Effect of trehalose and melibiose on crystallization of amorphous paracetamol.

This paper investigates the solid-state behavior of two-phase solid dispersions involving small molecules. The effect of two sugars, trehalose and melibiose, on the recrystallization of amorphous paracetamol, and vice versa, was investigated. The solid dispersions were prepared via heating and quench-cooling, and then stored at a temperature of 38.5 ± 0.5 °C and relative humidities of 3 ± 1% and 75 ± 1%. X-ray powder diffraction (XRPD) confirmed that the solid dispersions were amorphous, while Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) revealed that the solid dispersions were two-phase systems with drug-rich and excipient-rich regions. XRPD was used to qualitatively and quantitatively study the crystallization of the components, and revealed that, despite the existence of two phases, the sugars hindered the crystallization of paracetamol. In contrast, once the paracetamol crystallization started, it accelerated the crystallization of the sugars. Overall, the study demonstrates that small-molecule solid-dispersions need not be single-phase to observe mutual influences between the components in crystallization behavior, and that these effects are likely mediated through interactions at the phase interfaces, as well as alterations in water sorption and mechanical effects.

Erratum: Evolutionary dynamics and molecular epidemiology of West Nile virus in New York State: 1999-2015.

[This corrects the article DOI: 10.1093/ve/vez020.].

Genome-Wide Screen for Saccharomyces cerevisiae Genes Contributing to Opportunistic Pathogenicity in an Invertebrate Model Host.

Environmental opportunistic pathogens can exploit vulnerable hosts through expression of traits selected for in their natural environments. Pathogenicity is itself a complicated trait underpinned by multiple complex traits, such as thermotolerance, morphology, and stress response. The baker's yeast, Saccharomyces cerevisiae, is a species with broad environmental tolerance that has been increasingly reported as an opportunistic pathogen of humans. Here we leveraged the genetic resources available in yeast and a model insect species, the greater waxmoth Galleria mellonella, to provide a genome-wide analysis of pathogenicity factors. Using serial passaging experiments of genetically marked wild-type strains, a hybrid strain was identified as the most fit genotype across all replicates. To dissect the genetic basis for pathogenicity in the hybrid isolate, bulk segregant analysis was performed which revealed eight quantitative trait loci significantly differing between the two bulks with alleles from both parents contributing to pathogenicity. A second passaging experiment with a library of deletion mutants for most yeast genes identified a large number of mutations whose relative fitness differed in vivovs.in vitro, including mutations in genes controlling cell wall integrity, mitochondrial function, and tyrosine metabolism. Yeast is presumably subjected to a massive assault by the innate insect immune system that leads to melanization of the host and to a large bottleneck in yeast population size. Our data support that resistance to the innate immune response of the insect is key to survival in the host and identifies shared genetic mechanisms between S. cerevisiae and other opportunistic fungal pathogens.

Do you want to live to be 100? Answers from older people.

BACKGROUND: little is known about the oldest-olds' views on ageing. OBJECTIVE: to investigate older people's desire and the reasons they give for wanting to live to 100. DESIGN: a postal questionnaire, analysed both quantitatively and qualitatively. SETTING: population based in Helsinki, Finland. SUBJECTS: a random sample (response rate 64%; N = 1,405) of community-dwelling older people (aged 75-96). METHODS: a structured self-completed questionnaire with an open-ended question on the reasons why/why not participants wished/did not wish to live to 100. RESULTS: one-third (32.9%) of home-dwelling older people wanted to live to be 100. Those who did were older, more often male and self-rated their health better than those who did not. Often the desire for long life was conditional: 'Yes, if I stay healthy'. Among the reasons is that many were curious to see what would happen. Many stated that they loved life, they had twinkle in their eye or significant life roles. Those who did not want to live extremely long lives gave various rationales: they would become disabled, life would be meaningless, they were reluctant to become a burden to others or they feared loss of autonomy or suffering pain or loneliness. Some people also shared the view that they should not intervene in destiny or they felt that they had accomplished what they wanted in life. CONCLUSIONS: one-third of the oldest-old participants wanted to live to 100. Identifying what motivated them to desire long life could be a resource in their care plans.

