Low-field thermal mixing in [1-13C] pyruvic acid for brute-force hyperpolarization

We detail the process of low-field thermal mixing (LFTM) between 1H and 13C nuclei in neat [1-13C] pyruvic acid at cryogenic temperatures (4–15 K). Using fast-field-cycling NMR, 1H nuclei in the molecule were polarized at modest high field (2 T) and then equilibrated with 13C nuclei by fast cycling (∼300–400 ms) to a low field (0–300 G) that activates thermal mixing. The 13C NMR spectrum was recorded after fast cycling back to 2 T. The 13C signal derives from 1H polarization via LFTM, in which the polarized (‘cold’) proton bath contacts the unpolarised (‘hot’) 13C bath at a field so low that Zeeman and dipolar interactions are similar-sized and fluctuations in the latter drive 1H–13C equilibration. By varying mixing time (tmix) and field (Bmix), we determined field-dependent rates of polarization transfer (1/τ) and decay (1/T1m) during mixing. This defines conditions for effective mixing, as utilized in ‘brute-force’ hyperpolarization of low-γ nuclei like 13C using Boltzmann polarization from nearby protons. For neat pyruvic acid, near-optimum mixing occurs for tmix ∼ 100–300 ms and Bmix ∼ 30–60 G. Three forms of frozen neat pyruvic acid were tested: two glassy samples, (one well-deoxygenated, the other O2-exposed) and one sample pre-treated by annealing (also well-deoxygenated). Both annealing and the presence of O2 are known to dramatically alter high-field longitudinal relaxation (T1) of 1H and 13C (up to 102–103-fold effects). Here, we found smaller, but still critical factors of ∼(2–5)× on both τ and T1m. Annealed, well-deoxygenated samples exhibit the longest time constants, e.g., τ ∼ 30–70 ms and T1m ∼ 1–20 s, each growing vs. Bmix. Mixing ‘turns off’ for Bmix > ∼100 G. That T1m ≫ τ is consistent with earlier success with polarization transfer from 1H to 13C by LFTM

Mediation - the Malaysian Dental Association experience

Objectives: To study the success and failures of the mediation process of complaints from 2004-2007 by the Complaints Bureau Steering Committee and the Public Complaints Bureau Committee of the Malaysian Dental Association (MDA). Methods: Retrospective analysis of complaints from the records at the MDA. Results: There were 41 cases during the three years, most involved surgical procedures (9), endodontics (9), miscellaneous (8), orthodontics (6), restorative problems (6) and prosthetics (2). Chinese patients made the most complaints (67) followed by Malay patients (23), foreigners (7) and Indians (3). 69 of the cases where against dentists working in the KIang Valley, 14 in Johore and the rest in various part of the country. 69 of the cases were successfully mediated, 10 unresolved and the 21 are in various stages of mediation. Conclusion: It is heartening to report that the MDA has successfully mediated in a sizeable number of the complaints which are increasing yearly

Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions

To clarify the biochemical behavior of 2′-deoxyribonucleoside 5′-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (Co) and adenine N-oxide (Ao), we examined their base recognition ability in DNA duplex formation using melting temperature (Tm) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the Tm values of modified DNA–DNA duplexes incorporating 2′-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo−) and Vent (exo−) suggested that Co and Ao selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo−) toward Ao on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator

Craniofacial Corrective Surgery in Syndromic Craniosynostosis

This surgical field has now progressed and becoming an established subspecialty involving various surgical disciplines worldwide. Various complex CMF syndromes reported in syndromic craniosynostosis include Crouzon, Apert and Pfeiffer syndromes. These syndromes carry specific functional discrepancies associated with the affected structural anomaly and may therefore have functional issues involving the brain, eye and airway among others. As corrective surgery is often indicated depending on the affected vital functions, other factors that need to be considered are patient’s age, comorbidities, urgency, available expertise and patient’s overall prognosis based on the degree of anomaly. As such, the corrective surgery can be categorized into; (1) intermediate which is performed at an early phase and aimed to improve or salvage important vital functions such as the brain, eye, airway or feeding which are important for the child’s development and, (2) definitive treatment aimed at permanently correct the functional discrepancies. Intermediate corrective surgery may include invasive procedures such as ventriculo-peritoneal (VP) shunts, tarsorrhaphy, adenotonsillectomy and tracheostomy whereas definitive corrective surgery may include surgical procedures such as monobloc, Le Fort III osteotomy, posterior cranial vault expansion and mandibular advancement. This chapter will elaborate on the indications, types, challenges in the management and the proposed prevention measures in corrective surgery for specifically for syndromic craniosynostosis patients

Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for Tm and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct Tm's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher Tm closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function

