Evaluation of Acquisition Modes for Semi-Quantitative Analysis by Targeted and Untargeted Mass Spectrometry

RATIONALE: Analyte quantitation by mass spectrometry underpins a diverse range of scientific endeavors. The fast growing field of mass spectrometer development has resulted in several targeted and untargeted acquisition modes suitable for these applications. By characterizing the acquisition methods available on an ion mobility (IM) enabled orthogonal acceleration time-of-flight (oa-ToF) instrument, the optimum modes for analyte semi-quantitation can be deduced. METHODS: Serial dilutions of commercial metabolite, peptide, or crosslinked peptide analytes were prepared in matrices of human urine or E. coli digest. Each analyte dilution was introduced into an IM separation enabled oa-ToF mass spectrometer by reversed phase liquid chromatography and electrospray ionization. Data were acquired for each sample in duplicate using nine different acquisition modes, including four IM enabled acquisitions modes, available on the mass spectrometer. RESULTS: Five (metabolite) or seven (peptide/crosslinked peptide) point calibration curves were prepared for analytes across each of the acquisition modes. A non-linear response was observed at high concentrations for some modes, attributed to saturation effects. Two correction methods, one MS1 isotope-correction and one MS2 ion intensity-correction, were applied to address this observation, resulting in an up to two-fold increase in dynamic range. By averaging the semi-quantitative results across analyte classes, two parameters, linear dynamic range (LDR) and lower limit of quantitation (LLOQ), were determined to evaluate each mode. CONCLUSION: Comparison of the acquisition modes revealed that data independent acquisition and parallel reaction monitoring methods are most robust for semi-quantitation when considering achievable LDR and LLOQ. IM enabled modes exhibited sensitivity increases, but a simultaneous reduction in dynamic range which required correction methods to recover. These findings will assist users in identifying the optimum acquisition mode for their analyte quantitation needs, supporting a diverse range of applications and providing guidance for future acquisition mode developments

Localization of AQP5 during development of the mouse submandibular salivary gland

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs

Neutrophils in cancer: neutral no more

Neutrophils are indispensable antagonists of microbial infection and facilitators of wound healing. In the cancer setting, a newfound appreciation for neutrophils has come into view. The traditionally held belief that neutrophils are inert bystanders is being challenged by the recent literature. Emerging evidence indicates that tumours manipulate neutrophils, sometimes early in their differentiation process, to create diverse phenotypic and functional polarization states able to alter tumour behaviour. In this Review, we discuss the involvement of neutrophils in cancer initiation and progression, and their potential as clinical biomarkers and therapeutic targets

BPI-fold (BPIF) containing/plunc protein expression in human fetal major and minor salivary glands.

The aim of this study was to determine expression, not previously described, of PLUNC (palate, lung, and nasal epithelium clone) (BPI-fold containing) proteins in major and minor salivary glands from very early fetal tissue to the end of the second trimester and thus gain further insight into the function of these proteins. Early fetal heads, and major and minor salivary glands were collected retrospectively and glands were classified according to morphodifferentiation stage. Expression of BPI-fold containing proteins was localized through immunohistochemistry. BPIFA2, the major BPI-fold containing protein in adult salivary glands, was detected only in the laryngeal pharynx; the lack of staining in salivary glands suggested salivary expression is either very late in development or is only in adult tissues. Early expression of BPIFA1 was seen in the trachea and nasal cavity with salivary gland expression only seen in late morphodifferentiation stages. BPIFB1 was seen in early neural tissue and at later stages in submandibular and sublingual glands. BPIFA1 is significantly expressed in early fetal oral tissue but BPIFB1 has extremely limited expression and the major salivary BPIF protein (BPIFA2) is not produced in fetal development. Further studies, with more sensitive techniques, will confirm the expression pattern and enable a better understanding of embryonic BPIF protein function

