Cryptococcus neoformans rapidly invades the murine brain by sequential breaching of airway and endothelial tissues barriers, followed by engulfment by microglia.

Cryptococcus neoformans causes lethal meningitis and accounts for approximately 10%-15% of AIDS-associated deaths worldwide. There are major gaps in our understanding of how this fungus invades the mammalian brain. To investigate the dynamics of C. neoformans tissue invasion, we mapped fungal localization and host cell interactions in infected brain, lung, and upper airways using mouse models of systemic and airway infection. To enable this, we developed an in situ imaging pipeline capable of measuring large volumes of tissue while preserving anatomical and cellular information by combining thick tissue sections, tissue clarification, and confocal imaging. We confirm high fungal burden in mouse upper airway after nasal inoculation. Yeast in turbinates were frequently titan cells, with faster kinetics than reported in mouse lungs. Importantly, we observed one instance of fungal cells enmeshed in lamina propria of the upper airways, suggesting penetration of airway mucosa as a possible route of tissue invasion and dissemination to the bloodstream. We extend previous literature positing bloodstream dissemination of C. neoformans, by finding viable fungi in the bloodstream of mice a few days after intranasal infection. As early as 24 h post systemic infection, the majority of C. neoformans cells traversed the blood-brain barrier, and were engulfed or in close proximity to microglia. Our work presents a new method for investigating microbial invasion, establishes that C. neoformans can breach multiple tissue barriers within the first days of infection, and demonstrates microglia as the first cells responding to C. neoformans invasion of the brain.IMPORTANCECryptococcal meningitis causes 10%-15% of AIDS-associated deaths globally. Still, brain-specific immunity to cryptococci is a conundrum. By employing innovative imaging, this study reveals what occurs during the first days of infection in brain and in airways. We found that titan cells predominate in upper airways and that cryptococci breach the upper airway mucosa, which implies that, at least in mice, the upper airways are a site for fungal dissemination. This would signify that mucosal immunity of the upper airway needs to be better understood. Importantly, we also show that microglia, the brain-resident macrophages, are the first responders to infection, and microglia clusters are formed surrounding cryptococci. This study opens the field to detailed molecular investigations on airway immune response, how fungus traverses the blood-brain barrier, how microglia respond to infection, and ultimately how microglia monitor the blood-brain barrier to preserve brain function.

Cryptococcus neoformans rapidly invades the murine brain by sequential breaching of airway and endothelial tissues barriers, followed by engulfment by microglia.

The fungus Cryptococcus neoformans causes lethal meningitis in humans with weakened immune systems and is estimated to account for 10-15% of AIDS-associated deaths worldwide. There are major gaps in our understanding of how this environmental fungus evades the immune system and invades the mammalian brain before the onset of overt symptoms. To investigate the dynamics of C. neoformans tissue invasion, we mapped early fungal localisation and host cell interactions at early times in infected brain, lung, and upper airways using mouse models of systemic and airway infection. To enable this, we developed an in situ imaging pipeline capable of measuring large volumes of tissue while preserving anatomical and cellular information by combining thick tissue sections, tissue clarification, and confocal imaging. Made possible by these techniques, we confirm high fungal burden in mouse upper airway turbinates after nasal inoculation. Surprisingly, most yeasts in turbinates were titan cells, indicating this microenvironment enables titan cell formation with faster kinetics than reported in mouse lungs. Importantly, we observed one instance of fungal cells enmeshed in lamina propria of upper airways, suggesting penetration of airway mucosa as a possible route of tissue invasion and dissemination to the bloodstream. We extend previous literature positing bloodstream dissemination of C. neoformans, via imaging C. neoformans within blood vessels of mouse lungs and finding viable fungi in the bloodstream of mice a few days after intranasal infection, suggesting that bloodstream access can occur via lung alveoli. In a model of systemic cryptococcosis, we show that as early as 24 h post infection, majority of C. neoformans cells traversed the blood-brain barrier, and are engulfed or in close proximity to microglia. Our work establishes that C. neoformans can breach multiple tissue barriers within the first days of infection. This work presents a new method for investigating cryptococcal invasion mechanisms and demonstrates microglia as the primary cells responding to C. neoformans invasion.

Supplementation of protease to low amino acid diets containing superdose level of phytase for wean-to-finish pigs: effects on performance, postweaning intestinal health and carcass characteristics.