BOLD-fMRI of PTZ-induced seizures in rats.

PURPOSE: To develop a non-invasive method for exploring seizure initiation and propagation in the brain of intact experimental animals. METHODS: We have developed and applied a model-independent statistical method--Hierarchical Cluster Analysis (HCA)--for analyzing BOLD-fMRI data following administration of pentylenetetrazol (PTZ) to intact rats. HCA clusters voxels into groups that share similar time courses and magnitudes of signal change, without any assumptions about when and/or where the seizure begins. RESULTS: Epileptiform spiking activity was monitored by EEG (outside the magnet) following intravenous PTZ (IV-PTZ; n=4) or intraperitoneal PTZ administration (IP-PTZ; n=5). Onset of cortical spiking first occurred at 29+/-16 s (IV-PTZ) and 147+/-29 s (IP-PTZ) following drug delivery. HCA of fMRI data following IV-PTZ (n=4) demonstrated a single dominant cluster, involving the majority of the brain and first activating at 27+/-23s. In contrast, IP-PTZ produced multiple, relatively small, clusters with heterogeneous time courses that varied markedly across animals (n=5); activation of the first cluster (involving cortex) occurred at 130+/-59 s. With both routes of PTZ administration, the timing of the fMRI signal increase correlated with onset of EEG spiking. CONCLUSIONS: These experiments demonstrate that fMRI activity associated with seizure activity can be analyzed with a model-independent statistical method. HCA indicated that seizure initiation in the IV- and IP-PTZ models involves multiple regions of sensitivity that vary with route of drug administration and that show significant variability across animal subjects. Even given this heterogeneity, fMRI shows clear differences that are not apparent with typical EEG monitoring procedures, in the activation patterns between IV and IP-PTZ models. These results suggest that fMRI can be used to assess different models and patterns of seizure activation.

Do glia have heart? Expression and functional role for ether-a-go-go currents in hippocampal astrocytes.

Potassium homeostasis plays an important role in the control of neuronal excitability, and diminished buffering of extracellular K results in neuronal Hyperexcitability and abnormal synchronization. Astrocytes are the cellular elements primarily involved in this process. Potassium uptake into astrocytes occurs, at least in part, through voltage-dependent channels, but the exact mechanisms involved are not fully understood. Although most glial recordings reveal expression of inward rectifier currents (K(IR)), it is not clear how spatial buffering consisting of accumulation and release of potassium may be mediated by exclusively inward potassium fluxes. We hypothesized that a combination of inward and outward rectifiers cooperate in the process of spatial buffering. Given the pharmacological properties of potassium homeostasis (sensitivity to Cs(+)), members of the ether-a-go-go (ERG) channel family widely expressed in the nervous system could underlie part of the process. We used electrophysiological recordings and pharmacological manipulations to demonstrate the expression of ERG-type currents in cultured and in situ hippocampal astrocytes. Specific ERG blockers (dofetilide and E 4031) inhibited hyperpolarization- and depolarization-activated glial currents, and ERG blockade impaired clearance of extracellular potassium with little direct effect on hippocampal neuron excitability. Immunocytochemical analysis revealed ERG protein mostly confined to astrocytes; ERG immunoreactivity was absent in presynaptic and postsynaptic elements, but pronounced in glia surrounding the synaptic cleft. Oligodendroglia did not reveal ERG immunoreactivity. Intense immunoreactivity was also found in perivascular astrocytic end feet at the blood-brain barrier. cDNA amplification showed that cortical astrocytes selectively express HERG1, but not HERG2-3 genes. This study provides insight into a possible physiological role of hippocampal ERG channels and links activation of ERG to control of potassium homeostasis.

Administration of progesterone before oocyte retrieval negatively affects the implantation rate.

