Matrix-associated Laser Desorption Ionization Research Articles

Characterization of the effect of histone deacetylation inhibitors on CD8+ T cells in the context of aging

BackgroundPosttranslational protein modifications regulate essential cellular processes, including the immune cell activation. Despite known age-related alterations of the phenotype, composition and cytokine profiles of immune cells, the role of acetylation in the aging process of the immune system was not broadly investigated. Therefore, in the current study the effect of acetylation on the protein expression profiles and function of CD8+ T cells from donors of distinct age was analyzed using histone deacetylase inhibitors (HDACi).MethodsCD8+ T cells isolated from peripheral blood mononuclear cells of 30 young (< 30 years) and 30 old (> 60 years) healthy donors were activated with anti-CD3/anti-CD28 antibodies in the presence and absence of a cocktail of HDACi. The protein expression profiles of untreated and HDACi-treated CD8+ T cells were analyzed using two-dimensional gel electrophoresis. Proteins with a differential expression level (less than 0.66-fold decrease or more than 1.5-fold increase) between CD8+ T cells of young and old donors were identified by matrix-associated laser desorption ionization—time of flight mass spectrometry. Functional enrichment analysis of proteins identified was performed using the online tool STRING. The function of CD8+ T cells was assessed by analyses of cytokine secretion, surface expression of activation markers, proliferative capacity and apoptosis rate.ResultsThe HDACi treatment of CD8+ T cells increased in an age-independent manner the intracellular acetylation of proteins, in particular cytoskeleton components and chaperones. Despite a strong similarity between the protein expression profiles of both age groups, the functional activity of CD8+ T cells significantly differed with an age-dependent increase in cytokine secretion and expression of activation markers for CD8+ T cells from old donors, which was maintained after HDACi treatment. The proliferation and apoptosis rate of CD8+ T cells after HDACi treatment was equal between both age groups.ConclusionsDespite a comparable effect of HDACi treatment on the protein signature of CD8+ T cells from donors of different ages, an initial higher functionality of CD8+ T cells from old donors when compared to CD8+ T cells from young donors was detected, which might have clinical relevance.

Bovine mastitis in northeastern Brazil: Occurrence of emergent bacteria and their phenotypic and genotypic profile of antimicrobial resistance

The aim of this study was to identify emergent pathogens associated with bovine mastitis in northeastern Brazil and to characterize them for phenotypic and genotypic resistance to antimicrobials. A total of 321 milk samples from cows with subclinical mastitis were collected, and the isolates obtained in culture were identified using matrix-associated laser desorption-ionization - time of flight mass spectrometry. Phenotypic and genotypic antimicrobial resistance tests were performed. We identified 72 bacteria considered emergent in the study region: Enterococcus faecalis (26.3%; 19/72), Streptococcus agalactiae (22.2%; 16/72), Enterococcus faecium (20.0%; 15/72), Escherichia coli (6.9%; 5/72), 6.9% (5/72) Lactococcus garvieae (6.9%; 5/72), Acinetobacter baumannii (5.5%; 4/72), Bacillus subtilis (1.3%; 1/72), Kocuria marina (1.3%; 1/72), Macrococcus caseolyticus (1.3%; 1/72), Microbacterium resistens (1.3%; 1/72), Micrococcus luteus (1.3%; 1/72), Streptococcus dysgalactiae (1.3%; 1/72), Streptococcus hyovaginalis (1.3%; 1/72) and Streptococcus pluranimalium (1.3%; 1/72). The antibiogram revealed the following resistance profiles: ampicillin (77.7%; 56/72), cefoxitin (69.4%; 50/72), erythromycin (61.1%; 44/72), oxacillin (63.8%; 46/72), penicillin (79.1%; 57/72), tetracycline (63.8%; 46/72), gentamicin (25.0%; 18/72), and vancomycin (20.8%; 15/72). Of the isolates, 83.4% (60/72) showed multiple resistance to antimicrobials. The tetM gene was identified in 43.0% (31/72) of the isolates, followed by tetL (31.9%; 23/72), and blaZ (26.3%; 19/72). 83.4% (60/72) of the isolates presented a multiple antimicrobial resistance index higher than 0,2. Emergent bacteria with zoonotic and multiresistant potential occur in cows with mastitis in northeastern Brazil. It is necessary to monitor the occurrence of these and other bacteria in livestock environments and develop control strategies to prevent their spread.

