High-level semi-synthetic production of the potent antimalarial artemisinin.

In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

The needs of persons with lupus and health care providers: a qualitative study aimed toward the development of the Lupus Interactive Navigator™.

OBJECTIVE: Systemic lupus erythematosus is an inflammatory autoimmune disease associated with high morbidity and unacceptable mortality. A major challenge for persons with lupus is coping with their illness and complex care. Our objective was to identify the informational and resource needs of persons with lupus, rheumatologists, and allied health professionals treating lupus. Our findings will be applied toward the development of an innovative web-based technology, the Lupus Interactive Navigator (LIN™), to facilitate and support engagement and self-management for persons with lupus. METHODS: Eight focus groups were conducted: four groups of persons with lupus (n=29), three groups of rheumatologists (n=20), and one group of allied health professionals (n=8). The groups were held in British Columbia, Ontario, and Quebec. All sessions were audio-recorded and transcribed verbatim. Qualitative analysis was performed using grounded theory. The transcripts were reviewed independently and coded by the moderator and co-moderator using 1) qualitative data analysis software developed by Provalis Research, Montreal, Canada, and 2) manual coding. RESULTS: Four main themes emerged: 1) specific information and resource needs; 2) barriers to engagement in health care; 3) facilitators of engagement in health care; and 4) tools identified as helpful for the self-management of lupus. CONCLUSION: These findings will help guide the scope of LIN™ with relevant information topics and specific tools that will be most helpful to the diverse needs of persons with lupus and their health care providers.

Effect of quinidine on the digoxin receptor in vitro.

To investigate the basis for a clinically important digitalis-quinidine interaction that is characterized by increases in serums digoxin concentrations when quinidine is administered to digoxin-treated patients, we have studied in vitro the interaction of quinidine with the digoxin receptor. Evidence has been obtained that quinidine is capable of decreasing the affinity for digoxin of cardiac glycoside receptor sites on purified Na,K-ATPase and on intact human erythrocyte membranes. As others have shown, quinidine is capable of inhibiting Na,K-ATPase activity, and evidence has been obtained in the current study that, while quinidine can reduce the affinity of the enzyme for digoxin, it is also capable of acting together with digoxin in inhibiting enzyme activity to a degree greater than the inhibitory effect of digoxin alone. The concentrations of digoxin and quinidine used in this study were considerably greater than their therapeutic serum concentrations. Nevertheless, these observations are consistent with the hypothesis that the increases in serum digoxin concentrations and the decreases in volumes of digoxin distribution observed clinically when quinidine is administered to digoxin-treated patients may reflect, at least in part, a decrease in the affinity of tissue receptors for digoxin. The possibility must also be considered that enhanced cardiac effects of digoxin may occur clinically as the result of an augmentation, by quinidine, of digoxin effects, which more than compensates for the modest reduction in digoxin binding.

Comparative study of torsional and bending properties for six models of nickel-titanium root canal instruments with different cross-sections.

This study investigated the influence of cross-section profile on the mechanical behaviors of six commercial nickel-titanium (NiTi) root canal instruments using the finite element method. The nonlinear mechanical characteristics of the NiTi alloy were taken into account. The six root canal instruments studied were ProTaper, Hero642, Mtwo, ProFile, Quantec, and NiTiflex. Mathematical models for these instruments were constructed and their performances were analyzed under equal torque conditions. The ProTaper and Hero642 models achieved the lowest stress levels that made them the most torque-resistant while the NiTiflex model was the poorest. The maximum stress value and the stress distribution in a model were found strongly influenced by the cross-section profile. Factors affecting the stress distribution include the cross-sectional inertia, depth of the flute, area of the inner core, radial land, and peripheral surface ground. As the area of the inner core of the cross-section increased, the model was more torque-resistant.

Characterization of a promoter region supporting transcription of a novel human beta-galactoside alpha-2,6-sialyltransferase transcript in HepG2 cells.

In humans, two transcripts encoding beta-galactoside alpha-2,6-sialytransferase (EC 2.4.99.1.) have previously been described. One of the transcripts is widely expressed, whereas the other is restricted to mature B-cells. In this study we demonstrate the existence of a third transcript in the hepatoma cell-line HepG2. The expression of this transcript is controlled by a promoter region which efficiently supports transcription in HepG2 cells, and which harbours putative binding sites for liver-enriched and acute phase inducible transcription factors.