OBJECTIVE: To compare the efficacy of two clinically accepted methods of progesterone supplementation during IVF. DESIGN: Prospective randomized trial. SETTING: A university-based IVF program. PATIENT(S): Three hundred fourteen stimulated IVF cycles between January 1993 and October 1994. INTERVENTION(S): Patients were assigned to one of two luteal phase progesterone regimens by a random permuted block design. In protocol A, 12.5 mg of IM progesterone was given 12 hours before oocyte retrieval; in protocol B, 25 mg of IM progesterone was given on the day of oocyte retrieval. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): Patient demographic characteristics, including age, diagnosis, number of oocytes retrieved and fertilized, and number of embryos transferred, were not different between the two groups. There was no difference in the rate of cycle cancellation between the groups. One hundred forty ETs were performed in patients assigned to protocol A and 142 in patients assigned to protocol B. The clinical pregnancy rate in group A was 12.9% compared with 24.6% in group B. CONCLUSION(S): The administration of progesterone before oocyte retrieval is associated with a lower pregnancy rate than the administration of progesterone after oocyte retrieval.

A new model of the blood--brain barrier: co-culture of neuronal, endothelial and glial cells under dynamic conditions.

Developing in vitro blood-brain barrier (BBB) models that closely mimic the natural state is important for theoretical and practical applications, including drug development. We previously developed an in vitro BBB model based on co-culturing endothelial cells with glia in the presence of flow on hollow fiber tube culture substrates. We now report that this dynamic in vitro BBB (DIV-BBB) can be successfully used to co-culture differentiated serotonergic neurons in the presence of a BBB. These neurons demonstrated fluoxetine-sensitive serotonin (5HT) uptake and depolarization-induced release of [3H]5HT. Our results demonstrate that the DIV-BBB is a suitable model for culturing of neurons in a quasi-physiological microenvironment and in the presence of a high-resistance, stereoselective BBB.

Effects of intraaccumbens microinjections of quinopirole on head turning and circling movement in the rat.

This study was designed to evaluate whether nucleus accumbens dopamine D2 receptors are involved in the initiation of the movement, as distinguished from its execution. For this purpose, the effects of the quinpirole-induced increase of nucleus accumbens dopamine D2 receptor activity were observed on specific parameters of the circling behavior and of its first stage, the head-turning (HT) movement. The experiments were performed on rats with unilateral 6-hydroxydopamine (6-OHDA) lesion of the pars compacta of the substantia nigra and d-amphetamine i.p. (3 mg/kg). Bilateral intraaccumbens microinjections of quinpirole (1, 5, and 10 microg/0.5 microl), an agonist of the D2 receptor family, were performed on three groups of animals. Bilateral saline (0.5 microl) was injected in a fourth group as control. An additional control experiment, with quinpirole (10 microg/0.5 microl) bilaterally injected in accumbens without d-amphetamine i.p., was also performed in a further group of 6-OHDA-lesioned animals. By means of a videoanalysis system, HT duration, angle, and speed were analyzed. Modifications of the circling rate (increase), HT duration (decrease), HT angle (decrease or increase according to the dose), and HT speed (increase) were observed. Moreover, a very close head-to-tail position and a very short-diameter type of turn were also evidenced. Similar modifications, even if different in amplitude and in % distribution, were observed following bilateral quinpirole in accumbens without d-amphetamine i.p. The results indicate a close relationship among head-turning speed, type of turn, and position of the animal in the circling motor sequence. We conclude that D2 receptor family in nucleus accumbens is involved in the initiation of movement as distinguished from its execution.

Inhibitory effect of A10 dopaminergic neurons of the ventral tegmental area on the orienting response evoked by acoustic stimulation in the cat.

The effect of bilateral electric stimulation of A10 dopaminergic neurons of the ventral tegmental area (80-300 microA, 20-50 Hz, 0.1-0.5 ms, 2 s duration) on latency and duration of the orienting response, evoked by acoustic stimuli (4500-8000 Hz, 2 s), was studied in the cat. A10 neuron stimulation, simultaneous with the acoustic one, was performed with threshold parameters inducing minimal behavioral signs (head searching movement, sniffing, increase in alertness). By means of a videoanalysis system, a statistically significant increase, both of latency and duration of the response, was observed. The possible role of dopamine was studied administrating sulpiride (20 mg/kg i.p.), a dopaminergic antagonist prevalently acting on the mesolimbic-mesocortical system. In this condition, the disappearance of A10 neuron effect occurred. Sulpiride injection did not affect the parameters of the orienting response to acoustic stimulus alone, suggesting a direct effect on A10 dopaminergic neurons. Moreover, when saline administration was carried out, no significant modification of the effects, obtained following A10 neuron activation, was observed. The data suggest that A10 dopaminergic neurons, origin of the mesolimbic-mesocortical system, may be involved in the control of the response to sensory stimuli, likely by influencing sensorimotor integration processes. An involvement in the inhibitory regulation of the switching of attention is also discussed.

