Hyaluronan is organized into fiber-like structures along migratory pathways in the developing mouse cerebellum.

Hyaluronan is a free glycosaminoglycan which is abundant in the extracellular matrix of the developing brain. Although not covalently linked to any protein it can act as a backbone molecule forming aggregates with chondroitin sulfate proteoglycans of the lectican family and link proteins. Using neurocan-GFP as a direct histochemical probe we analyzed the distribution and organization of hyaluronan in the developing mouse cerebellum, and related its fine structure to cell types of specified developmental stages. We observed a high affinity of this probe to fiber-like structures in the prospective white matter which are preferentially oriented parallel to the cerebellar cortex during postnatal development suggesting a specially organized form of hyaluronan. In other layers of the cerebellar cortex, the hyaluronan organization seemed to be more diffuse. During the second postnatal week, the overall staining intensity of hyaluronan in the white matter declined but fiber-like structures were still present at the adult stage. This type of hyaluronan organization is different from perineuronal nets e.g. found in deep cerebellar nuclei. Double staining experiments with cell type specific markers indicated that these fiber-like structures are predominantly situated in regions where motile cells such as Pax2-positive inhibitory interneuron precursors and MBP-positive oligodendroglial cells are located. In contrast, more stationary cells such as mature granule cells and Purkinje cells are associated with lower levels of hyaluronan in their environment. Thus, hyaluronan-rich fibers are concentrated at sites where specific neural precursor cell types migrate, and the anisotropic orientation of these fibers suggests that they may support guided neural migration during brain development.     

Techniques for in utero,longitudinal MRI of fetal brain development in baboons at 3 T

Magnetic Resonance Imaging (MRI) is a promising tool for the noninvasive, longitudinal study of developing primate brains. We developed a protocol to scan pregnant baboons serially at 3 T for up to 3 h per session. This protocol includes procedures for animal preparation, anesthesia, MRI scanning, and post-scan animal care. We applied this protocol to scan 5 baboons multiple times across the latter 70% of gestation—from as early as 56 days post-conceptional age to as late as 185 days (term ～180 days). We successfully acquired high-resolution anatomical images and maps of relaxation times (T₁ and T₂) of the fetal brains at multiple time points across gestation. These images and maps demonstrated the convergence of gray and white matter contrast near term, and furthermore demonstrated that the convergence of contrast is a consequence of the continuous change in relaxation times during fetal brain development. We estimated the rates of decrease of T₁ and T₂ in white matter and gray matter, respectively. In addition, we measured the volumes of fetal brain at different gestational ages and calculated the growth rates of whole brain (0.91 ± 0.08 cm³/day) and cortical gray matter (0.40 ± 0.04 cm³/day). We also measured the mean diffusivity in white matter and deep gray matter using diffusion tensor imaging. In conclusion, in utero MRI of fetal baboon brains greatly enhances the use of nonhuman primate models to study fetal brain development longitudinally.     

Évaluation quantitative préliminaire du 18F-FDDNP en TEP/TDM dans la maladie d’Alzheimer,en utilisant différentes régions de référence

IntroductionAlzheimer's disease (AD) is the leading cause of dementia in the world. His current treatment is symptomatic. New therapies, aimed at slowing the neurodegenerative process, are actually under study. Early detection of AD as also the evaluation of new generation of neuroprotectors drugs is essential. For this reason, markers of amyloid plaques and neurofibrillary lesions have been developed including ¹⁸F-FDDNP.MethodsTwo patients and two controls were included in this preliminary study. They were submitted to a PET/CT with ¹⁸F-FDDNP. Data acquisition was performed in a list mode during 90 minutes. We used a cerebral atlas to create ROI, after co registration with a perfusion PET template, and we estimated the distribution volume ratio (DVR) using two different reference regions: the cerebellum and the white matter. We calculated a simplified index of quantification (SUVr) using a single 10 minutes acquisition frame.ResultsWe found an increased DVR in temporal lobes (amygdala) (1.23–1.40 in patients vs 1.06–1.31 in controls, with cerebellum as reference region) and fronto-basal regions (1.48–1.30 in patients vs 1.16–1.23 in controls with the same reference region and 1.25–1.18 in patients vs 1.13–1.19 in controls with white matter as reference region). SUVr calculated between 50 and 60 minutes after injection was highly correlated to DVR values (correlation coefficient 0.93). The SUVr difference between patients and controls was more important with cerebellum rather than white matter as reference region (26% in amygdala).ConclusionThis is a very preliminary study due to the low number of subjects included. However, the results obtained suggest the feasibility of brain ROI segmentation and the DVR-SUVr calculation using an anatomical atlas.     