Urinary excretion of reduced metabolites of digoxin.

The urinary excretion of the relatively cardioinactive reduced metabolites of digoxin, dihydrodigoxin and related compounds was measured by radioimmunoassay in 131 normal subjects during studies of the bioavailability of digoxin preparations. Digoxin reduction products (DRP) constitute more than 5 percent of the excretion of digoxin and its metabolites in one-third of the volunteers after the administration of single or multiple doses of digoxin. There was little or no output of DRP during the first 8 hours after a single dose, with maximal excretion usually occurring on the second day. Most subjects who excreted more than 5 percent DRP on one occasion did so with each subsequent exposure to digoxin. Six volunteers, however, in whom substantial amounts of DRP had previously been found, failed to excrete detectable quantities after subsequent doses. In two, this change occurred shortly after they took erythromycin. Urinary DRP were less after the intravenous administration compared to the oral administration of digoxin. After oral doses, DRP excretion tended to vary inversely with the bioavailability of the preparation. The findings are consistent with the hypothesis that DRP are formed as the result of the activity of a variable component of the intestinal flora. Prospective studies will be necessary to prove this hypothesis.

Immunocytologic analyses of 10 nm intermediate filaments in the nervous system of Myxicola.

Antibodies prepared in rabbits against Myxicola infundibulum neurofilaments have been employed to stain neurofilaments immunohistochemically in intact Myxicola infundibulum nervous tissue. Paraffin-embedded and frozen sections (5--6 mu) were examined at the light microscopic level with Sternberger's peroxidase-antiperoxidase method, and Vibratome (20--40 mu) sections were studied at the ultrastructural level with Nakane's conjugated peroxidase method. The neurofilament antibody stained only neurons and axons at the light microscopic level. The staining pattern at the electron microscopic level corresponded to the neurofilaments within axons and neurons. Glial cells, which surround the axons, contain large bundles of filaments that resemble astrocytic filaments in mammalian astrocytes. These filaments do not stain with the anti-neurofilament antibody. Neurons, neurofilaments, glial cells, glial filaments, and nonnervous tissue showed no peroxidase staining when specific antiserum absorbed with neurofilaments was used. These structures were also unstained when antiserum to the glial fibrillary acidic protein of mammalian central nervous system astrocytes was substituted for the neurofilament antiserum. Therefore, in Myxicola infundibulum, the antigenic determinants of the neurofilament protein, as recognized immunohistochemically by anti-neurofilament protein antibodies, are not shared with those of glial filaments.

Expression of glutamate uptake transporters after dibutyryl cyclic AMP differentiation and traumatic injury in cultured astrocytes.

Our findings indicate that differentiation of primary astrocytes by dibutyryl cyclic adenosine monophosphate (dBcAMP) and scratch injury together resulted in increased glutamate transporter gene expression. Confluent primary cultures were prepared from cerebral cortex of normal new born rat pups. The primary cultures were then divided into four groups each: control and scratch-injured, and dBcAMP-treated control and scratch-injured cultures. Total RNA was extracted at 0, 1, 2, 4, and 7 days after injury. Expression of the electrogenic glutamate transporters, GLAST, GLT-1, and EAAC-1, was quantitated by the reverse transcriptase-polymerase chain reaction method (RT-PCR) and slot blot hybridization followed by densitometric scanning. Triplicate cultures were analyzed for each time-point. Our studies indicate that all these astrocyte cultures expressed the two glial transporters, GLAST and GLT-1, while none of the cultures expressed the neuronal transporter, EAAC-1. The expression of the two transporters in the dBcAMP-treated primary cultures were markedly increased from the non-treated cultures. The dBcAMP-treated cultures had 2- to 4-times increase in levels of GLAST and GLT-1-mRNA expression both before and after scratch injury, as compared to untreated non-injured and injured primary cultures. All of the cultures expressed GLAST in greater proportion than GLT-1.

Three types of sodium channels in adult rat dorsal root ganglion neurons.