Failed fertilization after intracytoplasmic sperm injection: the extent of paternal and maternal chromatin decondensation.

OBJECTIVE: To determine the extent of paternal and maternal chromatin decondensation in unfertilized eggs after intracytoplasmic sperm injection (ICSI). DESIGN: Eggs that failed to show two pronuclei (2-PN) 48 hours after ICSI were studied at two different time intervals: at ICSI program inception (group A) and after 8 months (group B). PATIENT(S): Forty-nine patients undergoing IVF cycles. MAIN OUTCOME MEASURE(S): The unfertilized eggs were studied by chromatin staining. RESULT(S): The average fertilization rate from all ICSI cycles in these two groups was 45%. The fertilization rates in groups A and B were 35% and 59%, respectively. In group A, 65% of the unfertilized eggs were characterized by condensed sperm chromatin with 11% showing partial decondensation. In group B, only 28% of the unfertilized eggs demonstrated condensed sperm chromatin, whereas 45% were partially decondensed. In these two groups, no sperm chromatin was detected in 24% of the unfertilized eggs. The maternal chromatin remained at metaphase II in 84% of all unfertilized eggs analyzed. CONCLUSION(S): These observations suggest that the technical problem of deposition of the sperm inside the egg is not the major cause of failure of fertilization rates in ICSI cycles. Rather, it is likely to be the failure to complete both the maternal and paternal chromatin transitions that occur with normal fertilization.

Behavioral and neurochemical recovery from partial 6-hydroxydopamine lesions of the substantia nigra is blocked by daily treatment with D1/D5, but not D2, dopamine receptor antagonists.

To determine whether D1/D5 dopamine (DA) receptors play a role in normalization of DA extracellular levels of striatal DA and behavioral recovery after partial 6-OHDA lesions of the substantia nigra, animals were treated on days 1-8 after lesioning with the D1/D5 DA receptor antagonists SCH 23390 (0.1 mg/kg, s.c.) and SCH 39166 (1.0 mg/kg, s.c.), the inactive enantiomer SCH 23388 (0.1 mg/kg, s.c.), the D2 antagonist eticlopride (0.1 mg/kg, i.p.), or saline. Spontaneous turning behavior was assessed on days 3 and 15. Basal extracellular DA and metabolites were measured in both striata using microdialysis on days 16 and 17, 8-9 d after termination of drug treatments. On day 3, all animals turned ipsilateral to the lesion. On day 15, animals previously treated with either saline, eticlopride, or SCH 23388 showed no behavioral asymmetries, whereas animals treated with SCH 23390 or SCH 39166 turned ipsilaterally. On days 16 and 17, extracellular DA did not differ on the two sides in animals treated with saline or eticlopride and were higher on the lesioned side after SCH 23388. In animals treated with the D1/D5 receptor antagonists, however, basal levels of DA were lower on the lesioned side, showing no evidence of normalization. These results suggest a role for the D1/D5 DA receptor in the development of compensatory changes in the DA neurons that accompany behavioral recovery from partial lesions of nigrostriatal DA system.

Behavioral and neurochemical recovery from partial 6-hydroxydopamine lesions of the substantia nigra is blocked by daily treatment with glutamate receptor antagonists MK-801 and CPP.

