Breast-to-brain metastasis is exacerbated with chemotherapy through blood-cerebrospinal fluid barrier and induces Alzheimer's-like pathology.

Control of breast-to-brain metastasis remains an urgent unmet clinical need. While chemotherapies are essential in reducing systemic tumor burden, they have been shown to promote non-brain metastatic invasiveness and drug-driven neurocognitive deficits through the formation of neurofibrillary tangles (NFT), independently. Now, in this study, we investigated the effect of chemotherapy on brain metastatic progression and promoting tumor-mediated NFT. Results show chemotherapies increase brain-barrier permeability and facilitate enhanced tumor infiltration, particularly through the blood-cerebrospinal fluid barrier (BCSFB). This is attributed to increased expression of matrix metalloproteinase 9 (MMP9) which, in turn, mediates loss of Claudin-6 within the choroid plexus cells of the BCSFB. Importantly, increased MMP9 activity in the choroid epithelium following chemotherapy results in cleavage and release of Tau from breast cancer cells. This cleaved Tau forms tumor-derived NFT that further destabilize the BCSFB. Our results underline for the first time the importance of the BCSFB as a vulnerable point of entry for brain-seeking tumor cells post-chemotherapy and indicate that tumor cells themselves contribute to Alzheimer's-like tauopathy.

Storing and releasing rhodamine as a model hydrophobic compound in polydimethylsiloxane microfluidic devices.

Polydimethylsiloxane (PDMS) is a ubiquitous material used in soft lithography and microfluidics. Due to its hydrophobic nature, PDMS tends to absorb small hydrophobic molecules, and is seen as a major disadvantage of the material in pharmaceutical and cell culture studies. While there have been extensive reports of attempts to treat PDMS to limit or block this absorption, little attention has been given to using this property as a feature in microfluidic devices. In this work, we leverage the ability of PDMS to store hydrophobic molecules inside the PDMS matrix and release them over time in a sustained manner.

Attenuation of oxidative hemolysis of human red blood cells by the natural phenolic compound, allylpyrocatechol.

The protecting ability of the Piper betle leaves-derived phenol, allylpyrocatechol (APC) against AAPH-induced membrane damage of human red blood cells (RBCs) was investigated. Compared to control, AAPH (50 mM) treatment resulted in significant hemolysis (55%, p < 0.01), associated with increased malondialdehyde (MDA) (2.9-fold, p < 0.001) and methemoglobin (6.1-fold, p < 0.001) levels. The structural deformation due to membrane damage was confirmed from scanning electron microscopy (SEM) images and Heinz bodies formation, while the cell permeability was evident from the K(+) efflux (28.7%, p < 0.05) and increased intracellular Na(+) concentration (8%, p < 0.05). The membrane damage, due to the reduction of the cholesterol/phospholipids ratio and depletion (p < 0.001) of ATP, 2,3-DPG by ˜44-54% and Na(+)-K(+) ATPase activity (43.7%), indicated loss of RBC functionality. The adverse effects of AAPH on all these biochemical parameters and the resultant oxidative hemolysis of RBCs were significantly reduced by pretreating the cells with APC (7 µM) or α-tocopherol (50 µM) for 1 h, prior to incubation with AAPH.

Classical density functional theory and the phase-field crystal method using a rational function to describe the two-body direct correlation function.

We introduce a new approach to represent a two-body direct correlation function (DCF) in order to alleviate the computational demand of classical density functional theory (CDFT) and enhance the predictive capability of the phase-field crystal (PFC) method. The approach utilizes a rational function fit (RFF) to approximate the two-body DCF in Fourier space. We use the RFF to show that short-wavelength contributions of the two-body DCF play an important role in determining the thermodynamic properties of materials. We further show that using the RFF to empirically parametrize the two-body DCF allows us to obtain the thermodynamic properties of solids and liquids that agree with the results of CDFT simulations with the full two-body DCF without incurring significant computational costs. In addition, the RFF can also be used to improve the representation of the two-body DCF in the PFC method. Last, the RFF allows for a real-space reformulation of the CDFT and PFC method, which enables descriptions of nonperiodic systems and the use of nonuniform and adaptive grids.

