifications. [•] Unveils transcriptome-wide binding sites of a single RBP or multi-subunit RNPs. [•] Distinguishes between footprints of RNPs with overlapping yet distinct composition. [•] Suitable for all RBPs irrespective of their RNA recognition mode. [ABSTRACT FROM AUTHOR]

Abstract: Associations between proteins and RNA–RNA duplexes are important in post-transcriptional regulation of gene expression. The CLASH (Cross-linking, Ligation and Sequencing of Hybrids) technique captures RNA–RNA interactions by physically joining two RNA molecules associated with a protein complex into a single chimeric RNA molecule. These events are relatively rare and considerable effort is needed to detect a small number of chimeric sequences amongst millions of non-chimeric cDNA reads resulting from a CLASH experiment. We present the “hyb” bioinformatics pipeline, which we developed to analyse high-throughput cDNA sequencing data from CLASH experiments. Although primarily designed for use with AGO CLASH data, hyb can also be used for the detection and annotation of chimeric reads in other high-throughput sequencing datasets. We examined the sensitivity and specificity of chimera detection in a test dataset using the BLAST, BLAST+, BLAT, pBLAT and Bowtie2 read alignment programs. We obtained the most reliable results in the shortest time using a combination of preprocessing with Flexbar and subsequent read-mapping using Bowtie2. The “hyb” software is distributed under the GNU GPL (General Public License) and can be downloaded from https://github.com/gkudla/hyb. [Copyright &y& Elsevier]

The bonding behavior between hydrophobically modified alkaline-treated gelatin (hm-AlGltn) films and porcine blood vessels was evaluated under wet conditions. Hexanoyl (Hx: C6), decanoyl (Dec: C10), and stearyl (Ste: C18) chlorides were introduced into the amino groups of AlGltn to obtain HxAlGltn, DecAlGltn, and SteAlGltn, respectively, with various modification percentages. The hm-AlGltn was fabricated into films and thermally crosslinked to obtain water-insoluble films (t-hm-AlGltn). The 42% modified t-HxAlGltn (t-42HxAlGltn) possessed higher wettability than the 38% modified t-DecAlGltn (t-38DecAlGltn) and the 44% modified t-SteAlGltn (t-44SteAlGltn) films, and the t-42HxAlGltn film showed a high bonding strength with the blood vessel compared with all the hm-AlGltn films. Histological observations indicated that t-42HxAlGltn and t-38DecAlGltn remained on the blood vessel even after the bonding strength measurements. From cell culture experiments, the t-42HxAlGltn films showed significant cell adhesion compared to other films. These findings indicate that the Hx group easily interpenetrated the surface of blood vessels and effectively enhanced the bonding strength between the films and the tissue. [ABSTRACT FROM AUTHOR]

Fanconi Anemia (FA) is a rare genetic disorder associated with a bone-marrow failure, cancer predisposition and hypersensitivity to DNA crosslinking agents. Majority of the 15 FA genes and encoded proteins characterized so far are integrated into DNA repair pathways, however, other important functions cannot be excluded. FA cells are sensitive to oxidants, and accumulation of oxidized proteins has been characterized for several FA subgroups. Clinical phenotypes of both FA and other closely related diseases suggest altered functions of mitochondria, organelles responsible for cellular energetic metabolism, and also serving as an important producer and the most susceptible target from reactive oxidative species (ROS). In this study, we have shown that elevated level of mitochondrial ROS in FA cells is in parallel with the decrease of mitochondrial membrane potential, the decrease of ATP production, impaired oxygen uptake and pathological changes in the morphology of mitochondria. This is accompanied by inactivation of enzymes that are essential for the energy production (F1F0ATPase and cytochrome C oxidase) and detoxification of ROS (superoxide dismutase, SOD1). In turn, overexpression of SOD1 could rescue oxygen consumption rate in FA-deficient cells. Importantly, the depletion of mitochondria improved survival rate of mitomycin C treated FA cells suggesting that hypersensitivity of FA cells to chemotherapeutic drugs could be in part due to the mitochondria-mediated oxidative stress. On the basis of our results, we propose that deficiency in FA genes lead to disabling mitochondrial ROS-scavenging machinery further affecting mitochondrial functions and suppressing cell respiration. [ABSTRACT FROM AUTHOR]

Liquid chromatography-electrospray ionization-infrared multiphoton dissociation (IRMPD) mass spectrometry was developed to investigate the distributions of intrastrand crosslinks formed between cisplatin and two oligodeoxynucleotides (ODNs), d(ATGGGTACCCAT) (G3-D) and its analog d(ATGGGTTCCCAT) (G3-H), which have been reported to adopt different secondary structures in solution. Based on the formation of site-specific fragment ions upon IRMPD, two isobaric crosslink products were differentiated for each ODN. The preferential formation of GG and GG crosslinks was determined as a function of reaction conditions, including incubation temperature and presence of metal ions. G3-D consistently exhibited a greater preference for formation of the GG crosslink compared with the G3-H ODN. The ratio of GG:GG crosslinks increased for both G3-D and G3-H at higher incubation temperatures or when metal salts were added. Comparison of the IRMPD fragmentation patterns of the unmodified ODNs and the intramolecular platinated crosslinks indicated that backbone cleavage was significantly suppressed near the crosslink. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]