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.

Assessment of fibromyalgia & chronic fatigue syndrome: a new protocol designed to determine work capability--chronic pain abilities determination (CPAD).

The objective was to design a protocol to assess work ability in people suffering ill-defined painful and disabling disorders, the outstanding prototype of which is fibromyalgia/chronic fatigue syndrome (FM/CSF).Following an extensive literature search, the mos appropriate components of current methods of assessment of physical and cognitive abilities were incorporated into the protocol, occasionally with appropriate modification to suit the specific requirements of the individual. The initial part of the assessment consists of a standard history taking, principally focusing on the patient's self-reported physical and cognitive abilities and disabilities, as well as the completion of established pain and fatigue scales, and relevant disability questionnaires. Following this, physical and cognitive abilities are objectively assessed on two separate occasions, utilizing computerized hand-held dynamometers, inclinometers, algometers, and force dynamometers. Specific work simulation tests using the industrial standards Methods-Time-Measurement testing are availed of, as is aerobic testing using the Canadian Aerobic Fitness Test (CAFT). Objective computerised neuro-cognitive testing are also utilised as an integral component of the assessment. All results are then subject to specific computerized analysis and compared to normative and standardised work-based databases. The designed system produces reliable, consistent and reproducible results. It also proves capable of detecting any inconsistencies in patient input and results, in addition to being independent of any possible assessor bias. A new protocol has been designed to determine the working capability of individuals who suffer from various chronic disabling conditions, and represents a significant step forward in a difficult but rapidly expanding area of medical practice.

Optical organophosphate sensor based upon gold nanoparticle functionalized fumed silica gel.

We report on the creation of a high surface area, chemically selective material for the efficient adsorption of organophosphate and organophosphonate species. Using silica microparticles in conjunction with gold nanoparticles and surface modification chemistry, we have demonstrated a material with a binding constant for organophosphonates and organophosphates (OPPs) of K=2x10(6) M-1. The binding of OPPs to the modified gold nanoparticles appears as a spectral shift in the gold nanoparticle resonance. The sensitivity of this technique is limited by scattering losses of suspensions of the particles, and we report on how this sensitivity can be recovered to a significant extent by the use of solvents with a refractive index close to that of the silica particles.

Formation of gold nanoparticles using amine reducing agents.

We report on the use of amines as reducing agents in the formation of gold nanoparticles. We can predict whether the amines will function as reducing agents in this reaction based on their redox properties. The kinetics of AuNP formation can be understood in terms of Marcus electron transfer theory, where the slower reactions proceed in the inverted region owing to the difference between the Au reduction potential and the amine oxidation potential. For a certain number of the amine reducing agents, following reduction of HAuCl4, a subsequent reaction of the amine radical cation with other reducing agent molecules in solution can form poly(amine)s. These findings point collectively to the utility of amines as reducing agents in AuNP formation and provide information on the conditions under which these reactions will proceed.

Self-testing of blood glucose: biosensors and beyond.

There is a possible major shake-up on the way in blood glucose monitoring. Sales of disposable electrochemical biosensors now reach approximately US$2.5 billion per annum. However, there are numerous rival technologies on the horizon, which threaten this leading position. These offer the potential to monitor noninvasively or with minimal discomfort. Success in developing any of these has the potential to change the future of self-testing of glucose and many other analytes.

The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor.

The Rhodobacter sphaeroides extra cytoplasmic function sigma factor, sigma(E), directs transcription of promoters for the cycA gene (cycA P3) and the rpoEchrR operon (rpoE P1). These genes encode the periplasmic electron carrier cytochrome c(2) and sigma(E)/ChrR, respectively. Using in vitro transcription assays with purified R. sphaeroides core RNA polymerase and sigma(E), we show that ChrR is sufficient to inhibit sigma(E)-dependent transcription. Inhibition is proposed to proceed through a binding interaction, since sigma(E) and ChrR form a 1:1 complex that can be purified when expressed at high levels in Escherichia coli. Active preparations of ChrR and the sigma(E)/ChrR complex each contain stoichiometric zinc. Removal of zinc from ChrR or a single amino acid substitution that abolishes zinc binding, results in a protein that is incapable of inhibiting sigma(E) activity or forming a complex with the sigma factor, indicating that metal binding is important to ChrR activity. Treatment of ChrR with the thiol-modifying reagent p-hydroxymecuriphenylsulfonic acid results in the release of about one mole of zinc per mole of protein. Furthermore, two N-terminal cysteine residues are protected from reaction with the thiol-specific reagent dithionitrobenzoic acid until zinc is removed, suggesting that these residues may be involved in zinc binding. These data indicate that ChrR is a specific anti-sigma factor of sigma(E) that requires zinc for function. Based on amino acid sequence similarity, we propose that ChrR is part of a family of similar anti-sigma factors that are found in alpha and gamma proteobacteria.