This experiment investigated the effects of protease supplementation to low amino acid (AA) diets containing phytase on pig growth performance, postweaning intestinal health and carcass characteristics. A total of 936 weaned pigs (21 d of age, initial BW 5.87 ± 0.31 kg) were used in a 2 × 2 factorial design comparing the main effects of AA supply [standard feeding program: balanced for all nutrients with adjustment of Ca and P due to inclusion of phytase (2,500 FTU/kg in Phase 1 to 4; 500 FTU/kg in Phase 5 to 9) vs. low AA feeding program: 15% lower standardized ileal digestible lysine with relative reduction of all other essential AA] and protease level (0 vs. 0.0125%). Pens were assigned to dietary treatment according to a randomized complete block design with 26 pigs per pen and nine replicates per dietary treatment. Feed and water were provided on an ad libitum basis for all phases throughout the wean-to-finish period. Feed intake and body weight were determined every 2 wk during nursery period and monthly in the grow-finish period. Intestinal health in the first 17 d was assessed based on lactulose:mannitol ratio (L:M), serum IgA, and pen diarrhea assessment. Overall, pigs fed standard wean-to-finish diets had greater (P < 0.05) ADG and G:F than pigs fed low AA diets. Pig growth performance was not different throughout the wean-to-finish period with or without protease supplementation and with no interaction between AA supply and protease supplementation. There were no differences among dietary treatments for carcass characteristics. No difference was observed for urinary L:M and serum IgA; however, the L:M ratio was approximately 32% lower in pigs fed low AA diets + protease compared with pigs fed standard and low AA diets at d 5 and d 17 postweaning. Pigs fed protease supplemented diets had lower incidence of diarrhea (χ 2 < 0.05) compared with pigs fed diets without protease. Results of the experiment indicate that dietary protease supplementation benefits intestinal health of nursery pigs.

Evaluating dietary adjustment and collection times for total tract digestibility of Ca, P, and the essential microminerals with grower swine.

An experiment was conducted to determine 1) the length of time necessary for grower pigs to adjust to a new diet and 2) the consistency in excretion of urine and feces in 4 consecutive 5-d collection periods. The total tract excretion and digestibility values for Ca, P, and the essential microminerals were evaluated. The experiment was conducted in 6 replicates as a randomized complete block design. Pigs were fed a pretest diet from 20 to 40 kg BW that met the requirements. At 40 kg, 12 barrows were allotted to stainless-steel metabolism crates, where they continued being fed the pretest diet for a 7-d period for adjustment purposes. Treatment diets were then fed for the following 20-d period in four 5-d intervals. Treatment diets were a corn-soybean meal mixture and contained either 1) reduced Ca and P levels and no added microminerals (LOW) or 2) a diet with elevated Ca and P levels and supplemental microminerals that exceeded the pig's requirements (HIGH). The study collected urine and feces. Markers were added to the ration at the start of each period to distinguish between test intervals. Feces and urine were collected daily, frozen, and composited for each period. Analysis of diets and excrement was conducted using inductively coupled plasma mass spectrometry technology. In all cases, the excreted minerals and digestibility values were greater ( < 0.01) for the macrominerals when the HIGH diet was fed, whereas the digestibility values for the microminerals were often lower when the HIGH diet was fed. The macrominerals Ca and P both had consistent urine and fecal values for each of the final 3 collection periods within diet. The micromineral values were generally consistent for each 5-d collection period but varied between periods for several microminerals. These results indicate that a 5-d adjustment period was adequate for pigs to adjust to the treatment diets. A 5-d collection period was adequate for Ca and P, but the micromineral excretion and digestibility values were more variable, and a collection period of 10 d might be warranted. The results also indicated that the innate microminerals had a higher digestibility and bioavailability than thought previously and their digestibility is greater than that of inorganic microminerals. Thus, these results indicate that the innate microminerals should be an important factor in establishing the micromineral requirements for growing pigs.

Intestinal gene expression profiles of piglets benefit from maternal supplementation with a yeast mannan-rich fraction during gestation and lactation.

The objective was to study the effect of maternal supplementation with a yeast cell wall-based product containing a mannan-rich fraction (MRF) during gestation and lactation on piglet intestinal gene expression. First parity sows were fed experimental gestation and lactation diets with or without MRF (900 mg/kg). After farrowing, piglets were fostered within treatment, as necessary. Sow and litter production performance data were collected until weaning. On day 10 post farrowing, jejunum samples from piglets were collected for gene expression analysis using the Affymetrix Porcine GeneChip array. Most performance parameters did not differ between the treatments. However, protein (P<0.01), total solids less fat (P<0.03) and the concentration of immunoglobulin G (IgG) in milk were greater (P<0.05) in the MRF-supplemented group. Gene expression results using hierarchical clustering revealed an overall dietary effect. Further analysis elucidated activation of pathways involved in tissue development, functioning and immunity, as well as greater cell proliferation and less migration of cells in the jejunum tissue. In conclusion, feeding the sow MRF during pregnancy and lactation was an effective nutritional strategy to bolster colostrum and milk IgG that are essential for development of piglet immune system and gut. In addition, the gene expression patterns affected by the passive immunity transfer showed indicators that could benefit animal performance long term.