STUDY OF THE LIPID PIGMENT COMPOSITION IN THE WHITE SEA ALGAE SACCHARINA LATISSIMA USING TLC AND MALDI-MS

The qualitative composition of the most important biologically active lipid substances-carotenoids and chlorophyll derivatives of the White sea algaeSaccharina latissimahas been studied. The lipid extract was separated by thin-layer chromatography (TLC) and examined by matrix-associated laser desorption-ionization with mass-spectrometric analysis (MALDI-MS). The extract contained fucoxanthin, fucoxanthinol, pheophytin a, pheophorbide a, as well as other carotenoids and chlorophylls, including those not described in the literature. The results obtained, which significantly expand the information about the composition of S. latissima pigments, can be used to standardize raw materials and preparations based on these algae.

Ultrasound-Activated Cascade Effect for Synergistic Orthotopic Pancreatic Cancer Therapy.

SummaryIn some malignant tumor, especially for pancreatic tumor, poor solid-tumor penetration of nanotherapeutics impedes their treatment efficacy. Herein, we develop a polymer-peptide conjugate with the deep tissue penetration ability, which undergoes a cascade process under ultrasound (US), including (1) the singlet oxygen 1O2 is generated by P18, (2) the thioketal bond is cleaved by the 1O2, (3) the departure of PEG chains leads to the in situ self-assembly, and (4) the resultant self-assembled PK nanoparticles show considerable cellular internalization. Owing to the synergistic effect of US on increasing the membrane permeability, the endocytosis and lysosome escape of PK nanoparticles are further enhanced effectively, resulting in the improved therapeutic efficacy. Thanks to the high tissue-penetrating depth and spatial precision of US, PTPK presents enhanced tumor inhibition in an orthotopic pancreatic tumor model. Therefore, the US-activated cascade effect offers a novel perspective for precision medicine and disease theranostics.

The new era for diagnostic microbiology laboratories: ‘All things are ready, if our mind be so’

Identification and characterization of micro-organisms that cause infections are crucial for successful management of patients. For several decades, routine clinical microbiological diagnostic laboratories have been equipped with a growing panel of culture-dependent and culture-independent methods to investigate the microbial etiology of infectious diseases. Considering the disadvantages of traditional methods, multiplex PCR techniques have been routinely endorsed in clinical microbiology laboratories as rapid and sensitive diagnostic and prognostic tool including many PCR panels routinely used in microbial diagnostics. The great advance in medical biotechnology has been associated with development of Matrix-Associated Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI TOF MS). The power of MALDI TOF MS correctly identified 93.2% of organisms to the species level and 5.3% to the genus level. Recently, the application of next-generation sequencing (NGS) technology and its various methodological variants makes it possible to detect different types of microorganisms present within a microbial sample simultaneously, using a culture-independent approach and in a single sequencing run. Over the next 5 to 10 years, although it is unlikely to see NGS completely replacing the conventional culture and susceptibility methods, a wealth of NGS applications will be acquired in the vast majority of diagnostic microbiology laboratories worldwide, providing enhanced diagnostic capabilities and improving the quality of patients care. In Egyptian diagnostic microbiology laboratories, we have to ask ourselves; are we ready to subsist this new era? Sure the answer is: ‘All things are ready, if our mind be so’.

Avaliação da especificidade do ágar Violeta Vermelho Bile Glicose para o isolamento de Enterobacteriaceae em leite de cabra cru