Mealworm feeders as environmental enrichment for common marmosets.

The impact of a foraging enrichment device, the "mealworm feeder," on the behavior of the common marmoset was examined. In 3-h weekly exposures to the wormfeeder device, behavioral observations were conducted to compare the rates of feeder use, use of other enrichment devices, stereotyped behavior, and inactivity, to those of control sessions in which the enrichment device was not provided. Significantly decreased rates of pacing and time spent sitting still were observed in association with placement of the mealworm feeder. Feeder use declined over a period of 3 h, even if the feeders' contents were not fully depleted, and the effects of enrichment on activity waned in a like fashion. Use of other enrichment devices, comprised primarily of cage furniture, increased in the presence of the mealworm feeder. This effect did not change significantly over the 3 h of exposure even though use of the feeder declined. There was significant variation in feeder use among sex and housing condition, with females housed singly and in peer groups using the feeders significantly more than did males, whereas subadults used the feeder significantly more often than did either the dominant female or male in family groups. The results of this study suggest that the mealworm feeder is an effective form of environmental enrichment for the common marmoset, but interest wanes after approximately 3 h.

Prolactin levels are elevated after infant carrying in parentally inexperienced common marmosets.

Alloparental behavior (parental behavior directed to nondescendant young) is pervasive among human cultures but rare among other mammals. New World primates of the family Callitrichidae, including common marmosets (Callithrix jacchus jacchus), dwell in large families and many family members carry and/or provision infants. This study experimentally characterized alloparenting in common marmosets by measuring infant retrieval and carrying outside of the context of the family group. Prolactin was measured in samples collected before and after infant exposure to determine whether elevated prolactin levels were predictive of alloparental responsiveness. Seventeen nonbreeding, parentally inexperienced, singly or peer-housed common marmosets (10 females, 7 males, 6-66 months of age) were tested repeatedly in an infant retrieval paradigm. Infant retrieval was shown by 5 out of 17 monkeys (29%) in their first test. The rate of retrieval increased to 10 monkeys (59%) by the fourth test. No significant differences in age, sex, or housing condition existed between monkeys that retrieved infants and those not retrieving. Prolactin concentrations were significantly elevated in serum obtained after testing only in monkeys that retrieved infants. Prolactin levels after infant exposure were positively related to carrying duration. A separate experiment verified that neither prolactin nor cortisol was significantly elevated in response to prolonged handling. Cortisol levels were inversely proportional to handling time. Prolactin levels were not significantly related to handling time. Our results indicate that alloparental behavior is expressed at high rates after minimal infant experience, and prolactin levels rise concomitantly with the expression of alloparental behavior in this species.

Bromocriptine administration lowers serum prolactin and disrupts parental responsiveness in common marmosets (Callithrix j. jacchus).

The influence of prolactin on parental responsiveness was investigated in eight unpaired, parentally inexperienced common marmosets (Callithrix jacchus; 6 female, 2 male). The marmosets were prescreened with infants (1-10 days of age) and required to exhibit short latency retrieval and infant carrying in at least three consecutive pretests as criterion for inclusion in this study. The marmosets were then administered the dopamine agonist bromocriptine (0.5 mg/0.5ml vehicle subcutaneously) and the vehicle alone (0.5 ml 10% ethanol solution) twice daily for 3 consecutive days in random order. Bromocriptine treatment reduced circulating prolactin to nondetectable levels. Bromocriptine treatment eliminated infant retrieval in four of the eight marmosets and was associated with significantly increased retrieval latencies and significantly reduced carrying durations in the four monkeys that continued to retrieve following bromocriptine treatment. When given the vehicle alone, the marmosets retrieved infants significantly faster than during pretests, suggesting a handling effect of the injection series. Bromocriptine treatments were associated with significantly increased movement during the tests. The results of this study indicate that prolactin and/or its regulatory neurotransmitters are involved in the control of the spontaneous display of parental responsiveness in common marmosets.