Shelf-life evaluation of natural antimicrobials for Concord and Niagara grape juices.

This study was conducted to evaluate the effectiveness of natural antimicrobials for shelf-life extension of cold-filled still and carbonated Concord and Niagara grape juices, which have traditionally been preserved with chemical preservatives. Commercial juices were inoculated with a spoilage yeast cocktail of Dekkera, Kluveromyces, Brettanomyces, and Zygosaccharomyces at 10(2) and 10(4) CFU/ml. The following agents were added to still juices: no preservative (negative control), 0.05% potassium sorbate plus 0.05% sodium benzoate (positive control), 0.1 or 0.2% cultured dextrose, 250 ppm of dimethyldicarbonate (DMDC), 10 or 20 ppm of natamycin, and 250 ppm of DMDC plus 5 or 10 ppm of natamycin. Carbonated juice was treated with the negative control, positive control, and 250 ppm of DMDC plus 10 ppm of natamycin. Microbial stability of samples was assessed every 2 weeks during 6 months of storage at 21°C by yeast enumeration and measurement of turbidity, pH, and °Brix. Juices were deemed spoiled when yeast counts exceeded 10(6) CFU/ml. Cultured dextrose was not effective at levels tested in both types of juice. The most promising results were obtained with DMDC and natamycin combination treatments in still Niagara juice and in carbonated Concord and Niagara juices. In these treatments, shelf-life extension similar to that of the positive control (153 to 161 days) was achieved while maintaining similar turbidity, pH, and °Brix. Spoiled juices had lower pH and °Brix values and higher turbidity due to microbial activity and increased in microbial levels.

Impact of harvesting and processing conditions on green leaf volatile development and phenolics in Concord grape juice.

UNLABELLED: The disruption of plant cell walls during fruit juice processing results in the enzymatic formation of herbaceous-smelling green leaf volatiles (GLVs). Our objective was to assess the impact of thermal processing conditions on resulting levels of GLVs (hexanal, trans-2-hexenal, hexanol, cis-3-hexenol, and trans-2-hexenol), total phenols, monomeric anthocyanins, and percent polymeric color in Concord grape juice. The effects of fruit maturity and stage of juice processing on juice GLV content was also assessed. Of the GLVs studied, only trans-2-hexenal routinely exceeded its published sensory threshold in finished juice. We observed an inverse linear correlation between berry maturity (total soluble solids) and trans-2-hexenal levels in finished juice (P < 0.05, R(2)= 0.91). Trans-2-hexenal was at a maximum immediately following crushing (569 microg/kg, >30-fold over detection threshold [DT]), decreased to 100 microg/kg following depectinization, pressing, and pasteurization, and to 32 microg/kg following cold-stabilization. The loss of trans-2-hexenal could be explained primarily by its reduction to trans-2-hexenol, which increased from 53 microg/kg after crushing to 500 microg/kg after cold-stabilization. High temperature pretreatment of must immediately following crushing ("hot break") resulted in 5- to 6-fold higher concentrations of trans-2-hexenal in the final bottled juice as compared to conventional hot press. Contrary to expectations, no significant increase in phenolics and anthocyanins were observed in hot break conditions. These results indicate that hot break procedures may thermally inactivate enzymes responsible for transforming trans-2-hexenal under normal processing conditions and potentially alter the flavor qualities of the finished Concord juice. Different equivalent pasteurization regimes (82 to 93 degrees C) prior to bottling had no significant effect on GLV content of the finished Concord juices (P > 0.05). PRACTICAL APPLICATION: Introducing new processing techniques to fruit juice production can potentially result in undesirable changes to organoleptic properties. We have observed significantly higher levels of trans-2-hexenal, a potent herbaceous off-flavor, in Concord grape juice prepared with an initial high temperature heat treatment ("hot break"). Concord juice producers should be cautious in using hot break processing, especially with immature fruit, as it may result in persistence of green aromas in juice.