Organic trace mineral levels in the first 96-h post-hatch impact growth performance and intestinal gene expression in broiler chicks.

Alterations in nutrient intake in the avian neonatal posthatch period can impact development, performance, and metabolism in adulthood. Very little is known about how mineral levels during the post-hatch period affect or "program" gene expression patterns later in life. The objective of this study was to determine the effect of post-hatch (0 to 96 h) dietary mineral supplementation on performance, tissue mineral content, and intestinal gene expression profiles in 21-day-old broiler chicks. One-day-old chicks were randomly assigned to one of two treatment groups consisting of N (organic Zn, Cu, and Mn provided at 100 % of recommendations (National Research Council 1994)) and/or L (organic Zn, Cu, and Mn provided at 20 % of recommendations (National Research Council 1994)) diets fed in two intervals (days 1­4, days 5­21) as follows: (1)N­Lor (2)L­L. Performance parameters did not differ between treatments except that body weight gain was greater (P < 0.05) in L­L birds than N­L birds over the experimental period. Bone mineral content was similar for both treatments at day 21. Intestinal gene expression profiling was examined using the Affymetrix GeneChip Chicken genome array. Ingenuity pathway analysis revealed differences in gene expression profiles between N and L treatments at day 5. At day 21, profiles were unique between N­L and L­L, suggesting that the diet fed until day 4 had an impact on gene expression patterns at day 21 even when birds were fed the same diets day 5­day 21. In this study, we demonstrated that diets fed for the 96 h post-hatch had long-term effects on gene expression, providing unique information as to why post-hatch diets are so important for the longterm bird health and productivity.

Rearrangement of cyclotriveratrylene (CTV) diketone: 9,10-diarylanthracenes with OLED applications.

Electroluminescent 9,10-diaryl anthracenes have been shown to be promising host and hole-transporting materials in organic electroluminescence due to their high thermal stability, electrochemical reversibility, and wide band gap useful for organic light-emitting diodes (OLEDs), especially blue OLEDs. Oxidation of cyclotriveratrylene (CTV) to the corresponding diketone and subsequent bromination resulted in an unexpected rearrangement to a highly functionalized 9-aryl-10-bromoanthracene derivative, which was employed in Suzuki couplings to synthesize a series of 9,10-diaryl compounds that are structural analogues of anthracene derivatives used in the preparation of OLEDs but are more highly functionalized, including electron-donating methoxy groups in addition to substitution by a carboxylic acid moiety. The UV/fluorescence solution spectra show strong emissions at 446, 438, and 479 nm, respectively, for the anthracene 10-phenyl, 10-naphthyl, and 10-pyrenyl adducts containing a benzoic acid functional group, whereas the analogues bearing the hydroxymethylene moiety from reduction of the benzoic acid to the corresponding alcohols gave much shorter emission wavelengths of 408, 417, and 476 nm, respectively, and had somewhat higher quantum yields, suggesting they are better candidates for OLED applications.

Dietary lysine requirement of sows increases in late gestation.

Current AA recommendations for sows are to provide a fixed amount of AA intake throughout gestation; however, the demand for nutrients changes from maternal lean tissue in early gestation (EG) to fetal and mammary growth in late gestation (LG). The objective of this study was to determine the Lys requirement in EG (d 24 to 45) and LG (d 86 to 110) using the indicator AA oxidation method with simultaneous determination of heat production. Each of 7 Large White × Landrace sows received 6 diets in random order in both EG and LG. Three semisynthetic diets (14.0 MJ ME/kg) based on corn were formulated and mixed to produce a basal diet (60% of 1998 NRC Lys requirement) and high diets for EG and LG (150% and 185% of 1998 NRC Lys requirements, respectively). The 6 test diets provided Lys intakes of 7.5 to 19.3 g/d in EG and 8.1 to 23.7 g/d in LG. Sows were placed in respiration chambers, and expired air and blood were collected every 30 min for 5.5 h. The tracer AA, l-[1-(13)C]Phe, was given orally at a rate of 2 mg/(kg BW ⋅ h) over the last 4 h, divided into 8, 0.5-h meals. Expired air was measured for (13)CO(2) enrichment, and plasma was measured for l-[1-(13)C]Phe enrichment and free Lys concentration. Background (13)CO(2) was subtracted from plateau (13)CO(2) enrichment. Requirements were determined using a 2-phase nonlinear model. Mean maternal BW gain in gestation (43.7 kg; pooled SE, 1.2 kg), litter size (14.6 total born piglets; pooled SE, 0.8), and litter weight (19.4 kg; pooled SE, 0.9 kg) did not differ between parities. Sow weight gain and BW was greater (P = 0.001) in LG than EG. Lysine requirement was 9.4 and 17.4 g/d in EG and LG, respectively. Phenylalanine retention in LG was maximized at a Lys intake of 17.7 g/d. Heat production was more (P = 0.069) and energy retention less (P = 0.019) in LG than EG. Energy retention in LG was not different from 0. Quantitative Phe kinetics in EG were not affected by Lys intake. In LG, Phe retention increased with Lys intake (P = 0.004), whereas Phe oxidation decreased (P = 0.005). The Lys requirement was determined to be less than current recommendations in EG and more than current recommendations in LG. To meet the change in requirements, diets with increased lysine content are needed in LG. Increasing the feed allowance in LG is necessary to maintain a positive energy balance throughout gestation.