Artificial turf foraging boards as environmental enrichment for pair-housed female squirrel monkeys.

We investigated the use of artificial turf foraging boards to determine if providing captive squirrel monkeys an opportunity for semi-natural foraging behavior would 1) alter the monkeys' time budget to better approximate that seen in wild populations, 2) reduce the stereotypic, self-injurious, and aggressive behavior occasionally seen in captive squirrel monkeys, and 3) provide sustained enrichment. Five groups of pair-housed female squirrel monkeys were videotaped the week prior to, the week following, and for 2 weeks during the enrichment phase, when treat-enhanced boards were provided for 2 h daily. During the first 30 min of daily enrichment, inactivity declined 35.3%, locomotion increased 3.8%, and board-related behaviors occupied 36.3% of the activity budget; these changes were not evident after 1.5 h. Stereotypic behavior (pacing, headswinging, tailchewing) and aggression were not altered by the foraging opportunity. The foraging board retained the interest of the subjects across 2 weeks in the same daily pattern. Use of the foraging board altered the squirrel monkeys' time budget to become more like activity patterns seen in wild populations.

Feasibility of using fMRI to study mothers responding to infant cries.

While parenting is a universal human behavior, its neuroanatomic basis is currently unknown. Animal data suggest that the cingulate may play an important function in mammalian parenting behavior. For example, in rodents cingulate lesions impair maternal behavior. Here, in an attempt to understand the brain basis of human maternal behavior, we had mothers listen to recorded infant cries and white noise control sounds while they underwent functional MRI (fMRI) of the brain. We hypothesized that mothers would show significantly greater cingulate activity during the cries compared to the control sounds. Of 7 subjects scanned, 4 had fMRI data suitable for analysis. When fMRI data were averaged for these 4 subjects, the anterior cingulate and right medial prefrontal cortex were the only brain regions showing statistically increased activity with the cries compared to white noise control sounds (cluster analysis with one-tailed z-map threshold of P < 0.001 and spatial extent threshold of P < 0.05). These results demonstrate the feasibility of using fMRI to study brain activity in mothers listening to infant cries and that the anterior cingulate may be involved in mothers listening to crying babies. We are currently replicating this study in a larger group of mothers. Future work in this area may help (1) unravel the functional neuroanatomy of the parent-infant bond and (2) examine whether markers of this bond, such as maternal brain response to infant crying, can predict maternal style (i.e., child neglect), offspring temperament, or offspring depression or anxiety.

The Rhodobacter sphaeroides ECF sigma factor, sigma(E), and the target promoters cycA P3 and rpoE P1.

Rhodobacter sphaeroides rpoE encodes a 19.2 kDa protein, sigma(E), related to members of the extra-cytoplasmic function subfamily of eubacterial RNA polymerase sigma factors. We demonstrate that sigma(E) directs transcription from rpoE P1, the promoter for the rpoEchrR operon, and from cycA P3, a promoter for the cytochrome c2 structural gene. Comparison of these sigma(E)-dependent promoters reveals significant sequence conservation in their -35 and -10 regions; however, rpoE P1 is over 80-fold stronger than cycA P3. Both promoters contain identical -35 hexamers, (-36)TGATCC(-31), that appear to constitute the preferred sequence, since any single base mutation in this region of cycA P3 reduces promoter function. The higher activity of rpoE P1 appears to reflect a better -10 region, (-13)TAAGA(-9), as it contains four out of five of the nucleotides found to be important to sigma(E)-dependent transcription. We also propose that ChrR acts as an inhibitor of sigma(E), since these two proteins can form a complex, and DeltachrR mutations increase sigma(E)-dependent transcription. ChrR is believed to respond to a signal from tetrapyrrole biosynthesis because loss of function mutations in chrR lead to cohemin resistance. Based on our observations, we present a model in which cohemin resistance is conferred by increasing sigma(E) activity.

Context and sex differences exist in the acoustic structure of phee calls by newly-paired common marmosets (Callithrix jacchus).