Introduction: Coliforms have been used over the years as indicator microorganisms for microbiological quality. However, several studies report the lack of direct correlation between their presence and that of pathogens in the evaluation of food safety, leading to the suggestion of new analysis proposals, such as the search for total enterobacteria, which includes all members of the Enterobacteriaceae family. Objective: This study aimed to determine the population of enterobacteria in raw goat milk samples based on the microbiological criteria suggested by the National Health Surveillance Agency - Ministry of Health (Anvisa - MS) - Public Consultation Noo 542 of July 17, 2018 for Pasteurized milk samples. Method: 21 samples from small producers from different regions of the state of Rio de Janeiro were used. Isolates were obtained from inoculation on VRBG agar according to ISO 21528-2 and identified by Matrix Associated Laser Desorption-Ionization – Time of Flight (MALDI/TOF) mass spectrometry. Results: Of the 222 isolates identified, only 26.6% belonged to the Enterobacteriaceae family. Another 61.3% belonged to the Pseudomonas genus, 4.5% to the Acinetobacter genus and 7.6% were Stenotrophomonas maltophilia. Although there are no microbiological parameters in Anvisa-MS RDC No 12 of January 2, 2001 for raw milk, the results of isolation on VBRG agar led to the investigation of other studies with similar findings regarding isolation of VRBG bacteria from goat and bovine milk samples. Conclusions: Detection in VRBG agar of non-Enterobacteriaceae-negative oxidase bacteria, such as those of the genus Acinetobacter, suggests the need for a debate on the methodology indicated by ISO 21528-2 for enumeration of enterobacteria, as the new regulation on standard microbiological data for food will no longer use the coliform group, but the Enterobacteriaceae/g or mL count, as an indicator.

Short-term culture for rapid identification of anaerobic bacteria from blood cultures

Identification of anaerobic bacteria causing blood stream infections (BSI) is challenging. This study describes the epidemiology of anaerobic BSI at a tertiary care hospital and the performance of a rapid method for identification of anaerobic bacteria from blood cultures over three years, between June 2015 and June 2018. Short-term culturing is a low-cost user-friendly method and may be used for rapid identification of bacteria from positive blood cultures by matrix-associated laser desorption ionization time of flight (MALDI-TOF). Short-term culturing is performed after Gram staining on all positive blood culture bottles (BCBs) before 10:30 a.m. in our laboratory. Successful short-term cultures were defined as growth and reliable MALDI-TOF result in maximum 8 h after culture positivity. Data pertaining to unique anaerobic episodes and short-term cultures was collected retrospectively from our laboratory information system. Overall, during the three-year period, 692 unique anaerobic episodes (including Propionibacterium spp) were isolated. A total of 17 anaerobic bacterial genera were isolated in our laboratory, with 5 GNB genera and 12 GPB genera. The most prevalent bacteria were Bacteroides spp. 266/692 (38%), Propionibacterium spp. 128/692 (18%), Clostridium spp. 103/692 (15%), Fusobacterium spp. 34/692 (5%), and Actinomyces spp. 34/692 (5%). We performed short-term cultures on 270/564 (48%) clinically relevant episodes (excluding Propionibacterium spp) on chocolate agar plates. Growth within 8 h from culture positivity was detected in 33/270 (12%) short-term cultures. There were 22/33 (67%) gram negative (GNB) and 11/33 (33%) gram positive bacteria (GPB). Only two genera were identified: Bacteroides spp 22/33 (67%) and Clostridium spp 11/33 (33%). Thus, short-term culturing can function as a low-cost add-on to already existing protocols involving MALDI-TOF, where both GNB and GPB can be identified.

Clinical and microbiological features of bacteraemia with Gram-positive anaerobic cocci: a population-based retrospective study

ObjectivesGram-positive, anaerobic cocci (GPAC) can cause infections in humans. Only a few cases of bacteraemia with GPAC have been reported. We describe the clinical and microbiological characteristics of GPAC bacteraemia. MethodsA retrospective population-based study of GPAC bacteraemia 2012–2016 in southern Sweden was performed. GPAC were identified using matrix-associated laser desorption ionization time-of-flight mass spectrometry or 16S rRNA gene sequencing. Etests were used to determine antibiotic susceptibilities. Data on patient and infection characteristics, treatment, and outcome were collected from the medical records. ResultsA total of 226 episodes of GPAC bacteraemia in adults were studied; this corresponds to an annual incidence of 3.4 cases per 100,000 persons per year. The bacteria identified were Anaerococcus spp. (n = 43), Atopobium spp. (n = 7), Blautia spp. (n = 1), Finegoldia spp. (n = 15), Parvimonas spp. (n = 100), Peptoniphilus spp. (n = 52), Peptostreptococcus spp. (n = 2), and Ruminococcus spp. (n = 9) of which 200 isolates were identified to the species level. Resistance to imipenem and piperacillin was not identified, whereas resistance among the 229 isolates to penicillin was detected in four, to metronidazole in six, and clindamycin in 16 isolates. The median age of patients was 73 years (55–83, IQR), 57% were male and comorbidities were common. Fifty-one per cent of infections were polymicrobial. In 60% of cases a focus of infection was identified. Forty per cent of patients had either organ dysfunction or shock. The 30-day mortality was 11%, and nosocomial infections were over-represented among the deceased. ConclusionsGPAC bacteraemia is much more common than previously reported. GPAC-bacteraemia is a condition with significant mortality mainly affecting elderly persons with comorbidities.