Mechanical difference between white and gray matter in the rat cerebellum measured by scanning force microscopy

The mechanical properties of tissues are increasingly recognized as important cues for cell physiology and pathology. Nevertheless, there is a sparsity of quantitative, high-resolution data on mechanical properties of specific tissues. This is especially true for the central nervous system (CNS), which poses particular difficulties in terms of preparation and measurement. We have prepared thin slices of brain tissue suited for indentation measurements on the micrometer scale in a near-native state. Using a scanning force microscope with a spherical indenter of radius ～20 μm we have mapped the effective elastic modulus of rat cerebellum with a spatial resolution of 100 μm. We found significant differences between white and gray matter, having effective elastic moduli of K=294±74 and 454±53 Pa, respectively, at 3 μm indentation depth (n_g=245, n_w=150 in four animals, p<0.05; errors are SD). In contrast to many other measurements on larger length scales, our results were constant for indentation depths of 2–4 μm indicating a regime of linear effective elastic modulus. These data, assessed with a direct mechanical measurement, provide reliable high-resolution information and serve as a quantitative basis for further neuromechanical investigations on the mechanical properties of developing, adult and damaged CNS tissue.     

Viscoelastic properties of human cerebellum using magnetic resonance elastography

BackgroundThe cerebellum has never been mechanically characterised, despite its physiological importance in the control of motion and the clinical prevalence of cerebellar pathologies. The aim of this study was to measure the linear viscoelastic properties of the cerebellum in human volunteers using Magnetic Resonance Elastography (MRE).MethodsCoronal plane brain 3D MRE data was performed on eight healthy adult volunteers, at 80 Hz, to compare the properties of cerebral and cerebellar tissues. The linear viscoelastic storage (G′) and loss moduli (G″) were estimated from the MRE wave images by solving the wave equation for propagation through an isotropic linear viscoelastic solid. Contributions of the compressional wave were removed via application of the curl-operator.ResultsThe storage modulus for the cerebellum was found to be significantly lower than that for the cerebrum, for both white and grey matter. Cerebrum: white matter (mean±SD) G′=2.41±0.23 kPa, grey matter G′=2.34±0.22 kPa; cerebellum: white matter, G′=1.85±0.18 kPa, grey matter G′=1.77±0.24 kPa; cerebrum vs cerebellum, p<0.001. For the viscous behaviour, there were differences in between regions and also by tissue type, with the white matter being more viscous than grey matter and the cerebrum more viscous than the cerebellum. Cerebrum: white matter G″=1.21±0.21 kPa, grey matter G″=1.11±0.03 kPa; cerebellum: white matter G″=1.1±0.23 kPa, grey matter G″=0.94±0.17 kPa.DiscussionThese data represent the first available data on the viscoelastic properties of cerebellum, which suggest that the cerebellum is less physically stiff than the cerebrum, possibly leading to a different response to mechanical loading. These data will be useful for modelling of the cerebellum for a range of purposes.     

Synthesis of 18F-labelled 2-(4′-fluorophenyl)-1,3-benzothiazole and evaluation as amyloid imaging agent in comparison with [11C]PIB

2-(4′-[¹⁸F]fluorophenyl)-1,3-benzothiazole was synthesized as a fluorine-18 labelled derivative of the Pittsburg Compound-B (PIB), which has known affinity for amyloid β and promising characteristics as tracer for in vivo visualisation of amyloid deposits in patients suffering from Alzheimer’s disease (AD). Both the nitro-precursor 2-(4′-nitrophenyl)-1,3-benzothiazole and the non-radioactive reference compound were synthesized using a 1-step synthesis pathway. Labelling was achieved by direct aromatic nucleophilic substitution of the nitro-precursor using [¹⁸F]fluoride by heating for 20 min at 150 °C and with a radiochemical yield of 38%. The reference compound showed high affinity for amyloid in an in vitro competition binding study using human AD brain homogenates (K_i = 9.0 nM) and fluorescence imaging of incubated transgenic APP mouse brain slices confirmed binding to amyloid plaques. A biodistribution study in normal mice showed a high brain uptake at 2 min pi (3.20% ID/g) followed by a fast washout (60 min pi: 0.21% ID/g). A dynamic μPET study was performed in a transgenic APP and normal WT mouse, but, similar to [¹¹C]PIB, no difference was seen in tracer retention between both kind of mice. The new ¹⁸F-labelled 2-phenylbenzothiazole showed excellent preclinical characteristics comparable with those of the ¹¹C-labelled PIB.     