Captive common marmosets of all ages robustly produce a "separation" phee call during brief separations from their group. In contrast, a second structural variant, which may function as an intergroup call, is produced in the home cage primarily by the reproductive adults. A previous study found that postpubertal but nonreproductive offspring rarely produce phee calls when in the home cage with the natal group, yet these marmosets call frequently after pairing with an opposite-sex partner. The sudden increase in home cage phee calls may indicate the rapid onset of intergroup calling. Alternatively, marmosets may be producing the separation phee variant as a result of separation from the natal group. The present study investigated whether phee calls produced by recently paired individuals in the home cage were structurally distinguishable from their calls recorded in a separation paradigm. We also tested whether sex differences, known to exist in the calls of mature adults, could be found in calls recorded from younger, nonreproductive animals separated from their natal groups. We analyzed 18 acoustic parameters of phee calls produced in the home cage after pairing and of calls produced during separation both from the natal group and from a new mate. Discriminant function analyses found that home cage calls were clearly discriminable from separation calls (average 91.7% correctly classified), indicating that the rapid increase in home cage phee call production shortly after pairing is not a consequence of separation from the family group. Postpubertal marmosets appear to show a rapid behavioral adjustment to separation from their natal groups. Additionally, sex was clearly discriminable in calls recorded both before and after pairing (average 86.8% correctly classified). Like calls recorded from well-established paired marmosets, phee calls produced by recently paired, postpubertal marmosets are discriminable by context and sex.

Longitudinal association of cardiovascular reactivity and blood pressure in Samoan adolescents.

OBJECTIVE: The longitudinal association between blood pressure (BP) reactivity to a video game and resting BP 3 to 4 years later was investigated in 83 Samoan adolescents from American and (Western) Samoa as part of a multidisciplinary study of cardiovascular disease (CVD) risk in modern Samoans. METHODS: Participants ranged in age from 11 to 14 years at baseline, in 1992 to 1993, and 14 to 18 years at follow-up in 1996. Video game BP reactivity was defined as the residual score of the regression of the maximum BP during the video game on the minimum resting BP before the video game. The predictive effect of baseline video game BP reactivity on follow-up resting BP was tested using regression models with baseline resting BP, baseline body mass index (BMI), and age as covariates. RESULTS: Systolic BP reactivity to the video game at baseline was significantly, p=.04, and independently associated with resting systolic BP 3 to 4 years later. Samoan adolescents who had higher systolic BP reactivity scores at baseline had significantly higher resting systolic BP at follow-up after adjustment for the significant effects of baseline resting systolic BP, age, and BMI. There were no interactions between sex and reactivity or between residence, American Samoa or (Western) Samoa, and reactivity in the models, indicating that the effects of systolic BP reactivity in early adolescence on later adolescent resting systolic BP were similar in the entire study sample. CONCLUSIONS: Video game cardiovascular reactivity seems to assess aspects of psychophysiological arousal and prospective CVD risk in Samoan adolescents of both sexes residing in both Samoas, and may be useful for understanding the role of psychosocial stress and health in modernizing societies.

Effects of separation and novelty on distress vocalizations and cortisol in the common marmoset (Callithrix jacchus).

In socially-bonding species, separation from familiar attachment figures is widely known to stimulate a physiological and behavioral stress response. This study investigated the hormonal and vocal responses of adult common marmosets to separation from familiar group members and to 24 hr of cohabitation with an unfamiliar opposite-sex conspecific. All subjects were removed from their home cages and placed into a novel environment for 20 min. In one group, marmosets were exposed to an unfamiliar, opposite-sex partner in the novel environment and remained paired with this partner for the 24 hr test period. In three other groups, marmosets experienced the novel environment alone and subsequently were returned to their original social- or single-housing condition, or kept separate from their social groups for a 24 h period. Blood samples were collected the day before, and at 30 min, 90 min, and at 24 h after separation. Cortisol responses were differentially affected by the length of separation and the presence of unfamiliar conspecifics. Brief separation followed by the return to the social group had minimal effect on plasma cortisol levels. All marmosets produced high levels of separation calls in the novel environment, but there was no apparent relationship between calling and cortisol levels. The lack of a temporal relationship between the production of distress vocalizations and serum cortisol has previously been noted in squirrel monkey and rhesus monkey infant separation studies; the behavioral and physiological responses to separation appear to be similarly dissociated in the marmoset. Further, the characteristics of a separation environment can differentially affect the hormonal response by adult marmosets without differentially affecting their behavioral response.

Vasopressin in the forebrain of common marmosets (Callithrix jacchus): studies with in situ hybridization, immunocytochemistry and receptor autoradiography.