Non-invasive imaging of a transgenic mouse model using a prostate-specific two-step transcriptional amplification strategy.

Non-invasive assessment of transgenic animals using bioluminescence imaging offers a rapid means of evaluating disease progression in animal models of disease. One of the challenges in the field is to develop models with robust expression to image repetitively live intact animals through solid tissues. The prostate-specific antigen (PSA) promoter is an attractive model for studying gene regulation due to its hormonal response and tissue-specificity permitting us to measure signaling events that occur within the native tissues. The use of the GAL4-VP16 activator offers a powerful means to augment gene expression levels driven by a weak promoter. We have used a two-step transcriptional amplification (TSTA) system to develop a transgenic mouse model to investigate the tissue-specificity and developmental regulation of firefly luciferase (fl) gene expression in living mice using bioluminescence imaging. We employed an enhanced prostate-specific promoter to drive the yeast transcriptional activator, GAL4-VP16 (effector). The reporter construct carries five Gal4 binding sites upstream of the fl gene. We generated a transgenic mouse model using a single vector carrying the effector and reporter constructs. The transgenic mice show prostate-specific expression as early as three weeks of age. The bioluminescence signal in the prostate is significantly higher than in other organs. We also demonstrate that blocking androgen availability can downregulate the fl expression in the prostate. The transgenic mice display normal physical characteristics and developmental behavior, indicating that the high level of GAL4 driven expression is well tolerated. These findings suggest that the GAL4-VP16 transactivator can be used to amplify reporter gene expression from a relatively weak promoter in a transgenic mouse model. The transgenic TSTA model in conjunction with other transgenic cancer models should also help to detect and track malignancies. The strategies developed will be useful for transgenic research in general by allowing for amplified tissue specific gene expression.

Safety and cognitive effect of frontal DC brain polarization in healthy individuals.

BACKGROUND: Data from the human motor cortex suggest that, depending on polarity, direct current (DC) brain polarization can depress or activate cortical neurons. Activating effects on the frontal lobe might be beneficial for patients with frontal lobe disorders. This phase 1 study tested the safety of frontal DC, including its effects on frontal and other brain functions. METHODS: The authors applied 20 minutes of anodal, cathodal, or sham DC to the left prefrontal cortex in three groups of right-handed subjects and looked for effects on global measures of processing and psychomotor speed, emotion, and verbal fluency, a measure of local cortical function. In one experiment (n = 30), the authors tested before and after 1 mA DC and monitored EEG in 9 subjects. In two other experiments using 1 mA (n = 43) and 2 mA (n = 30), the authors tested before and then starting 5 minutes after the onset of DC. RESULTS: All subjects tolerated DC well. There were no significant effects on performance with 1 mA DC. At 2 mA, verbal fluency improved significantly with anodal and decreased mildly with cathodal DC. There were no clinically significant effects on the other measures. CONCLUSIONS: Limited exposure to direct current polarization of the prefrontal cortex is safe and can enhance verbal fluency selectively in healthy subjects. As such, it deserves consideration as a procedure to improve frontal lobe function in patients.

Optical imaging of transferrin targeted PEI/DNA complexes in living subjects.