A new single-photon emission computed tomography (SPECT) imaging agent for serotonin transporters: [125I]Flip-IDAM, (2-((2-((dimethylamino)methyl)-4-iodophenyl)thio)phenyl)methanol

New ligands for in vivo brain imaging of serotonin transporter (SERT) with single photon emission tomography (SPECT) were prepared and evaluated. An efficient synthesis and radiolabeling of a biphenylthiol, FLIP-IDAM, 4, was accomplished. The affinity of FLIP-IDAM was evaluated by an in vitro inhibitory binding assay using [¹²⁵I]-IDAM as radioligand in rat brain tissue homogenates (K_i = 0.03 nM). New [¹²⁵I]Flip-IDAM exhibited excellent binding affinity to SERT binding sites with a high hypothalamus to cerebellum ratio of 4 at 30 min post iv injection. The faster in vivo kinetics for brain uptake and a rapid washout from non-specific regions provide excellent signal to noise ratio. This new agent, when labeled with ¹²³I, may be a useful imaging agent for mapping SERT binding sites in the human brain.     

Exocytotic release of dopamine in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice

The present study used voltammetry to ascertain whether electrically stimulated somatodendritic dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice was due to exocytosis or dopamine transporter reversal, as has been debated. The maximal concentration of electrically evoked dopamine release was similar between ventral tegmental area slices from dopamine transporter knockout and C57BL/6 mice. Dopamine transporter blockade (10 μM nomifensine) in slices from C57BL/6 mice inhibited dopamine uptake but did not alter peak evoked dopamine release. In addition, dopamine release and uptake kinetics in ventral tegmental area slices from dopamine transporter knockout mice were unaltered by the norepinephrine transporter inhibitor, desipramine (10 μM), or the serotonin transporter inhibitor, fluoxetine (10 μM). Furthermore, maximal dopamine release in ventral tegmental area slices from both C57BL/6 and dopamine transporter knockout mice was significantly decreased in response to Na⁺ channel blockade by 1 μM tetrototoxin, removal of Ca²⁺ from the perfusion media and neuronal vesicular monoamine transporter inhibition by RO-04-1284 (10 μM) or tetrabenazine (10 and 100 μM). Finally, the glutamate receptor antagonists AP-5 (50 and 100 μM) and CNQX (20 and 50 μM) had no effect on peak somatodendritic dopamine release in C57BL/6 mice. Overall, these data suggest that similar mechanisms, consistent with exocytosis, govern electrically evoked dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice.     

An LED-based UV-B irradiation system for tiny organisms: System description and demonstration experiment to determine the hatchability of eggs from four Tetranychus spider mite species from Okinawa

We developed a computer-based system for controlling the photoperiod and irradiance of UV-B and white light from a 5 × 5 light-emitting diode (LED) matrix (100 × 100 mm). In this system, the LED matrix was installed in each of four irradiation boxes and controlled by pulse-width modulators so that each box can independently emit UV-B and white light at irradiances of up to 1.5 and 4.0 W m⁻², respectively, or a combination of both light types. We used this system to examine the hatchabilities of the eggs of four Tetranychus spider mite species (T. urticae, T. kanzawai, T. piercei and T. okinawanus) collected from Okinawa Island under UV-B irradiation alone or simultaneous irradiation with white light for 12 h d⁻¹ at 25 °C. Although no eggs of any species hatched under the UV-B irradiation, even when the irradiance was as low as 0.02 W m⁻², the hatchabilities increased to >90% under simultaneous irradiation with 4.0 W m⁻² white light. At 0.06 W m⁻² UV-B, T. okinawanus eggs hatched (15% hatchability) under simultaneous irradiation with white light, whereas other species showed hatchabilities <1%. These results suggest that photolyases activated by white light may reduce UV-B–induced DNA damage in spider mite eggs and that the greater UV-B tolerance of T. okinawanus may explain its dominance on plants in seashore environments, which have a higher risk of exposure to reflected UV-B even on the undersurface of leaves. Our system will be useful for further examination of photophysiological responses of tiny organisms because of its ability to precisely control radiation conditions.     