The distribution of vasopressin (AVP) producing cells, their projections and AVP receptors was examined in the brain of common marmosets (Callithrix jacchus) using in situ hybridization, immunocytochemistry and receptor autoradiography. Clusters of cells labeled for AVP mRNA or stained for AVP immunoreactivity (AVP-ir) were found in the paraventricular (PVN), supraoptic (SON) and suprachiasmatic nuclei (SCN) of the hypothalamus. Scattered AVP producing cells were also found in the lateral hypothalamus and the bed nucleus of the stria terminalis (BST). Neither AVP mRNA-labeled nor AVP-ir cells were detected in the amygdala. Although AVP-ir fibers were evident outside of the hypothalamic-neurohypophyseal tract, a plexus of fibers in the lateral septum, as observed in the rat brain, was not detected. Receptor autoradiography using 125I-linear-AVP revealed specific binding for AVP receptors in the nucleus accumbens, diagonal band, lateral septum, the BST, SCN, PVN, amygdala, anterodorsal and ventromedial nucleus of the hypothalamus, indicating sites for central AVP action in the marmoset brain. Together, these data provide a comprehensive picture of AVP pathways in the marmoset brain, demonstrating differences from rodents in the distribution of cell bodies, fibers and receptors.

Vasopressin and oxytocin immunoreactive neurons and fibers in the forebrain of male and female common marmosets (Callithrix jacchus).

Vasopressin (AVP) and oxytocin (OT) immunoreactive (ir) neurons and fibers were examined in the forebrain of male and female common marmosets (Callithrix jacchus). As expected from previous studies of cell distribution in the rodent and primate brain, AVP-ir cells were most evident in the paraventricularis, supraopticus, and suprachiasmaticus of the hypothalamus. AVP-ir cells were also widely distributed in the lateral hypothalamus and the bed nucleus of the stria terminalis. A sexually dimorphic pattern of AVP-ir cells was found in the bed nucleus of the stria terminalis, in which males had more AVP-ir cells than females. OT-ir cells were found in the paraventricularis and supraopticus of the hypothalamus as well as in the bed nucleus of the stria terminalis and the medial amygdala. Male and female marmosets did not differ in the distribution of OT-ir cells. Fibers for both AVP and OT were evident outside of the hypothalamic-neurohypophyseal tract, but a plexus of AVP-ir fibers in the lateral septum or lateral habenular nucleus, as seen in the rat brain, could not be detected for either peptide.

Neonatal ablations of the amygdala and inferior temporal cortex alter the vocal response to social separation in rhesus macaques.

Rhesus macaques that had received bilateral ablations to either the amygdala or area TE in inferior temporal cortex in the 1st week of life were briefly separated from familiar conspecifics at 10-14.5 months of age in order to assess the vocal response to this mild challenge. Sound spectrograms were subjected to quantitative analysis and compared with calls from normal, age-matched controls subjected to the same testing conditions. Animals with TE damage called at a higher rate than animals in the other two groups. TE subjects also produced more coos than controls. Males with TE lesions produced noisy calls at a higher rate than males of the other two groups. Females did not differ between groups in this measure. Analysis of the detailed acoustic structure of the 'coo' indicated significant differences in a measure of slope of the fundamental frequency (rate of frequency change over time) between amygdalectomized animals and those of the other 2 groups. The amygdalectomized monkeys produced calls with lower slope values, giving the calls a less inflected quality both in sonagrams and to the listener. These findings suggest an important role for the amygdala and inferior temporal cortex in regulating the vocal response to social separation during development.

Effects of administration regime on the psychotomimetic properties of d-amphetamine in the squirrel monkey (Saimiri sciureus).

Studies have reported that d-amphetamine can induce a schizophreniform psychosis in humans and can induce abnormal behaviour patterns in monkeys that resemble the psychotic symptoms observed in man. The purpose of the present study was to identify a drug administration regime that in squirrel monkeys reliably could induce such behaviours in order to use this as a model of schizophrenia. The behavioural effects of acute, subchronic and continuous administration of d-amphetamine were determined in male and female squirrel monkeys during short term separation from the colony and in the home cage. It was found that abnormal behaviours developed in both male and female subjects and that they were most evident in the home cage. The number of subjects responding was highest during continuous infusion followed by subchronic treatment. The study indicated that prolonged administration of high doses of d-amphetamine is necessary for the development of abnormal behaviours. These findings suggest that animal models of schizophrenia based on d-amphetamine should be based on chronic administration or continuous infusion of d-amphetamine instead of acute injections.