Noninvasive optical bioluminescence imaging systems are important tools for evaluating gene expression in vivo for study of individual and temporal variation in a living animal. In this report, we demonstrate that expression of the firefly luciferase reporter gene (fl) delivered by transferrin (Tf) targeted polyethylenimine (PEI) complexes with, or without, poly(ethylene glycol) (PEG) modifications can be imaged in living A/J mice bearing N2A tumors using a cooled charged coupled device (CCD) camera. Tf-PEI-PEG, Tf-PEI, and PEI (positive control) complexes were tail-vein injected and mice were imaged at 5, 24, 48, and 72 h after complex injection. After imaging, the organs were analyzed ex vivo for firefly luciferase protein (FL) activity. The Tf and PEG modified formulations show significantly (P<0.05) higher FL activity in vivo and ex vivo at the tumor as compared to other organs, including the lungs (a site of high expression with PEI, the positive control). Furthermore, the in vivo bioluminescent signal correlated well (R(2)=0.83) with ex vivo FL activity. These data support that noninvasive imaging of fl reporter expression can be used to monitor the specificity of Tf-PEI and Tf-PEI-PEG polyplex targeting of N2A tumors in A/J mice.

Immunological detection of Fusarium species in cornmeal.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect Fusarium species in foods. Antibodies to proteins extracted from the mycelia of Fusarium graminearum and Fusarium moniliforme (verticillioides) were produced in New Zealand white rabbits. These antibodies detected 13 Fusarium species in addition to the producer strains. Levels of Fusarium semitectum and Fusarium tricinctum strains were below the detection threshold. The specificity of the assay was tested against 70 molds and yeasts belonging to 23 genera. One strain of Monascus species and one strain of Phoma exigua were detected; however, these two molds are not common contaminants of cereal grains or foods and should not interfere with the assay. The indirect ELISA's detection limits for F. graminearum and F. moniliforme were 0.1 and 1 microg of mold mycelium per ml of a cornmeal mixture, respectively. When spores of each mold were added individually to cornmeal mixtures (at ca. 10 spores per g) and incubated at 25 degrees C, these spores were detected by the indirect ELISA when they reached levels of 10(2) to 10(3) CFU/ml after 24 to 36 h. The indirect ELISA developed here shows promise for the detection of Fusarium species in grains or foods.

Noninvasive quantitative imaging of protein-protein interactions in living subjects.

We are developing methods to image molecular and cellular events in living subjects. In this study, we validate imaging of protein-protein interactions in living mice by using bioluminescent optical imaging. We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible. We employ the NF-kappaB promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID). We modulate the NF-kappaB promoter through tumor necrosis factor alpha. Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy. We demonstrate the ability to detect this induced protein-protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells. The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein-protein interactions in living subjects. The approaches validated should have important implications for the study of protein-protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein-protein interactions.

Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography.

We have used copper-64-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM) to radiolabel cells ex vivo for in vivo positron-emission tomography (PET) imaging studies of cell trafficking in mice and for eventual application in patients. 2-[18F]-Fluoro-2-deoxy-d-glucose (FDG) cell labeling also was evaluated for comparison. 64Cu-PTSM uptake by C6 rat glioma (C6) cells increased for 180 min and then stabilized. The labeling efficiency was directly proportional to 64Cu-PTSM concentration and influenced negatively by serum. Label uptake per cell was greater with 64Cu-PTSM than with FDG. However, both 64Cu-PTSM- and FDG-labeled cells showed efflux of cell activity into supernatant. The 64Cu-PTSM labeling procedure did not interfere significantly with C6 cell viability and proliferation rate. MicroPET images of living mice indicate that tail-vein-injected labeled C6 cells traffic to the lungs and liver. In addition, transient splenic accumulation of radioactivity was clearly detectable in a mouse scanned at 3.33 h postinfusion of 64Cu-PTSM-labeled lymphocytes. In contrast, the liver was the principal organ of tracer localization after tail-vein administration of 64Cu-PTSM alone. These results indicate that in vivo imaging of cell trafficking is possible with 64Cu-PTSM-labeled cells. Given the longer t(1/2) of 64Cu (12.7 h) relative to 18F (110 min), longer cell-tracking periods (up to 24-36 h) should be possible now with PET.

Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters.