Mass spectrometry imaging for early discovery and development of cancer drugs

A drug delivery system (DDS) is a method for delivering a drug to its site of action in the body, with the goal of achieving therapeutic benefits while reducing adverse effects. Pharmacokinetics (PK) and pharmacodynamics (PD) studies have been conducted to evaluate drug delivery, but these approaches are rarely used in the early stages of drug discovery and development. We demonstrated that the tumor stromal barrier inhibits drug distribution within tumor tissue, especially in refractory cancers such as pancreatic cancer. This poses an obstacle to the discovery of new drugs, and is difficult to overcome using conventional in vitro drug discovery methods. In addition, we are also developing new DDS drugs and antibody-drug conjugates (ADCs). These agents act via four steps: Systemic circulation, the enhanced permeability and retention (EPR) effect, penetration within the tumor tissue, and action on cells including controlled drug release. Most of these activities can be evaluated by conventional biological or pharmacological assays. However, it is difficult to examine drug distribution and controlled drug release within targeted tissues. Recent advances in mass spectrometry imaging (MSI) allow examining drug delivery much more conveniently with the off-labeling. A mass microscope, a new type of matrix-associated laser desorption/ionization (MALDI)-MSI analyzer, is a microscope coupled with an atmospheric MALDI and quadruple ion trap time-of-flight (TOF) mass spectrometer, and can provide imaging data with enhanced resolution and high sensitivity. Using a mass microscope, we succeeded in visualizing the EPR effect of a polymeric micelle drug and controlled drug release by an ADC. Currently, we are developing a new drug imaging method using electrospray ionization (ESI)-MSI. Here, we review the use of MSI in early stages of drug discovery and development, as well as our related recent work.

Campylobacter colonization is not associated with proventricular dilatation disease in psittacines.

Psittacine proventricular dilatation disease (PDD) is a neurological disease caused by parrot bornaviruses. A competing theory suggests that intestinal colonization by Campylobacter species may also be a potential cause of PDD or that their presence may be required for disease development. This theory proposes that PDD results from the activities of antiganglioside antibodies on enteric neurons in a manner similar to the pathogenesis of Guillain–Barré syndrome in humans. We therefore cultured feces from domestic chickens as well as from multiple parrot species to determine whether Campylobacter spp. could be detected in the latter. We failed to detect Campylobacter in a flock of cockatiels known to be highly susceptible to experimental parrot bornavirus-induced PDD. Even in naturally infected psittacines suffering from clinical PDD, no Campylobacter species were detected. Conversely, Campylobacter was readily cultured from domestic poultry samples and confirmed by using matrix-associated laser desorption ionization mass spectroscopy/real-time polymerase chain reaction. We conclude that not only are Campylobacter infections of psittacines uncommon, but also that infection by Campylobacter species is not related to the etiology of PDD.

Enzymatic synthesis of chlorogenic acid glucoside using dextransucrase and its physical and functional properties.

Chlorogenic acid, a major polyphenol in edible plants, possesses strong antioxidant activity, anti-lipid peroxidation and anticancer effects. It used for industrial applications; however, this is limited by its instability to heat or light. In this study, we for the first time synthesized chlorogenic acid glucoside (CHG) via transglycosylation using dextransucrase from Leuconostoc mesenteroides and sucrose. CHG was purified and its structure determined by nuclear magnetic resonance and matrix-associated laser desorption ionization-time-of-flight mass spectroscopy. The production yield of CHG was 44.0% or 141mM, as determined by response surface methodology. CHG possessed a 65% increased water solubility and 2-fold browning resistance while it displayed stronger inhibition of lipid peroxidation and of colon cancer cell growth by MTT assay, compared to chlorogenic acid. Therefore, this study may expand the industrial applications of chlorogenic acid as water-soluble or browning resistant compound (CHG) through enzymatic glycosylation.