Several types of Na+ currents have previously been demonstrated in dorsal root ganglion (DRG) neurons isolated from neonatal rats, but their expression in adult neurons has not been studied. Na+ current properties in adult dorsal root ganglion (DRG) neurons of defined size class were investigated in isolated neurons maintained in primary culture using a combination of microelectrode current clamp, patch voltage clamp and immunocytochemical techniques. Intracellular current clamp recordings identified differing relative contributions of TTX-sensitive and -resistant inward currents to action potential waveforms in DRG neuronal populations of defined size. Patch voltage clamp recordings identified three distinct kinetic types of Na+ current differentially distributed among these size classes of DRG neurons. 'Small' DRG neurons co-express two types of Na+ current: (i) a rapidly-inactivating, TTX-sensitive 'fast' current and (ii) a slowly-activating and -inactivating, TTX-resistant 'slow' current. The TTX-sensitive Na+ current in these cells was almost completely inactivated at typical resting potentials. 'Large' cells expressed a single TTX-sensitive Na+ current identified as 'intermediate' by its inactivation rate constants. 'Medium'-sized neurons either co-expressed 'fast' and 'slow' current or expressed only 'intermediate' current. Na+ channel expression in these size classes was also measured by immunocytochemical techniques. An antibody against brain-type Na+ channels (Ab7493)10 labeled small and large neurons with similar intensity. These results demonstrate that three types of Na+ currents can be detected which correlate with electrogenic properties of physiologically and anatomically distinct populations of adult rat DRG neurons.

Functional differences between 4-aminopyridine and tetraethylammonium-sensitive potassium channels in myelinated axons.

Intracellular recordings from rat sciatic nerve fibers showed that the potassium channel blocking agents 4-aminopyridine (4-AP) and tetraethylammonium (TEA) had different effects on action potential waveform. When applied alone, TEA did not appreciably alter the waveform of an individual action potential, whereas 4-AP application resulted in action potential broadening and, in some axons, repetitive firing. A prolonged afterhyperpolarization which was blocked by TEA occurred subsequent to repetitive firing. These results indicate the presence of at least two pharmacologically defined potassium channels in mammalian peripheral nerve fibers. The 4-AP-sensitive potassium channels are important for rapid action potential repolarization and the TEA-sensitive potassium channels may serve to modulate axonal excitability during repetitive firing.

Brachial plexus catheter reservoir for the treatment of upper-extremity cancer pain: technical case report.

OBJECTIVE AND IMPORTANCE: Infiltration of the brachial plexus with anesthetics can provide relief of upper-extremity pain from invasive cancer. Because the analgesia is short-lived, however, repeated invasive treatments are necessary. We describe the implantation of a catheter reservoir system, in which anesthetic injections through a subcutaneous port resulted in anesthetic infiltration of the brachial plexus. CLINICAL PRESENTATION: A 47-year-old Hispanic man with squamous cell carcinoma of the larynx had undergone surgical resection, radiation treatment, and chemotherapy. Two years later, he had locally recurrent disease involving the brachial plexus, neck, and chest wall. The patient's pain was minimally responsive to narcotics, which also caused severe nausea and anorexia. TECHNIQUE: The brachial plexus was localized percutaneously with a needle electrode stimulator. Contrast injection under fluoroscopy confirmed entry into the plexus sheath. With use of the Seldinger technique, two Silastic catheters were placed within the brachial plexus and attached with a "Y" connector to a reservoir. The patient experienced complete relief of upper-extremity pain after a test injection with xylocaine. Thereafter, serial injections of bupivacaine with triamcinolone at 1-week intervals provided complete pain relief. After the treatments were initiated, the patient reported improved sleep and an improvement in his quality of life. CONCLUSION: A catheter reservoir system for brachial plexus analgesia can provide safe and effective analgesia for upper-extremity pain. This technique negates the need for repeated invasive procedures and avoids the complications of neurolysis.

Craniospinal dissemination of central neurocytoma. Report of two cases.

Central neurocytoma was first described in the literature in 1982 and has been noted to be a benign neuronal tumor usually located in the ventricular system. Of the more than 100 reported cases, only seven recurrences have been reported, all of which have been local. The authors report two cases of recurrent central neurocytoma that disseminated through the ventricular system with seeding to the spine, as evidenced by magnetic resonance images and positive cerebrospinal fluid cytology. The histological appearance of these two tumors was typical for the lesion and lacked evidence of malignant change. Central neurocytoma may not be as benign as previously thought, and the recognition of this more malignant behavior has implications for patient follow up and therapy.

