A TdT-driven amplification loop increases CRISPR-Cas12a DNA detection levels.

Recent findings on CRISPR-Cas enzymes with collateral DNAse/RNAse activity have led to new and innovative methods for pathogen detection. However, many CRISPR-Cas assays necessitate DNA pre-amplification to boost sensitivity, restricting their utility for point-of-care applications. Achieving higher sensitivity without DNA pre-amplification presents a significant challenge. In this study, we introduce a Terminal deoxynucleotidyl Transferase (TdT)-based amplification loop, creating a positive feedback mechanism within the CRISPR-Cas12a pathogen detection system. Upon recognizing pathogenic target DNA, Cas12a triggers trans-cleavage of a FRET reporter and a specific enhancer molecule oligonucleotide, indicated by the acronym POISER (Partial Or Incomplete Sites for crRNA recognition). POISER comprises half of a CRISPR-RNA recognition site, which is subsequently elongated by TdT enzymatic activity. This process, involving pathogen recognition-induced Cas12a cleavage and TdT elongation, results in a novel single-stranded DNA target. This target can subsequently be recognized by a POISER-specific crRNA, activating more Cas12a enzymes. Our study demonstrates that these POISER-cycles enhance the signal strength in fluorescent-based CRISPR-Cas12a assays. Although further refinement is desirable, POISER holds promise as a valuable tool for the detection of pathogens in point-of-care testing, surveillance, and environmental monitoring.

Are women always better able to recognize faces? The unveiling role of exposure time.

A longer exposure time generally improves individuals' ability to recognize faces. The current research investigates whether this effect varies between genders and whether it is influenced by the gender of the exposed faces. Based on a set of four experimental studies, we advance our knowledge of face recognition, gender, gender distribution of exposed faces, and exposure time in three main ways. First, the results reveal that women are more likely than men to suffer from a decrease in face recognition ability due to a lower exposure time. Second, the findings show that when exposure time is short (vs. long) women recognize a larger proportion of same gender faces and also recognize a larger proportion of same gender faces as compared with the proportion of same gender faces recognized by men. Third, findings reveal that when individuals are only exposed to same gender faces, women recognize more faces than men regardless whether exposure time is short, or long. In short, the findings of this research suggest that insight into the interplay between gender and exposure time length is critical to appropriately determine human beings' ability to recognize faces.

Specific T Cell Responses against Minor Histocompatibility Antigens Cannot Generally Be Explained by Absence of Their Allelic Counterparts on the Cell Surface.

Allogeneic stem cell transplantation has emerged as immunotherapy in the treatment of a variety of hematological malignancies. Its efficacy depends on induction of graft versus leukemia by donor lymphocytes. Both graft versus leukemia and graft versus host disease are induced by T cells reactive against polymorphic peptides, called minor histocompatibility antigens (MiHA), which differ between patient and donor and are presented in the context of self-HLA (where HLA is human leukocyte antigen). The allelic counterpart (AC) of the MiHA is generally considered to be absent at the cell surface, based on the absence of immune responses directed against the AC. To study this in detail, we evaluate the recognition, HLA-binding affinity, and cell surface expression of three selected MiHA. By quantitative MS, we demonstrate the similarly abundant expression of both MiHA and AC at the cell surface. We conclude that the absent recognition of the AC cannot generally be explained by insufficient processing and presentation at the cell surface of the AC.

A Study of the Variation in the Salivary Peptide Profiles of Young Healthy Adults Acquired Using MALDI-TOF MS.

A cross-sectional observational study was conducted to evaluate the inter-individual variation in the MALDI-TOF MS peptide profiles of unstimulated whole saliva in a population of 268 systemically healthy adults aged 18-30 yr (150 males and 118 females) with no apparent caries lesions or periodontal disease. Using Spectral Clustering, four subgroups of individuals were identified within the study population. These subgroups were delimited by the pattern of variation in 9 peaks detected in the 2-15 kDa m/z range. An Unsupervised Feature Selection algorithm showed that P-C peptide, a 44 residue-long salivary acidic proline-rich protein, and three of its fragments (Fr. 1-25, Fr. 15-35 and Fr. 15-44) play a central role in delimiting the subgroups. Significant differences were found in the salivary biochemistry of the subgroups with regard to lysozyme and chitinase, two enzymes that are part of the salivary innate defense system (p < 0.001). These results suggest that MALDI-TOF MS salivary peptide profiles may relate information on the underlying state of the oral ecosystem and may provide a useful reference for salivary disease biomarker discovery studies.