To determine whether glutamate plays a role in the recovery from lesions of the substantia nigra, measures of behavioral functioning and extracellular levels of striatal dopamine (DA) were made after partial unilateral 6-OHDA lesions in adult male rats. In experiments 1 and 2, animals were treated on days 1-8 after lesioning with the noncompetitive NMDA receptor antagonist dizocilpine maleate (MK-801; 0.25 mg/kg, i.p.) or saline, and in experiment 3 with the competitive antagonist 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP; 1.0 mg/kg, i.p.) or saline. In experiment 1, behavior was assessed 3 and 8 d after lesioning before daily drug treatment; on days 9 and 10, basal extracellular DA and metabolites were measured in both striata using microdialysis. In experiments 2 and 3, behavior was assessed on days 3 and 15 and microdialysis on days 16 and 17, 8-9 d post-termination of drug treatments. On day 3, all animals turned ipsilateral to the lesion. On days 8 or 15, saline-treated animals showed no behavioral asymmetries, whereas MK-801- and CPP-treated animals turned ipsilaterally. In antagonist-treated animals, basal levels of extracellular DA were lower on the lesioned side whether measured 9-10 or 16-17 d after lesioning, whereas in saline-treated animals DA levels on the two sides did not differ. These results suggest that glutamate plays a role in the development of compensatory changes in the DA neurons that accompany behavioral recovery from partial lesions of nigrostriatal DA system.

A zona biochemical change and spontaneous cortical granule loss in eggs that fail to fertilize in in vitro fertilization.

OBJECTIVE: To determine the extent of biochemical changes in the zona pellucida (ZP) and cortical granule release in eggs failing to fertilize in IVF. DESIGN: After insemination, unactivated eggs without two pronuclei (PN) were studied by high resolution microscopy and fluorescent probes to determine cortical granule density and meiotic stage. After ZP isolation, proteins from individual ZPs were biotinylated, electrophoresed, and visualized by western blots with avidin-chemiluminescence. Controls included mouse unfertilized and fertilized eggs and human germinal vesicle stage oocytes and > or = 3PN eggs. SETTING: University medical center and hospital tertiary care IVF-ET program. RESULTS: Many of the unfertilized eggs were in metaphase and had relatively low cortical granule densities indicative of cortical granule loss. Approximately one half of the ZPs showed evidence of biochemical hardening with a modification of a 90 to 100 x 10(3) molecular (weight) ratio (M(r)) ZP protein. The 3PN eggs had few cortical granules and their ZPs had a pronounced 90 to 100 x 10(3) M(r) modification that was not detected in germinal vesicle stage ZP controls. CONCLUSION: The observed changes in ZP biochemistry and cortical granule quantitation demonstrate that failed fertilization is frequently associated with spontaneous cytoplasmic, but not nuclear, activation. In affected eggs, these changes could prevent fertilization in routine IVF and in cases of reinsemination. The relationship of changes in the ZP and cortical granules to infertility and in vitro culture requires further investigation. The 90 to 100 x 10(3) M(r) ZP protein modification can be detected biochemically with 1/10 of a human ZP.

Modifications of head turning and circling movement following sulpiride microinjections into nucleus accumbens in the rat.

The aim of this study was to determine whether a relationship exists between nucleus accumbens D2 receptors, circling behavior, and its first stage, the head turning. Rats were unilaterally lesioned in the substantia nigra with 6-hydroxydopamine and afterward treated with d-amphetamine IP following bilateral intraaccumbens microinjections (1, 5, 10 micrograms/0.5 microliters) of sulpiride, a D2 receptor antagonist. Computer-assisted video analysis allowed the study of some parameters (number of turns, type of turn, head turning duration, degree and speed) characterizing rotatory activity. Sulpiride microinfusion resulted in a dose-dependent decrease of the number of turns and head rotation speed and in a dose-dependent increase of head-turning duration. Two turn types were observed in relation to the animal's position: a large head-to-tail position with a short-diameter turn type following sulpiride microinjection, and a close head-to-tail position in relation to a wide diameter turn type in the control condition (saline). The results show a relationship between head turning parameters, circling behavior, and D2 receptors in nucleus accumbens, which may be also involved in the regulation of some mechanisms related to sensory-motor integration in the rat.

Ultrasound prediction of follicle volume: is the mean diameter reflective?