Multiparameter ranking of carbazoles for anti-trypanosome lead discovery

The criteria for the progression of hits in the discovery of leads for human African trypanosomiasis (HAT), a neglected disease caused by the microbial eukaryote Trypanosoma brucei, are not standardized. Hits are advanced upon meeting thresholds for drug-like molecules. Following those principles, pharmacokinetics (Cmax and AUC0–6h) and anti-trypanosome characteristics predicted the arrest of T. brucei proliferation in mice by three curaxins. Unexpectedly, while CBL0137 cured HAT in a mouse model, CBL0174 and CBL0187—structural analogs of CBL0137 with similar drug-like properties—failed to control T. brucei division. We here propose an alternative strategy that integrates physicochemical, metabolic, pharmacokinetic, pharmacodynamic, tissue distribution, and trypanocidality parameters into calculating a score for ranking compounds in hit-to-lead campaigns. Data from our studies of curaxins support the feasibility of this goal. Serum dropped the anti-trypanosome potency of CBL0174 and CBL0187 considerably. Delayed trypanocidal concentrations (DTC25 and DTC90) were used to study modes of curaxin actions in trypanosomes. Efficacy of CBL0137 in mice correlated with (i) a high AUC0–6h:DTC90 ratio, (ii) blocking of transferrin endocytosis, and (iii) the inhibition of protein synthesis. Hydroxylation of the carbazole prevented CBL0137 from inhibiting endocytosis of transferrin. The multiparametric score “Curaxin HAT lead efficacy (CHLE)” score was calculated using pharmacokinetic, physicochemical, metabolic, brain exposure, and pharmacodynamic data; CBL0137 was the highest scoring hit. Complementing these observations and predictive of performance of curaxins in mice, CBL0137, but not CBL0174 or CBL0187, was trypanocidal after the exposure of trypanosomes to AUC0–6h amounts of the hits for 6 hours in vitro. We discuss a role for CHLE scores in ranking curaxins for anti-HAT lead discovery. The principles used to develop CHLE scores may be used to calculate new ones for other scaffolds during the discovery of leads for HAT or other infectious diseases

Comparative study of the pharmacokinetics and biodistribution of various topical formulations of carbazole derivative CBL0100 in guinea pigs

An experimental drug CBL0100 is developed as an antifungal agent for topical application. In order to assess the effect of composition of the formulation on transdermal absorption, a comparative absorption study of its pharmacokinetics and biodistribution after a single topical application on the guinea pigs' skin. Two different formulations of CBL0100 (prototypes of final dosage forms for topical applications) - cream emulsion and polyethylene glycol vehicles with a content of active substance 1, 0,2 and 0,02% mass. in each of these formulations were used in the study. CBL0100 concentration in blood plasma and homogenates of organs and tissues of animals was analyzed by HPLC - MS/MS. During the preliminary blood-to-plasma partitioning in vitro experiment it was determined that CBL0100 is found mostly in the cell fraction of the blood, which, based upon the pharmacokinetic curves shape affects elimination from plasma. It was revealed that after a single topical application active compound enters the systemic circulation and intensively distributed into the spleen, liver, kidneys and lungs of guinea pigs. At the same time, its content in the muscular tissue and the heart is negligible; CBL0100 moderately penetrates into the brain. In general, it was shown that the emulsion composition provides improved bioavailability of CBL0100 compared to the polyethylene glycol composition

N-Acyl Homoserine Lactone Production by Klebsiella pneumoniae Isolated from Human Tongue Surface

Bacteria communicate by producing quorum sensing molecules called autoinducers, which include autoinducer-1, an N-hexanoyl homoserine lactone (AHL), and autoinducer-2. Bacteria present in the human oral cavity have been shown to produce autoinducer-2, but not AHL. Here, we report the isolation of two AHL-producing Klebsiella pneumoniae strains from the posterior dorsal surface of the tongue of a healthy individual. Spent culture supernatant extracts from K. pneumoniae activated the biosensors Agrobacterium tumefaciens NTL4(pZLR4) and Escherichia coli [pSB401], suggesting the presence of both long and short chain AHLs. High resolution mass spectrometry analyses of these extracts confirmed that both K. pneumoniae isolates produced N-octanoylhomoserine lactone and N-3-dodecanoyl-l-homoserine lactone. To the best of our knowledge, this is the first report of the isolation of K. pneumoniae from the posterior dorsal surface of the human tongue and the production of these AHLs by this bacterium

Microbial Contamination of Orthodontic Buccal Tubes from Manufacturers

This study aimed to test the sterility of new unused orthodontic buccal tubes received from manufacturers. Four different types of buccal tubes were used straight from the manufactures package without any additional sterilizing step. Of these buccal tubes tested, three genera of bacteria, implicated as opportunistic pathogens, namely Micrococcus luteus, Staphylococcus haemolyticus and Acinetobacter calcoaceticus were recovered from these buccal tubes. Our data showing microbial contamination on buccal tubes highlights the need of sterilization before clinical use. We also suggest that manufacturers should list the sterility state of orthodontic buccal tubes on their packaging or instructions stating the need for sterilization