Rapid and reliable discrimination between Shigella species and Escherichia coli using MALDI-TOF mass spectrometry.

E. coli-Shigella species are a cryptic group of bacteria in which the Shigella species are distributed within the phylogenetic tree of E. coli. The nomenclature is historically based and the discrimination of these genera developed as a result of the epidemiological need to identify the cause of shigellosis, a severe disease caused by Shigella species. For these reasons, this incorrect classification of shigellae persists to date, and the ability to rapidly characterize E. coli and Shigella species remains highly desirable. Until recently, existing matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assays used to identify bacteria could not discriminate between E. coli and Shigella species. Here we present a rapid classification method for the E. coli-Shigella phylogroup based on MALDI-TOF MS which is supported by genetic analysis. E. coli and Shigella isolates were collected and genetically characterized by MLVA. A custom reference library for MALDI-TOF MS that represents the genetic diversity of E. coli and Shigella strains was developed. Characterization of E. coli and Shigella species is based on an approach with Biotyper software. Using this reference library it was possible to distinguish between Shigella species and E. coli. Of the 180 isolates tested, 94.4% were correctly classified as E. coli or shigellae. The results of four (2.2%) isolates could not be interpreted and six (3.3%) isolates were classified incorrectly. The custom library extends the existing MALDI-TOF MS method for species determination by enabling rapid and accurate discrimination between Shigella species and E. coli.

Rapid and generic identification of influenza A and other respiratory viruses with mass spectrometry.

The rapid identification of existing and emerging respiratory viruses is crucial in combating outbreaks and epidemics. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid and reliable identification method in bacterial diagnostics, but has not been used in virological diagnostics. Mass spectrometry systems have been investigated for the identification of respiratory viruses. However, sample preparation methods were laborious and time-consuming. In this study, a reliable and rapid sample preparation method was developed allowing identification of cultured respiratory viruses. Tenfold serial dilutions of ten cultures influenza A strains, mixed samples of influenza A virus with human metapneumovirus or respiratory syncytial virus, and reconstituted clinical samples were treated with the developed sample preparation method. Subsequently, peptides were subjected to MALDI-TOF MS and liquid chromatography tandem mass spectrometry (LC-MS/MS). The influenza A strains were identified to the subtype level within 3h with MALDI-TOF MS and 6h with LC-MS/MS, excluding the culturing time. The sensitivity of LC-MS/MS was higher compared to MALDI-TOF MS. In addition, LC-MS/MS was able to discriminate between two viruses in mixed samples and was able to identify virus from reconstituted clinical samples. The development of an improved and rapid sample preparation method allowed generic and rapid identification of cultured respiratory viruses by mass spectrometry.

OmpU as a biomarker for rapid discrimination between toxigenic and epidemic Vibrio cholerae O1/O139 and non-epidemic Vibrio cholerae in a modified MALDI-TOF MS assay.

BACKGROUND: Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effective response to cholera outbreaks. RESULTS: The use of ferulic acid as a matrix in a new MALDI-TOF MS assay increased the measurable mass range of existing MALDI-TOF MS protocols for bacterial identification. The assay enabled rapid discrimination between epidemic V. cholerae O1/O139 strains and other less pathogenic V. cholerae strains. OmpU, an outer membrane protein whose amino acid sequence is highly conserved among epidemic strains of V. cholerae, appeared as a discriminatory marker in the novel MALDI-TOF MS assay. CONCLUSIONS: The extended mass range of MALDI-TOF MS measurements obtained by using ferulic acid improved the screening for biomarkers in complex protein mixtures. Differences in the mass of abundant homologous proteins due to variation in amino acid sequences can rapidly be examined in multiple samples. Here, a rapid MALDI-TOF MS assay was developed that could discriminate between epidemic O1/O139 strains and other less pathogenic V. cholerae strains based on differences in mass of the OmpU protein. It appeared that the amino acid sequence of OmpU from epidemic V. cholerae O1/O139 strains is unique and highly conserved.

Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD).