We are developing assays to image tissue-specific reporter gene expression in living mice by using optical methods and positron emission tomography. Approaches for imaging reporter gene expression depend on robust levels of mRNA and reporter protein. Attempts to image reporter gene expression driven by weak promoters are often hampered by the poor transcriptional activity of such promoters. Most tissue-specific promoters are weak relative to stronger but constitutively expressing viral promoters. In this study, we have validated methods to enhance the transcriptional activity of the prostate-specific antigen promoter for imaging by using a two-step transcriptional amplification (TSTA) system. We used the TSTA system to amplify expression of firefly luciferase (fl) and mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) in a prostate cancer cell line (LNCaP). We demonstrate approximately 50-fold (fl) and approximately 12-fold (HSV1-sr39tk) enhancement by using the two-step approach. The TSTA system is observed to retain tissue selectivity. A cooled charge-coupled device optical imaging system was used to visualize the amplified fl expression in living mice implanted with LNCaP cells transfected ex vivo. These imaging experiments reveal a approximately 5-fold gain in imaging signal by using the TSTA system over the one-step system. The TSTA approach will be a valuable and generalizable tool to amplify and noninvasively image reporter gene expression in living animals by using tissue-specific promoters. The approaches validated should have important implications for study of gene therapy vectors, cell trafficking, transgenic models, as well as studying development of eukaryotic organisms.

Monitoring gene therapy with reporter gene imaging.

Rapid advances in imaging technologies and gene transfer strategies offer a great opportunity to optimize clinical trials of human gene therapy. Reporter genes are emerging as very powerful tools to monitor the delivery, magnitude, and time variation of therapeutic gene transfer in vivo. Several reporter genes, such as the herpes simplex virus type 1 thymidine kinase, the dopamine type 2 receptor, and the somatostatin receptor type 2, are currently being successfully used with gamma camera, single photon emission computed tomography, and positron emission tomography imaging. These reporter genes can be coupled with a therapeutic gene of interest to indirectly monitor the expression of the therapeutic gene. Finally, applications of the reporter gene technology to other areas, such as cell trafficking studies and transgenic animal models, are now possible.

Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice.

The ability to monitor reporter gene expression noninvasively offers significant advantages over current techniques such as postmortem tissue staining or enzyme activity assays. Here we demonstrate a novel method of repetitively tracking in vivo gene expression of firefly luciferase (FL) in skeletal muscles of mice using a cooled charged coupled device (CCD) camera. We first show that the cooled CCD camera provides consistent and reproducible results within +/-8% standard deviation from mean values, and a detection sensitivity (range tested: 1 x 10(4) - 1 x 10(9) plaque form-ing units (pfu)) of 1 x 10(6) pfu of E1-deleted adenovirus expressing FL driven by a cytomegalovirus promoter (Ad-CMV-FL). The duration and magnitude of adenoviral mediated (1 x 10(9) pfu) FL gene expression were then followed over time. FL gene expression in immunocompetent Swiss Webster mice peaks within the first 48 hours, falls by 98% after 20 days, and persists for >150 days. In contrast, FL activity in nude mice remains elevated for >110 days. Finally, transduced Swiss Webster and nude mice were sacrificed to show that the in vivo CCD signals correlate well with in vitro luciferase enzyme assays (r(2)=0.91 and 0.96, respectively). Our findings demonstrate the ability of the cooled CCD camera to sensitively and noninvasively track the location, magnitude, and persistence of FL gene expression. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.

Human pharmacokinetic and dosimetry studies of [(18)F]FHBG: a reporter probe for imaging herpes simplex virus type-1 thymidine kinase reporter gene expression.

