Matrix-associated Laser Desorption Ionization-time 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.

Impact of early antimicrobial stewardship intervention in patients with positive blood cultures: results from a randomized comparative study

Antimicrobial stewardship intervention (ASI) appears to be necessary to realize the full benefits of rapid diagnostic technologies in clinical practice. This study aimed to compare clinical outcomes between early ASI paired with matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) compared with MALDI-TOF with standard of care (SOC) reporting in patients with positive blood cultures. Adult patients with positive blood cultures and organism speciation via MALDI-TOF admitted between February 2015 and September 2015 were randomized to ASI or SOC in a 1:1 fashion. Patients admitted for at least 48 h following positive culture were included in analyses. ASI was defined as a clinical assessment by a stewardship team member with non-binding treatment recommendations offered to the primary team. The primary outcome was time to definitive therapy. Secondary outcomes included post-culture length of stay (LOS), time to first change in antibiotics, and in-hospital mortality. In total, 149 patients were included in the analyses (76 in the ASI group and 73 in the SOC group). ASI and SOC arms did not differ according to age, sex, comorbidities or severity of illness. Gram-positive organisms were common in both SOC and ASI arms (74.0 vs. 61.8%, P=0.11). Time to definitive therapy was reduced, on average, by 30.3 h in the ASI group (71.6 vs. 41.3 h, P=0.01). Hospital LOS following the first positive blood culture was significantly shorter in the ASI group (8.7 vs. 11.2 days, P=0.049). ASI combined with MALDI-TOF reduced the time to definitive therapy and time to first change in antibiotics, and was associated with a shorter post-culture LOS.

Structural Characterization of Glycerophosphorylated and Succinylated Cyclic β-(1→2)-d-Glucan Produced by Sinorhizobium mliloti 1021.

Rhizobia produces different types of surface polysaccharides. Among them, cyclic β-(1→2)-d-glucan is located in the periplasmic space of rhizobia and plays an important role in the adaptation of bacteria to osmotic adaptation. Cyclic β-(1→2)-d-glucan (CG), synthesized from Sinorhiozbbium meliloti 1021, has a neutral and anionic form. In the present study, we characterized the exact chemical structures of anionic CG after purification using size exclusion s (Bio-Gel P-6 and P-2) chromatography, and DEAE-Sephadex anion exchange chromatography. The exact structure of each isolated anionic CG was characterized using various analytical methods such as nuclear magnetic resonance (NMR), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and matrix associated laser desorption ionization-time of Flight (MALDI-TOF) mass spectrometry. The precise chemical structures of novel anionic CG molecules were elucidated by various NMR spectroscopic analyses, including 1H, 13C, 31P, and 2D HSQC NMR spectroscopy. As a result, we discovered that anionic CG molecules have either glycerophosphoryl or succinyl residues at C6 positions of a neutral CG. In addition, the results of MALDI-TOF mass spectrometric analysis confirmed that there are two types of patterns for anionic CG peaks, where one type of peak was the succinylated CG (SCG) and the other was glycerophospholated CG (GCG). In addition, it was revealed that each anionic CG has one to four substituents of the succinyl group of SCG and glycerophosphoryl group of GCG, respectively. Anionic CG could have potential as a cyclic polysaccharide for drug delivery systems and a chiral separator based on the complexation with basic target molecules.

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’.