The identification of peptides presented by human leukocyte antigen (HLA) class I is tremendously important for the understanding of antigen presentation mechanisms under healthy or diseased conditions. Currently, mass spectrometry-based methods represent the best methodology for the identification of HLA class I-associated peptides. However, the HLA class I peptide repertoire remains largely unexplored because the variable nature of endogenous peptides represents difficulties in conventional peptide fragmentation technology. Here, we substantially enhanced (about threefold) the identification success rate of peptides presented by HLA class I using combined electron-transfer/higher-energy collision dissociation (EThcD), reporting over 12,000 high-confident (false discovery rate <1%) peptides from a single human B-cell line. The direct importance of such an unprecedented large dataset is highlighted by the discovery of unique features in antigen presentation. The observation that a substantial part of proteins is sampled across different HLA alleles, and the common occurrence of HLA class I nested sets, suggest that the constraints of HLA class I to comprehensively present the health states of cells are not as tight as previously thought. Our dataset contains a substantial set of peptides bearing a variety of posttranslational modifications presented with marked allele-specific differences. We propose that EThcD should become the method of choice in analyzing HLA class I-presented peptides.

Bordetella pertussis proteins dominating the major histocompatibility complex class II-presented epitope repertoire in human monocyte-derived dendritic cells.

Knowledge of naturally processed Bordetella pertussis-specific T cell epitopes may help to increase our understanding of the basis of cell-mediated immune mechanisms to control this reemerging pathogen. Here, we elucidate for the first time the dominant major histocompatibility complex (MHC) class II-presented B. pertussis CD4(+) T cell epitopes, expressed on human monocyte-derived dendritic cells (MDDC) after the processing of whole bacterial cells by use of a platform of immunoproteomics technology. Pertussis epitopes identified in the context of HLA-DR molecules were derived from two envelope proteins, i.e., putative periplasmic protein (PPP) and putative peptidoglycan-associated lipoprotein (PAL), and from two cytosolic proteins, i.e., 10-kDa chaperonin groES protein (groES) and adenylosuccinate synthetase (ASS). No epitopes were detectable from known virulence factors. CD4(+) T cell responsiveness in healthy adults against peptide pools representing epitope regions or full proteins confirmed the immunogenicity of PAL, PPP, groES, and ASS. Elevated lymphoproliferative activity to PPP, groES, and ASS in subjects within a year after the diagnosis of symptomatic pertussis suggested immunogenic exposure to these proteins during clinical infection. The PAL-, PPP-, groES-, and ASS-specific responses were associated with secretion of functional Th1 (tumor necrosis factor alpha [TNF-α] and gamma interferon [IFN-γ]) and Th2 (interleukin 5 [IL-5] and IL-13) cytokines. Relative paucity in the natural B. pertussis epitope display of MDDC, not dominated by epitopes from known protective antigens, can interfere with the effectiveness of immune recognition of B. pertussis. A more complete understanding of hallmarks in B. pertussis-specific immunity may advance the design of novel immunological assays and prevention strategies.

Mixed-bed ion exchange chromatography employing a salt-free pH gradient for improved sensitivity and compatibility in MudPIT.

In proteomics, comprehensive analysis of peptides mixtures necessitates multiple dimensions of separation prior to mass spectrometry analysis to reduce sample complexity and increase the dynamic range of analysis. The main goal of this work was to improve the performance of (online) multidimensional protein identification technology (MudPIT) in terms of sensitivity, compatibility and recovery. The method employs weak anion and strong cation mixed-bed ion exchange chromatography (ACE) in the first separation dimension and reversed phase chromatography (RP) in the second separation dimension (Motoyama et.al. Anal. Chem 2007, 79, 3623-34.). We demonstrated that the chromatographic behavior of peptides in ACE chromatography depends on both the WAX/SCX mixing ratio as the ionic strength of the mobile phase system. This property allowed us to replace the conventional salt gradient by a (discontinuous) salt-free, pH gradient. First dimensional separation of peptides was accomplished with mixtures of aqueous formic acid and dimethylsulfoxide with increasing concentrations. The overall performance of this mobile phase system was found comparable to ammonium acetate buffers in application to ACE chromatography, but clearly outperformed strong cation exchange for use in first dimensional peptide separation. The dramatically improved compatibility between (salt-free) ion exchange chromatography and reversed phase chromatography-mass spectrometry allowed us to downscale the dimensions of the RP analytical column down to 25 µm i.d. for an additional 2- to 3-fold improvement in performance compared to current technology. The achieved levels of sensitivity, orthogonality, and compatibility demonstrates the potential of salt-free ACE MudPIT for the ultrasensitive, multidimensional analysis of very modest amounts of sample material.