OBJECTIVE: To evaluate the relationship between 2 dimensional sonographic measurement of ovarian follicles and their actual volume. DESIGN: Prospective clinical study. SETTING: The in vitro fertilization (IVF) program of a University based, tertiary care hospital. PATIENTS AND INTERVENTIONS: Sonographic categorization by shape, and measurement of 96 individual ovarian follicles immediately prior to aspiration for IVF. Each follicle was aspirated under direct ultrasound guidance and the volume recorded. The 96 follicles were visualized in a total of 14 patients from whom 2 to 27 oocytes were obtained. MAIN OUTCOME MEASURE: Total volume of each follicle. RESULTS: Round and polygonal follicles exhibited a highly significant relationship between sonographically measured mean diameter and total follicle volume. The volume of follicles that were categorized as ellipsoid was not predicted by measurement of the longest diameter, shortest diameter or mean diameter. CONCLUSION: The mean diameter of round and polygonal follicles accurately predicts total follicular volume. However, clinical decisions in ovulation induction should be modified when the follicle shape is predominantly ellipsoid because the traditionally held belief that the sonographic measurement of the follicular diameter correlates with the follicular volume does not apply in those circumstances.

Accumbens microinjection of LY 171555 and sulpiride: effects on circling behaviour, in the rat.

In rats with unilateral 6-hydroxydopamine lesions of the substantia nigra (pars compacta) and amphetamine i.p. some parameters of the rotational activity, following intra-accumbens microinjection of D2 agonist (LY 171555) and antagonist (sulpiride), have been analyzed. An increase in the number of turns was observed with LY 171555, whereas sulpiride determined a decrease. The type of circling was also analyzed: D2 agonist induced a very close turn around the central axis of the animal and D2 antagonist a close turn in the centre of the cage. Data were compared with control condition, which exhibited a large turn around the walls of the cage. The results show that pharmacological manipulation of nucleus accumbens influences rotational behaviour with a specific role for D2 receptors.

Gonadotropin-releasing hormone agonist administration in early human pregnancy is associated with normal outcomes.

OBJECTIVE: To evaluate the reproductive outcomes of early human gestations exposed to GnRH agonist (GnRH-a). Gonadotropin-releasing hormone agonist is commonly used as an adjunct to ovulation induction regimens, and sometimes early pregnancies are inadvertently exposed to the drug. There has been concern regarding possible teratogenic effects of GnRH-a on these gestations, but there is a paucity of data regarding human subjects. DESIGN: Identified pregnancies were followed for obstetric and fetal outcomes. PATIENTS, PARTICIPANTS: Over 4 years 18 early pregnancies among 17 women were found to be exposed to GnRH-a. During the month of conception, daily injections were begun on menstrual day 22 after documenting an ovulatory P level on day 21. Gonadotropin-releasing hormone agonist was discontinued from 10 to 24 days later, immediately after pregnancy was recognized. Obstetric and fetal outcomes were ascertained by chart review and telephone follow-up with mothers. RESULTS: Five first trimester losses were observed in 18 hCG confirmed pregnancies for a loss rate of 28%. Thirteen pregnancies produced apparently normal babies. One normal fetus was lost at 20 weeks' gestation because of cervical incompetence. CONCLUSION: There does not appear to be an increased risk of birth defects or pregnancy wastage in human pregnancies exposed to daily low-dose GnRH-a therapy in the first weeks of gestation.

Influence of ventral tegmental area (A10 region) on flight behaviour elicited by hypothalamic stimulation in the cat.

The influence of the ventral tegmental area (VTA) (dopaminergic A10 group neurons) on flight behaviour, induced by hypothalamic stimulation, was studied in the cat. Co-stimulation of hypothalamus and VTA (ipsi- or contralateral) induced an increase of the flight latency. Slow-motion analysis of flight behaviour showed that this increase was due to the augmentation of the fixation latency (FL) whereas the upright latency (UL) was not modified. Sulpiride injection (50 mg/kg i.p.) provoked the disappearance of VTA effect without affecting the basal behavioural sequence. The results show that DA A10 group neurons increase the attentive component of the flight reaction, suggesting a possible influence of the DA A10 system on the mechanisms modulating focused attention in the animal.