Bovine Mastitis Caused by Multidrug-Resistant Nocardia farcinica

Background: Mastitis caused by Nocardia is characterized by pyogranulomatous inflammation related to inadequate hygiene conditions and is difficult to treat. Prompted by the absence of documentation of Nocardia farcinica associated to bovine mastitis in the Northeast region of Brazil, this is the first report to describe bovine mastitis caused by multidrug-resistant N. farcinica. Case: Four milk samples (one from each teat) obtained from a 3-year-old Jersey cow raised on a property located in the metropolitan region of Recife, Pernambuco state, Brazil, were submitted to the Laboratory of Infectious-Contagious Diseases of the Veterinary Hospital at Universidade Federal Rural de Pernambuco. At the laboratory, samples were cultured in base agar enriched with 7% sheep blood (blood agar) in a microbiological incubator at 37°C under aerobic conditions for 72 h. After only 48 h, however, pure bacterial colony growth was observed in all samples. Macroscopic analysis revealed small colonies, with an irregular shape, dry aspect, and greyish in color. Gram-positive rods forming filaments and/or ramifications were observed using a Gram staining method. Nocardia spp. were identified according to morphotinctorial characteristics. Susceptibility testing using the disc-diffusion method in agar (antibiogram) was performed using the following antibiotics: penicillin (10 IU), tetracycline (30 µg), amoxicillin (10 µg), gentamicin (10 µg), cephalexin (30 µg), erythromycin (15 µg), cephalothin (30 µg) and ampicillin (30 µg). However, the organism exhibited resistance to all drugs; as such, a new milk sample was obtained at the same location the initial samples were collected. Samples (approximately 5 mL) were collected aseptically and separately from all four teats in sterile bottles, during which the presence of granular material was noted. Bacterial culture was performed as previously described and, after 48 h, colony growth with the same characteristics as the first isolation were observed, and with same morphotinctorial characteristics in the Gram stain. A resistance profile was observed for 14 of the antimicrobial drugs tested; sensitivity was verified only for ciprofloxacin and amoxicillin with clavulanic acid. One bacterial colony was selected and sent to the Center of Strategic Technology of Northeast (CETENE-PE) for species identification using a matrix-associated laser desorption ionization-time of flight (MALDI-TOF/MS) technique, which confirmed the species to be N. farcinica. Molecular characterization of 16s ribosomal DNA was performed using polymerase chain reaction with universal prokaryotic primers 516F-13R. Subsequently, the amplified product was subjected to sequencing, and the result was analyzed for quality using Phred base calling software; bases with a Phred value &gt; 20 were kept. The sequence was evaluated using GenBank, in which the isolate exhibited 99% similarity to N. farcinica. Discussion: Clinical findings and animal history associated with microbiological culture and bacterial identification using the MALDI-TOF technique, as well as DNA sequencing, confirmed the case of clinical mastitis to be caused by N. farcinica. These bacteria are considered saprophytes, and their occurrence is associated with deficiencies in hygienic-sanitary management, such as not using pre- and post-dipping, which may favor mammary gland infection. Treatment of N. farcinica mastitis is effective only when properly performed, with agent identification and antibiotic sensitivity tests in vitro associated with the adoption of hygienic-sanitary measures. This is the first description of bovine mastitis caused by N. farcinica in the northeast of Brazil. Multidrug resistance should raise awareness of producers searching for laboratory aids in agent identification as well as antibiotic sensitivity tests, and to develop a proper therapeutic protocol based on results obtained in laboratory examinations.

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.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid identification of Mycobacterium abscessus

BackgroundNontuberculous mycobacteria are increasingly being implicated in infections and have become an important cause of health care associated infections. Mycobacterium abscessus, a rapidly growing mycobacteria, is of particular concern as it tends to be resistant to commonly used therapeutic options. Conventional phenotypic methods for speciation of mycobacteria are time consuming, labor intensive and not always reliable. Molecular methods require expertise and are expensive. The study was used to evaluate the use of matrix associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a means of rapid identification of M. abscessus. Methods35 isolates of rapidly growing Mycobacterium from an outbreak of surgical site infections at a tertiary care hospital were identified using phenotypic methods. The mycobacterial isolates were inactivated and an extraction protocol was followed. These isolates were then analyzed by MALDI biotyper (Bruker Daltonics) using biotyper software 4.0 and the mycobacterial reference database v 2.0. ResultsAll 35 isolates were identified as M. abscessus by MALDI biotyper but the scores obtained according to guidelines of the company were lower than previous studies with only 23 out of the 35 isolates having scores of greater 1.8 which was described as the minimum score to be achieved for reliable identification. ConclusionMALDI-TOF MS offers a rapid and inexpensive method for identification of Mycobacteria; however, the scores obtained in our study were lower than reported in other studies.

Association between polymorphisms in tumor suppressor genes and oncogenes and risk of hepatocellular carcinoma: a case-control study in an HCC epidemic area within the Han Chinese population.