A rare case of aerococcus urinae infective endocarditis

ABSTRACTIntroduction: Aerococcus urinae is a rare cause of infective endocarditis. Aerococcus is a gram positive cocci that is easily misidentified as Staphylococci or Streptococci. The true incidence rate of this pathogen is likely underestimated. Recent advances in laboratory diagnostic methods with matrix-associated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) have lead to increased recognition of this pathogen in the clinical microbiology lab, and awareness as a cause of infective endocarditis in the infectious disease community.Case reports: Aerococcus usually affects males with underlying urinary tract conditions. Herein, we report a case of prosthetic aortic valve endocarditis caused by Aerococcus urinae.Discussion: Our patient was considered high risk for cardiac surgery and was treated successfully with intravenous antibiotics alone for six weeks.Conclusion: Infective endocarditis should be considered in all cases of Aerococcus bacteremia and appropriate diagnostic evaluations pursued.Abbreviations: AV: Aortic valve; IE: Infective endocarditis

Alteration of Energy Metabolism and Antioxidative Processing in the Hippocampus of Rats Reared in Long-Term Environmental Enrichment

Environmental enrichment (EE) is a typical experimental method that promotes levels of novelty and complexity that enhance experience-dependent neuroplasticity and cognitive behavior function in laboratory animals. Early EE is associated with resilience in the face of later-life challenges. Since increased synaptic activity enhances endogenous neuronal antioxidant defenses, we hypothesized that long-term EE beginning at an early stage may alter the levels of oxidative stress. We investigated global protein expression and oxidative stress in hippocampal proteins from rats nurtured for a 6-month EE beginning in the prenatal period. The analysis of protein expression was carried out using 2-dimensional gel electrophoresis with matrix-associated laser desorption ionization time-of-flight mass spectrometry. Proteins with altered expression were involved in energy metabolism (phosphoglycerate mutase 1, α-enolase isoform 1, adenylate kinase 1, and triose phosphate isomerase) and antioxidant enzymes (superoxide dismutase 1, glutathione S-transferase ω type 1, peroxiredoxin 5, DJ-1, and glial maturation factor β). Using Western blot assays, some of the proteins with altered expression and NADPH oxidase 2 were confirmed to be decreased. Further confirmation was demonstrated with attenuated expression of 7,8-dihydro-8-oxo-deoxyguanine, a DNA oxidative stress marker, in the hippocampus of EE group rats. Our data demonstrate that a long-term EE program beginning in the prenatal and early postnatal phase of development modulates energy metabolism and reduced oxidant stress possibly through enhanced synaptic activity. We provide evidence that EE can be developed as a tool to protect the brain from oxidative stress-induced injury.

Determination of Mode of Chitosanase Action by Matrix Associated Laser Desorption Ionization - Time of Flight Mass Spectrometry Analysis

A detailed knowledge about the mode of enzyme action is critical to get better understanding the productivity of target molecules. The present study was then performed to elucidate the mode of enzyme action and hydrolysis pattern of chitosanase toward high molecular weight chitosan. Hydrolysates derived from the reaction condition were then analyzed by Matrix Associated Laser Desorption Ionization - Time of Flight mass spectrometry (MALDI-TOF MS) analysis to define the real size of hydrolysates. Also, the sequence of linking pattern of hydrolysates derived from the action of chitosanase was determined based on the identification of both glucosamine (GlcN) and N-acetylglucosamine (GlcNAc). The results demonstrate that both homogeneous and heterogeneous chitooligosaccharides, designated as active molecular chitosan (AMC) can be produced without such a big differences. According to these results, we suggest that GlcNAc may not be a terminator for enzymatic hydrolysis processing as a key molecule in chitosanase reaction to determine the length of AMC. Upon identifying the type of linkage and size of AMC, nevertheless, these can be used as potent materials in various research fields including cosmeceutical and pharmaceutical applications.