Quantitative proteomics reveals distinct differences in the protein content of outer membrane vesicle vaccines.

At present, only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. These vaccines however require detergent-extraction to remove endotoxin, which changes immunogenicity and causes production difficulties. To investigate this in more detail, the protein content of detergent-extracted OMV is compared with two detergent-free alternatives. A novel proteomics strategy has been developed that allows quantitative analysis of many biological replicates despite inherent multiplex restrictions of dimethyl labeling. This enables robust statistical analysis of relative protein abundance. The comparison with detergent-extracted OMV reveales that detergent-free OMV are enriched with membrane (lipo)proteins and contain less cytoplasmic proteins due to a milder purification process. These distinct protein profiles are substantiated with serum blot proteomics, confirming enrichment with immunogenic proteins in both detergent-free alternatives. Therefore, the immunogenic protein content of OMV vaccines depends at least partially on the purification process. This study demonstrates that detergent-free OMV have a preferred composition.

Unbiased selective isolation of protein N-terminal peptides from complex proteome samples using phospho tagging (PTAG) and TiO(2)-based depletion.

A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO(2)) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formaldehyde at the protein level, after which the proteins are digested and the newly formed internal peptides modified with the PTAG reagent glyceraldhyde-3-phosphate in nearly perfect yields (> 99%). The resulting phosphopeptides are depleted through binding onto TiO(2), keeping exclusively a set of N-acetylated and/or N-dimethylated terminal peptides for analysis by liquid chromatography-tandem MS. Analysis of peptides derivatized with differentially labeled isotopic analogs of the PTAG reagent revealed a high depletion efficiency (> 95%). The method enabled identification of 753 unique N-terminal peptides (428 proteins) in N. meningitidis and 928 unique N-terminal peptides (572 proteins) in S. cerevisiae. These included verified neo-N termini from subcellular-relocalized membrane and mitochondrial proteins. The presented PTAG approach is therefore a novel, versatile, and robust method for mass spectrometry-based N-proteome analysis and identification of protease-generated cleavage products.

Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS.

BACKGROUND: The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays. RESULTS: In this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA) data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences. CONCLUSIONS: MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.

Road to help-seeking among (dedicated) human service professionals with burnout.

OBJECTIVE: Describe and interpret the process of help-seeking among human service professionals with burnout. METHODS: Semi-structured interviews were conducted with 14 participants. Analysis was conducted using principles of grounded theory. RESULTS: All participants were dedicated and responsible workers, selflessly giving themselves to their work. Work was demanding, and often included some form of organisational change. After a period of time the problems of ill health appeared, since persons were overstretching their resources. However, the symptoms were denied, since the image of the ideal worker has been internalised and persons expected maximum performance from themselves. They kept on working hard and delayed the help-seeking process. Eventually, help was sought for medical symptoms or by talking to the supervisors. If postponed for too long, persons experienced a breaking point. CONCLUSION: Human service professionals with burnout internalise the ideal image of their professional role. They strive to keep this ideal image at the cost of their own needs, taking a long time to seek help for the obstacles they encounter. PRACTICE IMPLICATIONS: More awareness raising is needed in order to recognise early burnout symptoms. Particularly crucial in this process are supervisors and doctors, who have an authority role over employees.

Age-differences in work motivation and job satisfaction. The influence of age on the relationships between work characteristics and workers' outcomes.

This study investigated the influence of age on the relationship between work characteristics and workers' work motivation and job satisfaction. In total, 1036 workers of a Dutch division of a multinational organization participated. Data were collected by a digital questionnaire. Two interaction terms in the regression on work motivation were significant. The first interaction showed that the positive correlation between Motivating Potential Score (MPS) and motivation was much stronger for older than for younger employees. So, to remain motivated, older employees seem more in need of intrinsic challenging and fulfilling jobs. The second significant interaction indicated that the positive association between career opportunities and motivation was much stronger for younger employees than for older employees. This means that, especially, younger workers' motivation increases as they are offered more career opportunities. Careful career mentoring by the supervisor as part of an aging policy can contribute to the maintenance of workers of any age.