Data concerning the risk of hepatocellular carcinoma (HCC) and specific single nucleotide polymorphisms (SNPs) in an HBV-free population are currently limited. Therefore, we performed a case-control study to investigate the association between SNPs and the risk of HCC in individuals without chronic HBV infection. A total of 160 Han Chinese patients with HCC and an identical number of healthy controls were enrolled in this study. rs1042522, rs10814325, rs17401966, and rs2279744 genotypes were determined using matrix-associated laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). CG and GG genotypes in rs1042522 and heterozygote and homozygote in rs2279744 were significantly associated with an elevated risk of HCC. Homozygous mutation of rs1081432 conferred a 2.68-fold risk of HCC (95% CI 1.35-5.34); however heterozygosity was not statistically significant. rs17401966 heterozygosity or homozygosity was not significantly associated with a increased risk of HCC. Several polymorphisms associated with a significantly increased risk of HCC were identified. These may serve as biomarkers in evaluating HCC risk in the general population.

Phenotypic and molecular characteristics of carbapenem-resistant Enterobacteriaceae in a health care system in Los Angeles, California, from 2011 to 2013.

Carbapenem-resistant Enterobacteriaceae (CRE) are a concern for health care in the United States but remain relatively uncommon in California. We describe the phenotype, clonality, and carbapenemase-encoding genes present in CRE isolated from patients at a Californian tertiary health care system. CRE for this study were identified by evaluating the antibiograms of Enterobacteriaceae isolated in the UCLA Health System from 2011 to 2013 for isolates that were not susceptible to meropenem and/or imipenem. The identification of these isolates was subsequently confirmed by matrix-associated laser desorption ionization-time of flight, and broth microdilution tests were repeated to confirm the CRE phenotype. Real-time PCR for bla(KPC), bla(SME), bla(IMP), bla(NDM-1), bla(VIM), and bla(OXA-48) was performed. Clonality was assessed by repetitive sequence-based PCR (repPCR) and multilocus sequence typing (MLST). Of 15,839 nonduplicate clinical Enterobacteriaceae isolates, 115 (0.73%) met the study definition for CRE. This number increased from 0.5% (44/8165) in the first half of the study to 0.9% (71/7674) in the second (P = 0.004). The most common CRE species were Klebsiella pneumoniae, Enterobacter aerogenes, and Escherichia coli. A carbapenemase-encoding gene was found in 81.7% (94/115) of CRE and included bla(KPC) (78.3%), bla(NDM-1) (0.9%), and bla(SME) (2.6%). The majority of bla(KPC) genes were in K. pneumoniae isolates, which fell into 14 clonal groups on typing. bla(KPC) was identified in more than one species of CRE cultured from the same patient in four cases. Three bla(SME)-carrying Serratia marcescens isolates and one bla(NDM-1) carrying Providencia rettgeri isolate were detected. CRE are increasing in California, and carbapenemases, particularly KPC, are a common mechanism for carbapenem resistance in this region.

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.

Highly expressed recombinant human follicle-stimulating hormone from Chinese hamster ovary cells grown in serum-free medium and its effect on induction of folliculogenesis and ovulation

To develop efficient Chinese hamster ovary (CHO) cells that express recombinant human FSH (rhFSH) in serum-free conditions and to investigate the effect of this newly synthesized rhFSH on folliculogenesis and ovulation. Experimental study. Seoul National University, South Korea. Forty immature hypophysectomized rats and 40 androgen-sterilized mice. A stable single CHO cell that expresses rhFSH at a high level was obtained by introducing the human chorionic gonadotropin (hCG) alpha-subunit and FSH beta-subunit genes. After purification processing, we investigated the effect of this newly synthesized rhFSH on folliculogenesis in hypophysectomized rats and ovulation in androgen-sterilized mice. The ovary weight, uterine weight, number of follicles, and ovarian morphology were evaluated in immature hypophysectomized rats. The number of ovulated oocytes and ovarian morphology were examined in androgen-sterilized mice. After purification processing, we analyzed the new rhFSH using matrix-associated laser desorption ionization-time of flight and found that this new rhFSH increased both ovarian weight and uterine weight in hypophysectomized rats and induced ovulation in androgen-sterilized mice. This newly synthesized rhFSH might be safely used in anovulatory infertile woman as well as in ovulation induction protocols for subfertile women.