Effects of Different Maturation Systems on Bovine Oocyte Quality, Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming.

The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P<0.05) at D7 (MII = 62.4±17.5% and FSH = 58.8±16.1%) compared to those obtained from unstimulated animals (CONT = 37.9±8.5% and IMA = 50.6±14.4%). However, the maturation system did not affect the resistance of oocytes to vitrification because the blastocyst rate at D7 was similar (P>0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]+), which was more highly expressed in MII compared to FSH (P<0.05). The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification.

Biocontrol Activity of Bacillus subtilis Isolated from Agaricus bisporus Mushroom Compost Against Pathogenic Fungi.

Bacillus subtilis strain B154, isolated from Agaricus bisporus mushroom compost infected by red bread mold, exhibited antagonistic activities against Neurospora sitophila. Antifungal activity against phytopathogenic fungi was also observed. The maximum antifungal activity was reached during the stationary phase. This antifungal activity was stable over a wide pH and temperature range and was not affected by proteases. Assay of antifungal activity in vitro indicated that a purified antifungal substance could strongly inhibit mycelia growth and spore germination of N. sitophila. In addition, treatment with strain B154 in A. bisporus mushroom compost infected with N. sitophila significantly increased the yield of bisporus mushrooms. Ultraviolet scan spectroscopy, tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-associated laser desorption ionization time-of-flight mass spectrometry, and electrospray ionization tandem mass spectrometry analyses revealed a molecular weight consistent with 1498.7633 Da. The antifungal compound might belong to a new type of lipopeptide fengycin.

Multimodality Imaging Probe for Positron Emission Tomography and Fluorescence Imaging Studies

Our goal is to develop multimodality imaging agents for use in cell tracking studies by positron emission tomography (PET) and optical imaging (OI). For this purpose, bovine serum albumin (BSA) was complexed with biotin (histologic studies), 5(6)-carboxyfluorescein, succinimidyl ester (FAM SE) (OI studies), and diethylenetriamine pentaacetic acid (DTPA) for chelating gallium 68 (PET studies). For synthesis of BSA-biotin-FAM-DTPA, BSA was coupled to (+)-biotin N-hydroxysuccinimide ester (biotin-NHSI). BSA-biotin was treated with DTPA-anhydride and biotin-BSA-DTPA was reacted with FAM. The biotin-BSA-DTPA-FAM was reacted with gallium chloride 3 to 5 mCi eluted from the generator using 0.1 N HCl and was passed through basic resin (AG 11 A8) and 150 μCi (100 μL, pH 7-8) was incubated with 0.1 mg of FAM conjugate (100 μL) at room temperature for 15 minutes to give 68Ga-BSA-biotin-DTPA-FAM. A shaved C57 black mouse was injected with FAM conjugate (50 μL) at one flank and FAM-68Ga (50 μL, 30 μCi) at the other. Immediately after injection, the mouse was placed in a fluorescence imaging system (Kodak In-Vivo F, Bruker Biospin Co., Woodbridge, CT) and imaged (λex: 465 nm, λem: 535 nm, time: 8 seconds, Xenon Light Source, Kodak). The same mouse was then placed under an Inveon microPET scanner (Siemens Medical Solutions, Knoxville, TN) injected (intravenously) with 25 μCi of 18F and after a half-hour (to allow sufficient bone uptake) was imaged for 30 minutes. Molecular weight determined using matrix-associated laser desorption ionization (MALDI) for the BSA sample was 66,485 Da and for biotin-BSA was 67,116 Da, indicating two biotin moieties per BSA molecule; for biotin-BSA-DTPA was 81,584 Da, indicating an average of 30 DTPA moieties per BSA molecule; and for FAM conjugate was 82,383 Da, indicating an average of 1.7 fluorescent moieties per BSA molecule. Fluorescence imaging clearly showed localization of FAM conjugate and FAM-68Ga at respective flanks of the mouse, whereas only a hot spot at the expected flank (FAM-68Ga injection site) was observed in microPET imaging. Our results suggest that BSA-biotin-DTPA-FAM may function as a multiprobe for PET and fluorescence imaging. Experiments are currently in progress to demonstrate cell tracking using both optical and nuclear imaging.