Effect of antioxidants on microscopic semen parameters,lipid peroxidation and antioxidant activities in Angora goat semen following cryopreservation

The aim of this study was to determine the effects of the antioxidants glutamine and hyaluronan and the inclusion of different levels on microscopic semen parameters, lipid peroxidation and the antioxidant activities following the freeze–thawing of Angora goat semen. Ejaculates collected from three Angora goat bucks, were evaluated and pooled at 37 °C. The semen samples which were diluted with a Tris-based extender containing additives including glutamine (2.5; 5 mM) and hyaluronan (500; 1000 μl/ml), and an extender containing no antioxidants (control) were cooled to 5 °C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually (37 °C) for 20 s in a water bath for microscopic evaluation. Freezing extenders supplemented with 2.5 and 5 mM glutamine led to higher sperm motility and hypo-osmotic swelling test (HOST) values, compared to the control (P < 0.05) following the freeze–thawing process. The addition of 500 μl/ml hyaluronan resulted in a higher HOST percentage, compared to the addition of 1000 μl/ml hyaluronan and the control (P < 0.001). No significant difference was recorded in the percentage acrosome and total sperm abnormalities, following supplementation with antioxidants. The addition of antioxidants did not prevent malondialdehyde (MDA) formation, compared to the controls. Antioxidant treatment however decreased (P < 0.01) the superoxide dismutase (SOD) activity. The maintenance of catalase (CAT) activity was demonstrated to be insignificant following addition of antioxidants. Further studies are required to obtain more repeatable results regarding the characterization of the enzymatic and non-enzymatic antioxidant systems in cryopreserved goat sperm.     

Microbial–meiofaunal interrelationships in coastal sediments of the Red Sea

Population density and biomass of bacteria and meiofauna were investigated seasonally in the sediments of the north-western bank of Red Sea. Samples of sediments were collected seasonally from three different stations to determine microphytobenthic biomass (chlorophyll a), protein, lipid, carbohydrate, and total organic matter concentrations. These investigations revealed that microbial components tended to increase their dominancy, whereas sensitive meiofauna were extremely reduced during the entire study period. Thus a very low density of the total meiofauna (with an annual average of 109 ± 26 ind./10 cm²) was recorded whilst the benthic microbial population densities exhibited higher values (ranging from 0.31 ± 0.02 × 10⁸ to 43.67 ± 18.62 × 10⁸/g dry sediment). These changes in the relative importance analysis of benthic microbial components versus meiofaunal ones seem to be based on the impact of organic matter accumulation on the function and structure of these benthic communities. Proteins, lipids and carbohydrates showed very low concentration values, and the organic matter mostly consisted of carbohydrates, reflecting lower nutritional values for benthic fauna in general and meiofauna in particular. The distribution of microbial and meiofaunal communities seems to be dependent on the quality of the organic matter rather than on its quantity. Total organic matter concentrations varied between 5.8 and 7.6 mg/g, with organic carbon accounting for only 32% of the total organic matter. Chlorophyll a attained very low values, fluctuating between 0.11 and 0.56 μg/g, indicating the oligotrophy of the studied area. The very low concentration of chlorophyll a in the Red Sea sediment suggests that the sedimentary organic matter, heterotrophic bacteria and/or protozoa constitute an alternative resource that is consumed by meiofauna when algae are less abundant. Protozoa, therefore, represent the “missing link in bacteria–meiofauna interaction in the Red Sea marine sediment ecosystem.     