Tumor response to interferon inducer and radiation effect of serum interferon levels.

The effect of interferon inducing complex of polyriboinosinic, polyribocytidylic acid with poly-L-lysine and carboxymethylcellulose - Poly (ICLC) - on the response of Lewis lung carcinoma in C57B1 mice to radiation treatments was studied. Improved tumor response was obtained in mice receiving 1.5 mg/m2 or higher of Poly (ICLC). Such doses have induced more than 1000 mu/ml of serum interferon. The same doses of Poly (ICLC) potentiated the epilation effect of radiation. The injection of 0.15 mg/m2 of Poly (ICLC) led to protection of the tumor and the stimulation of it's growth. It also did not potentiate the epilation effect. In this study, one weekly administration of Poly (ICLC) was as effective as three times per week treatment. The cellular mechanism of the increased radiosensitivity caused by Poly (ICLC) was reflected in the reduction of the width of the shoulder on the cell survival curve, which was dependent on the dose of the drug. In cell cultures, doses of 100 micrograms/ml synchronized the cell division, thus contributing to the increase in radiosensitivity.

Robotic training and kinematic analysis of arm and hand after incomplete spinal cord injury: a case study.

Regaining upper extremity function is the primary concern of persons with tetraplegia caused by spinal cord injury (SCI). Robotic rehabilitation has been inadequately tested and underutilized in rehabilitation of the upper extremity in the SCI population. Given the acceptance of robotic training in stroke rehabilitation and SCI gait training, coupled with recent evidence that the spinal cord, like the brain, demonstrates plasticity that can be catalyzed by repetitive movement training such as that available with robotic devices, it is probable that robotic upper-extremity training of persons with SCI could be clinically beneficial. The primary goal of this pilot study was to test the feasibility of using a novel robotic device for the upper extremity (RiceWrist) and to evaluate robotic rehabilitation using the RiceWrist in a tetraplegic person with incomplete SCI. A 24-year-old male with incomplete SCI participated in 10 sessions of robot-assisted therapy involving intensive upper limb training. The subject successfully completed all training sessions and showed improvements in movement smoothness, as well as in the hand function. Results from this study provide valuable information for further developments of robotic devices for upper limb rehabilitation in persons with SCI.

Activity-dependent changes in extracellular potassium and excitability in turtle olfactory nerve.

The excitability properties of turtle olfactory nerve (o.n.) were studied in vitro using potassium-sensitive microelectrodes (KSM), a modified sucrose gap chamber, and a standard nerve chamber to measure conduction velocity. A pronounced supernormal period (SNP), as indicated by increased conduction velocity of the o.n. fiber volley, lasting up to several seconds, was observed following a single stimulus. The compound action potential recorded in the sucrose gap chamber showed a prolonged depolarization with a similar time course to the SNP. When stimulation intensity was submaximal the response amplitude, and the extracellular potassium concentration [K+]o, continuously increased during repetitive stimulation. In contrast, when supramaximal stimuli were applied, the amplitude of the o.n. fiber volley was reduced during a high-frequency stimulus train for all responses after the initial one even though latency was maximally reduced, i.e., during supernormal conduction. Superfusion with various levels of K+ elicited changes in the excitability of the o.n. fibers. Small increases in [K+]o above the resting concentration of 2.6 mM led to an increase in resting excitability, whereas larger increases resulted in decreased excitability and conduction block. The SNP was eliminated when extracellular potassium was elevated between 3 and 4 mM above resting levels. Microstimulation of a small bundle of o.n. fibers led to an increase in [K+]o along the bundle but also around adjacent nonactivated fibers. The excitability of these neighboring nonactivated fibers was increased, further indicating the importance of activity-dependent changes in [K+]o in modulating axonal excitability. These results demonstrate the importance of activity-dependent increases in extracellular potassium in modulating nonmyelinated o.n. fiber excitability. They also indicate that increases in [K+]o and an associated membrane depolarization contribute to the increased excitability observed during fiber recruitment and the supernormal period.