Expression of serpin B5 in colorectal cancer: Its relationship with CEA expression.

e14606 Background: Serpin B5 is a candidate tumor suppressor but its oncogenic activity has been also reported. Its function may be affected by interacting proteins. The aim of this study was to assess the relationship between serpin B5 and carcinoembryonic antigen (CEA) expressions in CRC. We also analyzed the clinicopathological significance of serpin B5 expression in colorectal cancer (CRC) patients. Methods: Two-dimensional gel electrophoresis (2-DE) analysis and matrix-associated laser desorption ionization mass spectrometry (MALDI-MS) were used to identify differentially expressing proteins according to CEA suppression. Serpin B5 and CEA expressions were evaluated by western blot and ELISA assays. Immunohistochemical expression of serpin B5 was assessed retrospectively in tissues from 377 CRC patients, and correlations between serpin B5 and clinicopathological parameters were analyzed. Results: Downregulation of serpin B5 was identified in CEA-suppressed LoVo cell line using 2-DE and MALDI-MS analyses, and further significant positive correlations between serpin B5 and CEA levels were also found in human CRC cell lines and in blood of CRC patients. Tissue expression of serpin B5 in CRC was significantly associated with serum CEA, histologic grade, stage, lymph node metastasis, liver metastasis, lymphatic and perineural invasion, and infiltrative border. High expression of serpin B5 was also associated with a reduced DFS (P = 0.001) and OS (P = 0.017). Conclusions: This is the first study describing relationship between serpin B5 and CEA expression in CRC. High expression of serpin B5 was also associated with worse prognosis in patients with CRC, and its expression may correlate with CEA levels in CRC.

Fabrication, structure and surface charges of albumin-chitosan hybrids

Protein-polymer hybrids consisting of protein and natural polymers or synthetic polymers exhibit superior properties to unmodified proteins, generating a high demand for these materials in the fields of medicine, biotechnology, and nanotechnology. Herein, protein-polysaccharide hybrids were fabricated via the formation of an amide bond between bovine serum albumin (BSA) and chitosan (CS) using N-ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as the couple reagent. FTIR spectrum reveals that the carboxyl group of BSA conjugated with the amino group of chitosan backbone. The molecular weight of BSA-CS hybrids was identified by matrix-associated laser desorption ionization time of flight mass spectra (MALDI-TOF MS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The maximum number of chitosan chains binding to each BSA molecule was estimated as 6, and the optimal number was estimated as 2. In addition, the secondary structure and surface property of BSA were dependent upon the number of polymer conjugating on protein. The secondary structure of BSA was not significantly changed, if a few chitosans were coupled with BSA. By further increasing the molar ratio of chitosan to BSA, the secondary structure of BSA was markedly damaged. The surface’s negative charges of modified BSA also decreased. The result of native polyacrylamide gel electrophoresis (native-PAGE) also demonstrated the changes in surface charges and molecular weight of BSA-CS hybrids.

Characterization of cAMP Response Element-Like Sequence in Gonadal Specific Aromatase Promoter Sequence ofXenopusEmbryos

The p450 aromatase gene has a tissue-specific promoter that is regulated by specific transcriptional factors. In rats and humans, a cAMP response element-like sequence (CLS) and an NR5A1/NR5A2 binding sequence have been identified as cis elements in the aromatase promoter; these cis elements mediate cAMP-induced expression in the ovaries and testes. CLS is recognized by a cAMP-responsive element binding protein (CREB) as the principal component. In this study, we performed a gel shift assay to analyze the proteins that interact with the cis-element in Xenopus aromatase. An electrophpretic mobility gel shift assay (EMSA) and matrix-associated laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis of the proteins responsible for retarding the mobility of CLS revealed that ATF4 interacted in vitro with CLS in gonadal specific aromatase promoter sequence of Xenopus embryos. Although a significant difference was observed in aromatase mRNA expression between male and female gonads, no difference in the expression of ATF4 was observed between them at stage 50. With regard to aromatase expression in the gonad of Xenopus embryos, ATF4 might act in combination with multiple transcription factors as a trans-element of CLS in place of CREB.