Exploring the human leukocyte phosphoproteome using a microfluidic reversed-phase-TiO2-reversed-phase high-performance liquid chromatography phosphochip coupled to a quadrupole time-of-flight mass spectrometer.

The study of protein phosphorylation events is one of the most important challenges in proteome analysis. Despite the importance of phosphorylation for many regulatory processes in cells and many years of phosphoprotein and phosphopeptide research, the identification and characterization of phosphorylation by mass spectrometry is still a challenging task. Recently, we introduced an approach that facilitates the analysis of phosphopeptides by performing automated, online, TiO(2) enrichment of phosphopeptides prior to mass spectrometry (MS) analysis. The implementation of that method on a "plug-and-play" microfluidic high-performance liquid chromatography (HPLC) chip design will potentially open up efficient phosphopeptide enrichment methods enabling phosphoproteomics analyses by a broader research community. Following our initial proof of principle, whereby the device was coupled to an ion trap, we now show that this so-called phosphochip is capable of the enrichment of large numbers of phosphopeptides from complex cellular lysates, which can be more readily identified when coupled to a higher resolution quadrupole time-of-flight (Q-TOF) mass spectrometer. We use the phosphochip-Q-TOF setup to explore the phosphoproteome of nonstimulated primary human leukocytes where we identify 1012 unique phosphopeptides corresponding to 960 different phosphorylation sites providing for the first time an overview of the phosphoproteome of these important circulating white blood cells.

Determinants of early retirement intentions among Belgian nurses.

AIM: This paper is a report of a study to gain insight into older nurses' retirement intentions and to establish factors determining early retirement intention in these individuals. BACKGROUND: In many developed countries, the working population is ageing. This will lead to a structural labour shortage in the near future. In nursing, this is already taking place. To retain nurses in employment, information on the determinants of their early retirement intentions are imperative. METHOD: A cross-sectional study was carried out in 2005 in one Belgian hospital. Data were collected by questionnaire with 100 nurses aged 45 or older. The response rate was 69.9%. FINDINGS: No fewer than 77% of the nurses wanted to stop working before the age of 65 years. The following individual, work-related, and organizational factors contributed to older nurses' intention to retire early: perceived health, marital status, gender, opportunities for change and development, workload, and negative stereotyping of older employees. CONCLUSION: Our findings offer insight regarding the influencing factors of early retirement intentions in nurses. This information may be useful to human resource managers and may enable them to successfully prevent early retirement in nurses. More research on this topic is needed as this will enable the development, implementation and evaluation of well-founded measures for retaining older nurses in the workplace.

Identifying the epitope-specific T cell response to virus infections.

Virus infection induces an adaptive immune response by T cells that is specific for defined viral epitopes. The epitope-specific analysis of T cells has become an important tool for investigating the anti viral response following infection or vaccination. In this review, the inherent differences in the procedures to identify the epitopes are discussed. Specifically, the screening of lymphocytes for epitope specific responses and the usage of mass spectrometry for sequencing of viral epitopes are evaluated.

Identification of outer membrane proteins of Yersinia pestis through biotinylation.

The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with biotin. Tagged proteins were visualised through streptavidin probing of Western blots. Seven biotinylated proteins of Y. pestis were identified including two porins and the putative virulence factor catalase peroxidase.

Chemostat-based proteomic analysis of toluene-affected Pseudomonas putida S12.

The aim of this study was to assess the cellular response of the solvent-tolerant Pseudomonas putida S12 to toluene as the single effector. Proteomic analysis (two-dimensional difference-in-gel-electrophoresis) was used to assess the response of P. putida S12 cultured in chemostats. This approach ensures constant growth conditions, both in the presence and absence of toluene. A considerable negative effect of toluene on the cell yield was found. The need for energy in the defence against toluene was reflected by differentially expressed proteins for cell energy management. In toluene-stressed cells the balance between proton motive force (PMF) enforcing and dissipating systems was shifted. NAD(P)H generating systems were upregulated whereas the major proton-driven system, ATP synthase, was downregulated. Other differentially expressed proteins were identified: outer membrane proteins, transport proteins, stress-related proteins and translation-related proteins. In addition, a protein with no assigned function was found. This study yielded a more detailed view of the effect of toluene on the intracellular energy management of P. putida S12 and several novel leads have been obtained for further targeted investigations.