UNLABELLED: 9-[4-[(18)F]fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]FHBG) has been used as a reporter probe to image expression of herpes simplex virus type-1 thymidine kinase (HSV1-tk) reporter gene in living animals. Our aim was to study the kinetics, biodistribution, stability, dosimetry, and safety of [(18)F]FHBG in healthy human volunteers, preparatory to imaging patients undergoing HSV1-tk gene therapy. METHODS: [(18)F]FHBG was synthesized with a specific activity of 37,000--444,000 GBq/mmol and a radiochemical purity > 99%. Ten healthy volunteers consented to participate in the study. A transmission scan was obtained before bolus injection of 70.3--229.4 MBq [(18)F]FHBG into a hand vein, followed by dynamic PET imaging with 4 consecutive emission scans. Warmed hand-vein blood was withdrawn at various times after injection for blood time--activity measurements. Electrocardiography, blood pressure, and blood and urine pharmacologic parameters were measured before and after injection of the [(18)F]FHBG tracer (n = 5). The stability of [(18)F]FHBG in the urine was analyzed. Attenuation-corrected images were reconstructed using the ordered-subsets expectation maximization algorithm. Image region-of-interest time-activity data were used with the MIRD program to estimate absorbed radiation dosages. RESULTS: [(18)F]FHBG had rapid blood clearance; only 8.42% +/- 4.76% (mean +/- SD) of the peak blood activity remained at approximately 30 min. The average ratio of plasma activity to whole-blood activity during the study was 0.91 +/- 0.04. Penetration of [(18)F]FHBG across the blood-brain barrier was not observed. The primary routes of clearance were renal and hepatobiliary. High activities were observed in the bladder, gut, liver, and kidneys, but <0.0002% of the injected dose per gram was observed in other tissues. In the urine, 83% of activity 180 min after injection was stable [(18)F]FHBG. Blood and urine pharmacologic parameters did not change significantly after injection of the [(18)F]FHBG tracer. The bladder absorbed the highest radiation dose. CONCLUSION: [(18)F]FHBG has the desirable in vivo characteristics of stability, rapid blood clearance, low background signal, biosafety, and acceptable radiation dosimetry in humans. This study forms the foundation for using [(18)F]FHBG in applications to monitor HSV1-tk reporter gene expression.

Post-traumatic endophthalmitis involving Clostridium tetani and Bacillus spp.

PURPOSE: To report a case of post-traumatic infectious endophthalmitis caused by Clostridium tetani and Bacillus spp. METHODS: Case report. RESULTS: A 25-year-old man developed endophthalmitis after a traumatic corneoscleral laceration of his right eye by a concrete reinforcement bar. He underwent pars plana lensectomy and vitrectomy with aspiration of vitreous fluid and a conjunctival swab for cultures. Cultures from the conjunctival swab were negative for organisms. Cultures of the vitreous aspirate were positive for Bacillus species and C. tetani. He had received a tetanus toxoid booster at the emergency department. By the time the culture results became available, he had developed severe eye pain associated with marked orbital congestion, increased swelling and erythema of the lids, marked injection and chemosis of the conjunctiva, and subsequently underwent evisceration. The inflammation resolved after evisceration of the right eye, and he was discharged to home on doxycycline 100 mg orally two times daily for 10 days. CONCLUSION: We are unaware of previous reports of endophthalmitis involving C tetani and could find none in a computerized MEDLINE search. Patients with penetrating eye injury should be assessed for tetanus immunization status, and early intervention with tetanus toxoid booster and/or tetanus immune globulin should be considered if cultures are positive.

Rate dependence of human visual cortical response due to brief stimulation: an event-related fMRI study.

The human brain response to a wide range of visual stimulus rates presented over a prolonged time period has been investigated by various neuroimaging techniques. However, to date, no imaging study has been performed to study the dynamic human brain response to various stimulus rates when presented in a short time. This report describes activation in the human brain due to brief visual stimulus presentation (1 s) for stimulus rates varying from 1 to 20 Hz using event-related functional MRI (fMRI). Our results show that the amplitude of the fMRI response increases with the stimulus frequency and plateaus at 6 Hz. This finding differs slightly from the results of previous blocked task paradigm experiments (with a longer time of stimulus presentation), in which the response peaks at approximately 8 Hz and then decreases. Our results are in close agreement with previously published psychophysical studies, suggesting that the fMRI signal in this experiment is indicative of cortical activity related to visual processing.