Highlights of the Annual Meeting of the Italian Association for Cell Cultures (AICC): new drug delivery strategies and technological platforms for diagnosis and therapy of tumors (Part I)

The application of computational analysis to biochemistry and molecular biology techniques has allowed the design of advanced technological platforms such as DNA microarrays, proteomic analysis and tissue microarrays. These are useful in the diagnosis of tumors and in driving the choice of opportune therapeutic options. The use of target-based agents requires the determination of the molecular features of the tumors. During the AICC meeting four new advanced technological platforms were analyzed: DNA microarrays, ProtoArrays, tissue microarrays and proteomic analysis with differential in-gel electrophoresis (DIGE) and matrix-associated laser desorption ionization-time of flight (MALDI–TOF). The use of DNA microarray in the study of neo-angiogenic components in tumor tissues was described and the ex vivo analysis in humans of circulating endothelial cells was proposed. The role of both genomic and pharmacogenomic profiles in the diagnosis and prediction of response to therapy and toxicity of both breast and colorectal cancer was described. A developmental model for the optimization of zoledronic acid (ZOL) antitumor activity based on either nanotechnological modification or molecular target fishing with DNA microarrays was discussed. The interdisciplinary network between pharmacologists, cellular and molecular biologists, biochemists and translational and clinical oncologists was encouraged and approved during the meeting in order to improve the efficacy of antitumor strategies.

Insulin-like growth factor-I downregulates embryonic myosin heavy chain (eMyHC) in myoblast nuclei

The obscure ability of the insulin-like growth factors (IGF-I & -II) to stimulate both myoblast proliferation and differentiation suggests that the latter effect may be mediated locally, possibly by IGF binding proteins (IGFBPs). In some cells, the growth inhibitory actions of IGFBP-5 require plasma membrane translocation and nuclear localization. Immunoreactivity of presumably endogenous IGFBP-5 was identified within proliferating rat L6 myoblast nuclei using fluorescent and confocal microscopy in separate experiments and was reduced by 100 ng/ml IGF-I in a time-dependent manner. Western blotting of nuclear and cytosolic protein identified a single anti-IGFBP-5 immunoreactive protein of approximately 200 kDa, primarily in nuclear fractions, that was downregulated in cells treated with IGF-I for 12 h. The unknown protein was immunopurified from nuclear fractions and identified as the rat homologue for embryonic myosin heavy chain (eMyHC) using matrix-associated laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy. Cross-reactivity of the IGFBP-5 antiserum with eMyHC was confirmed by blotting anti-IGFBP-5 nuclear immunoprecipitates with eMyHC monoclonal antibodies (F1.652). These data indicate that eMyHC is located predominantly within the nuclei of proliferating L6 myoblasts and suggest that IGF-stimulated differentiation is associated with the rapid downregulation of nuclear eMyHC as these cells stop expressing this myosin II isoform as they differentiate. Myosin Iβ has been identified within the nuclei of non-muscle cells where it helps to regulate gene transcription. Thus, eMyHC may serve a similar role in myoblasts that is specific only to the undifferentiated state.

Plasmodium berghei ookinetes bind to Anopheles gambiae and Drosophila melanogaster annexins

Using a proteomic approach we identified polypeptides from Anopheles gambiae and Drosophila melanogaster protein extracts that selectively bind purified Plasmodium berghei ookinetes in vitro; these were two and three distinct polypeptides, respectively, with an apparent molecular weight of about 36 kDa. Combining two-dimensional electrophoresis and MALDI-TOF (matrix-associated laser desorption ionization time of flight) mass spectrometry we determined that the polypeptides correspond to isomorphs of the annexin B11 protein of the fruit fly. When protein extracts derived from A. gambiae and D. melanogaster tissue culture cells were further fractionated, the binding activity matching the annexin protein could be localized in the fraction derived from cell membranes in both diptera. Antibody staining showed that annexin also binds to ookinetes during the invasion of the mosquito midgut. Finally, inclusion of antiannexin antisera in a mosquito blood meal impaired parasite development, suggesting a facilitating role for annexins in the infection of the mosquito by Plasmodium.