Inhibition of serotonin outflow by nociceptin/orphaninFQ in dorsal raphe nucleus slices from normal and stressed rats: Role of corticotropin releasing factor

In the dorsal raphe nucleus (DRN) many inputs converge and interact to modulate serotonergic neuronal activity and the behavioral responses to stress. The effects exerted by two stress-related neuropeptides, corticotropin releasing factor (CRF) and nociceptin/orphaninFQ (N/OFQ), on the outflow of [³H]5-hydroxytryptamine were investigated in superfused rat dorsal raphe nucleus slices.Electrical stimulation (100 mA, 1 ms for 2 min) evoked a frequency-dependent peak of [³H]5-hydroxytryptamine outflow, which was sodium and calcium-dependent. Corticotropin releasing factor (1–100 nM), concentration-dependently inhibited the stimulation (3 Hz)-evoked [³H]5-hydroxytryptamine outflow; the inhibition by 30 nM corticotropin releasing factor (to 68 ± 5.7%) was prevented both by the non selective CRF receptor antagonist alpha-helicalCRF(9-41) (α-HEL) (300 nM) and by the CRF₁ receptor antagonist antalarmin (ANT) (100 nM). The CRF₂ agonist urocortin II (10 nM) did not modify [³H]5-hydroxytryptamine outflow, ruling out the involvement of CRF₂ receptors. Bicuculline (BIC), a GABA_A antagonist (10 μM), prevented the inhibitory effect of corticotropin releasing factor (30 nM), supporting the hypothesis that the inhibition was mediated by increased γ-aminobutyric acid (GABA) release. Nociceptin/orphaninFQ (1 nM–1 μM) exerted an antalarmin- and bicuculline-insensitive inhibition on [³H]5-hydroxytryptamine outflow, with the maximum at 100 nM (to 63 ± 4.2%), antagonized by the NOP receptor antagonist UFP-101 (1 μM). Dorsal raphe nucleus slices prepared from rats exposed to 15 min of forced swim stress displayed a reduced [³H]5-hydroxytryptamine outflow, in part reversed by antalarmin and further inhibited by nociceptin/orphaninFQ. These findings indicate that (i) both corticotropin releasing factor and nociceptin/orphaninFQ exert an inhibitory control on dorsal raphe nucleus serotonergic neurons; (ii) the inhibition by corticotropin releasing factor involves γ-aminobutyric acid neurons; (iii) nociceptin/orphaninFQ inhibits dorsal raphe nucleus serotonin system in a corticotropin releasing factor- and γ-aminobutyric acid-independent manner; (iv) nociceptin/orphaninFQ modulation is still operant in slices prepared from stressed rats. The nociceptin/orphaninFQ-NOP receptor system could represent a new target for drugs effective in stress-related disorders.     

Co-activation of synaptic and extrasynaptic NMDA receptors by neuronal insults determines cell death in acute brain slice

Overactivation of NMDA receptors is linked to cell death during neuronal insults. However the precise role of synaptic and extrasynaptic NMDA receptors remains to be further determined. In this study, we used the acute brain slice to examine the contributions of synaptic and extrasynaptic NMDA receptors to neuronal death. By activation of synaptic NMDA receptors with bath application of 100 μM bicuculline in acute brain slices, we observed a significant up-regulation in activation of neuronal survival-related signaling (p-CREB, p-ERK1/2 and p-AKT), without an obvious increase of LDH release and neuronal death. Interestingly, activation of extrasynaptic NMDA receptors alone by high dose of glutamate (200 μM) following blockade of synaptic NMDA receptors with co-application of 20 μM MK801 and 100 μM bicuculline, we failed to observe inhibition of neuronal survival signaling and neuronal damage. In contrast, co-activation of synaptic and extrasynaptic NMDA receptors by applying 200 μM glutamate or oxygen–glucose deprivation (OGD) to acute brain slices for 30 min, we observed a significant inhibition of CREB, ERK1/2 and AKT activation, an increase of LDH release and neuronal condensation. Together, co-activation of synaptic and extrasynaptic NMDA receptors by neuronal insults contributes to cell death in acute brain slice.     

Male infertility: Decreased levels of selenium,zinc and antioxidants

In this study, we aimed to compare the level of zinc, selenium, glutathione peroxidase activity and antioxidant status in following populations of men: severe inflammation in prostate (>10⁶ white blood cells in prostate secretion; n = 29), severe leukocytospermia, (>10⁶ white blood cells in semen; n = 31), mild inflammation, (0.2–1 M white blood cells in semen or prostate secretion; n = 24), non-inflammatory oligozoospermia (n = 32) and healthy controls (n = 27). Male partners of infertile couples had reduced level of antioxidative activity, selenium and zinc in their seminal plasma. Most importantly, reduced selenium levels were evident in all patient groups regardless of inflammation status. Therefore, these patients might gain some benefit from selenium supplementation.     