Semiautomated detection of cerebral microbleeds in magnetic resonance images.

Cerebral microbleeds (CMBs) are increasingly being recognized as an important biomarker for neurovascular diseases. So far, all attempts to count and quantify them have relied on manual methods that are time-consuming and can be inconsistent. A technique is presented that semiautomatically identifies CMBs in susceptibility weighted images (SWI). This will both reduce the processing time and increase the consistency over manual methods. This technique relies on a statistical thresholding algorithm to identify hypointensities within the image. A support vector machine (SVM) supervised learning classifier is then used to separate true CMB from other marked hypointensities. The classifier relies on identifying features such as shape and signal intensity to identify true CMBs. The results from the automated section are then subject to manual review to remove false-positives. This technique is able to achieve a sensitivity of 81.7% compared with the gold standard of manual review and consensus by multiple reviewers. In subjects with many CMBs, this presents a faster alternative to current manual techniques at the cost of some lost sensitivity.

Impact of the transcriptional regulator, Ace2, on the Candida glabrata secretome.

Candida glabrata is a major fungal pathogen of humans, and the virulence of C. glabrata is increased by inactivation of the transcription factor, Ace2. Our previous examination of the effects of Ace2 inactivation upon the intracellular proteome suggested that the hypervirulence of C. glabrata ace2 mutants might be caused by differences in the secretome. Therefore in this study we have characterised the C. glabrata secretome and examined the effects of Ace2 inactivation upon this extracellular proteome. We have identified 31 distinct proteins in the secretome of wild-type C. glabrata cells by MS/MS of proteins that were precipitated from the growth medium and enriched by affinity chromatography on concanavalin A. Most of these proteins are predicted to be cell wall proteins, cell wall modifying enzymes and aspartyl proteinases. The endochitinase Cts1 and the endoglucanase Egt2 were not detected in the C. glabrata secretome following Ace2 inactivation. This can account for the cell separation defect of C. glabrata ace2 cells. Ace2 inactivation also resulted in the detection of new proteins in the C. glabrata secretome. The release of such proteins might contribute to the hypervirulence of ace2 cells.

Imaging the vessel wall in major peripheral arteries using susceptibility-weighted imaging.

PURPOSE: To demonstrate a novel contrast mechanism for imaging the vessel wall and vessel wall calcification using susceptibility-weighted imaging (SWI). MATERIALS AND METHODS: Eighteen subjects were imaged with multidetector computed tomography (MDCT) and high-resolution SWI at 3T. The SWI imaging parameters were optimized to allow for the best visualization of the femoral artery lumen and the arterial wall in magnitude and phase images, respectively. SWI-filtered phase data were used to evaluate the diamagnetic susceptibility of vessel wall and of putative vessel wall calcification. Imaging was performed using TE = 15.6 msec (in-phase for fat); TR = 25 msec, flip angle (FA) = 10 degrees , bandwidth (BW) = 80 Hz/pixel, resolution = 0.5 x 0.5 mm in-plane and 1.0 mm through-plane, an acquisition matrix of 512 x 384 x 64 (for read, phase, and slice-select directions), and a total scan time of 8 minutes. RESULTS: Nineteen calcifications were identified in CT and SWI and they correlated well in both size and position. The contrast-to-noise ratio between the blood signal in the lumen of the artery and arterial wall was 11.7:1 and 7.4:1 in magnitude and in phase images, respectively. CONCLUSION: SWI provides a novel means to visualize vessel wall and recognize the presence of calcification.

Susceptibility-weighted imaging: clinical angiographic applications.

By combining filtered phase and magnitude information to create a novel and intrinsic source of contrast, susceptibility-weighted imaging (SWI) has shown great promise in clinical angiography and venography. SWI has contributed to new insights into traumatic brain injury, the role of calcification in atherosclerosis, and the possible relationship between blood settling and deep venous thrombosis. A further contribution from SWI to deep venous thrombosis research (and also stroke) involves its application to the noninvasive measurement of oxygen saturation in the brain and in other tissues. Altogether, SWI offers manifold and diverse avenues for further research using angiographic and venographic techniques.

Congestive heart failure with preserved systolic function: is it a woman's disease?

Congestive heart failure with preserved systolic function is increased in prevalence with advancing age, especially in women, indicating the strong impact of gender on this common disease.