[11C]PIB-amyloid binding and levels of Aβ40 and Aβ42 in postmortem brain tissue from Alzheimer patients

β-Amyloid (Aβ) deposits are one of the major histopathological hallmarks of Alzheimer's disease (AD). The amyloid-imaging positron emission tomography (PET) tracer [¹¹C]PIB (N-methyl[¹¹C]2-(4′-methylaminophenyl)-6-hydroxy-benzothiazole) is used in the assessment of Aβ deposits in the human brain. [¹¹C]PIB-amyloid interaction and insoluble Aβ40 and Aβ42 peptide levels in the brain were quantified in postmortem tissue from nine AD patients and nine age-matched control subjects in the temporal, frontal and parietal cortices and the cerebellum. Autoradiographical studies showed significantly higher densities of specific [¹¹C]PIB-amyloid binding in gray matter in the temporal and parietal cortex (62 fmol/mg tissue) in AD patients as compared to control subjects, whereas the density was somewhat lower in the frontal cortex (56 fmol/mg tissue). No specific binding could be detected in the AD cerebellum or in the tissues from the control subjects (≤5 fmol/mg tissue). Insoluble Aβ40 and total Aβ levels (i.e. sum of Aβ40 and Aβ42) were significantly higher in patients than in controls in all measured cortical regions as determined using ELISA, which was confirmed using immunohistochemistry. The present findings show a more regional selective distribution of [¹¹C]PIB amyloid binding than previously reported. Moreover, it is suggested that some of the [¹¹C]PIB binding and insoluble Aβ seen in control subjects may be amyloid in the blood vessels.     

Comparative study on the influence of fluoride on lipid peroxidation and antioxidants levels in the different brain regions of well-fed and protein undernourished rats

Effects of fluoride on the levels of Lipid peroxidation (LP) and antioxidant enzymes in the brain regions of protein undernourished (PU) and well-fed rats (WF) rats exposed to 100 ppm fluoride in drinking water were investigated. The results indicate that the mean body weights and the total brain weights of PU rats as well as those given fluoride (both WF and PU) were significantly (P < 0.05) lower than their respective controls. The weights of different brain regions were also significantly reduced (P < 0.05) in PU rats compared to WF rats except in the brain stem. Fluoride ingestion diminished the weights of WF and PU rats affecting the cerebrum only (in the case of PU rats) and the cerebellum of both WF and PU rats without an effect on the brain stem of both WF and PU. Additionally, increased LP was observed in the cerebrum and cerebellum of PU rats but after fluoride ingestion, 30% increase in LP was observed only in the cerebrum. In the brain stem however, protein undernutrition was accompanied with a significant reduction in LP but the region seems insensitive to fluoride. There were significant reductions (P < 0.05) in CAT, SOD and GSH in all the brain regions (except the GSH level in the brain stem only) of PU rats. Fluoride induced reduction in the activity of CAT in the three brain regions and on SOD activity in cerebrum only for WF rats but no effect of fluoride on all the antioxidants studied in the three brain regions for PU rats. It is concluded that WF and PU rats responded differently to fluoride toxicity. However, it seems that at the dosage used, fluoride toxicity may be a direct effect on the antioxidant enzymes.     

Management of white mold in processing tomatoes by Trichoderma spp. and chemical fungicides applied by drip irrigation

Field trials were carried out to evaluate six treatments combining biological agents and chemical fungicides applied via chemigation against white mold (Sclerotinia sclerotiorum) on processing tomatoes. The experiment was performed in Goiânia, Brazil, with tomato hybrid Heinz 7155 in 2009 and 2010 in a field previously infested with S. sclerotiorum sclerotia. Treatments were arranged in a randomized complete block design in a 2 × 3 factorial structure (with and without Trichoderma spp. 1.0 × 10⁹ viable conidia mL⁻¹ ha⁻¹) × fluazinam (1.0 L ha⁻¹), procimidone (1.5 L ha⁻¹) and control, applied by drip irrigation. Treatments were applied three times 10 days apart, starting one month after transplanting. Each treatment consisted of plots with three 72-meter rows with four plants m⁻¹ and 1.5 m spacing between rows, with three replications. Based on disease incidence evaluated weekly, the area under the disease progress curve (AUDPC) was obtained. Yield and its components were evaluated in addition to fruit pH and °Brix. Results were subjected to ANOVA, Scott-Knott (5%), and regression analysis. Biocontrol using Trichoderma spp. via chemigation singly or in combination with synthetic fungicides fluazinam and procimidone reduced AUDPC and increased fruit yield up to 25 t ha⁻¹. The best treatment for controlling white mold also increased pulp yield around 1.0 and 7.0 t ha⁻¹ in 2009 and 2010, respectively. The present work demonstrated the advantages of white mold biological control in processing tomato crops, where drip irrigation favored Trichoderma spp. delivery close to the plants and to the inoculum source.     

Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168

Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01 g L⁻¹ to 3.16 g L⁻¹, with a molecular weight range of 1.40×10⁶–1.83×10⁶ Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10⁶ U mL⁻¹), the production of HA was substantially increased from 5.96 g L⁻¹ to 19.38 g L⁻¹. The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10³–1.42×10⁶ Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.     

Development of a novel fluorine-18 labeled deuterated fluororasagiline ([18F]fluororasagiline-D2) radioligand for PET studies of monoamino oxidase B (MAO-B)

The objective of this study was to synthesize and evaluate a novel fluorine-18 labeled deuterium substituted analogue of rasagiline (9, [¹⁸F]fluororasagiline-D₂) as a potential PET radioligand for studies of monoamine oxidase B (MAO-B).The precursor compound (6) and reference standard (7) were synthesized in multi-step syntheses. Radiolabeling of 9 was accomplished by a two-step synthesis, compromising a nucleophilic substitution followed by hydrolysis of the sulfamidate group. The incorporation radiochemical yield from fluorine-18 fluoride was higher than 30%, the radiochemical purity was >99% and the specific radioactivity was >160 GBq/μmol at the time of administration.In vitro compound 7 inhibited the MAO-B activity with an IC₅₀ of 173.0 ± 13.6 nM. The MAO-A activity was inhibited with an IC₅₀ of 9.9 ± 1.1 μM. The fluorine-18 version 9 was characterized in the cynomolgus monkey brain where a high brain uptake was found (275% SUV at 4 min). There was a higher uptake in the striatum and thalamus compared to the cortex and cerebellum. A pronounced blocking effect (50% decrease) was observed in the specific brain regions after administration of l-deprenyl (0.5 mg/kg) 30 min prior to the administration of 9. Radiometabolite studies demonstrated 40% of unchanged radioligand at 90 min post injection.An efficient radiolabeling of 9 was successfully established and in the monkey brain 9 binds to MAO-B rich regions and its binding is blocked by the selective MAO-B compound l-deprenyl. The radioligand 9 is a potential candidate for human PET studies.     

Mitochondrial calcium uniporter blocker effectively prevents brain mitochondrial dysfunction caused by iron overload

AimsAlthough iron overload induces oxidative stress and brain mitochondrial dysfunction, and is associated with neurodegenerative diseases, brain mitochondrial iron uptake has not been investigated. We determined the role of mitochondrial calcium uniporter (MCU) in brain mitochondria as a major route for iron entry. We hypothesized that iron overload causes brain mitochondrial dysfunction, and that the MCU blocker prevents iron entry into mitochondria, thus attenuating mitochondrial dysfunction.Main methodsIsolated brain mitochondria from male Wistar rats were used. Iron (Fe^2 + and Fe^3 +) at 0–286 μM were applied onto mitochondria at various incubation times (5–30 min), and the mitochondrial function was determined. Effects of MCU blocker (Ru-360) and iron chelator were studied.Key findingsBoth Fe^2 + and Fe^3 + entered brain mitochondria and caused mitochondrial swelling in a dose- and time-dependent manner, and caused mitochondrial depolarization and increased ROS production. However, Fe^2 + caused more severe mitochondrial dysfunction than Fe^3 +. Although all drugs attenuated mitochondrial dysfunction caused by iron overload, only an MCU blocker could completely prevent ROS production and mitochondrial depolarization.SignificanceOur findings indicated that iron overload caused brain mitochondrial dysfunction, and that an MCU blocker effectively prevented this impairment, suggesting that MCU could be the major portal for brain mitochondrial iron uptake.