Bayesian inference for the information gain model

One of the most popular paradigms to use for studying human reasoning involves the Wason card selection task. In this task, the participant is presented with four cards and a conditional rule (e.g., “If there is an A on one side of the card, there is always a 2 on the other side”). Participants are asked which cards should be turned to verify whether or not the rule holds. In this simple task, participants consistently provide answers that are incorrect according to formal logic. To account for these errors, several models have been proposed, one of the most prominent being the information gain model (Oaksford & Chater, Psychological Review, 101, 608–631, 1994). This model is based on the assumption that people independently select cards based on the expected information gain of turning a particular card. In this article, we present two estimation methods to fit the information gain model: a maximum likelihood procedure (programmed in R) and a Bayesian procedure (programmed in WinBUGS). We compare the two procedures and illustrate the flexibility of the Bayesian hierarchical procedure by applying it to data from a meta-analysis of the Wason task (Oaksford & Chater, Psychological Review, 101, 608–631, 1994). We also show that the goodness of fit of the information gain model can be assessed by inspecting the posterior predictives of the model. These Bayesian procedures make it easy to apply the information gain model to empirical data. Supplemental materials may be downloaded along with this article from www.springerlink.com

Local Increase of Arginase Activity in Lesions of Patients with Cutaneous Leishmaniasis in Ethiopia

The leishmaniases are a complex of diseases caused by Leishmania parasites. Currently, the diseases affect an estimated 12 million people in 88 countries, and approximately 350 million more people are at risk. The leishmaniases belong to the most neglected tropical diseases, affecting the poorest populations, for whom access to diagnosis and effective treatment are often not available. Leishmania parasites infect cells of the immune system called macrophages, which have the capacity to eliminate the intracellular parasites when they receive the appropriate signals from other cells of the immune system. In nonhealing persistent leishmaniasis, lymphocytes are unable to transmit the signals to macrophages required to kill the intracellular parasites. The local upregulation of the enzyme arginase has been shown to impair lymphocyte effector functions at the site of pathology. In this study, we tested the activity of this enzyme in skin lesions of patients presenting with localized cutaneous leishmaniasis. Our results show that arginase is highly upregulated in these lesions. This increase in arginase activity coincides with lower expression of a signalling molecule in lymphocytes, which is essential for efficient activation of these cells. These results suggest that increased arginase expression in the localized cutaneous lesions might contribute to persistent disease in patients presenting with cutaneous leishmaniasis

Rational Design of a New Trypanosoma rangeli Trans-Sialidase for Efficient Sialylation of Glycans

This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197-203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous sialidase, and we hypothesised that this motif is important for trans-sialidase activity. The 197-203 motif is strongly positively charged with a marked change in hydrogen bond donor capacity as compared to the sialidase. To investigate the role of this motif, we expressed and characterised a T. rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13's acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose. Sialic acid is important in association with human milk oligosaccharides, and Tr13 was shown to sialylate a number of established and potential prebiotics. Initial evaluation of prebiotic potential using pure cultures demonstrated, albeit not selectively, growth of Bifidobacteria. Since the 197-203 motif stands out in the native trans-sialidase, is markedly different from the wild-type sialidase compared to previous mutants, and is shown here to confer efficient and broad trans-sialidase activity, we suggest that this motif can serve as a framework for future optimization of trans-sialylation towards prebiotic production

The prevalence of anemia and its association with 90-day mortality in hospitalized community-acquired pneumonia

<p>Abstract</p> <p>Background</p> <p>The prevalence of anemia in the intensive care unit is well-described. Less is known, however, of the prevalence of anemia in hospitalized patients with lesser illness severity or without organ dysfunction. Community-acquired pneumonia (CAP) is one of the most frequent reasons for hospitalization in the United States (US), affecting both healthy patients and those with comorbid illness, and is typically not associated with acute blood loss. Our objective was to examine the development and progression of anemia and its association with 90d mortality in 1893 subjects with CAP presenting to the emergency departments of 28 US academic and community hospitals.</p> <p>Methods</p> <p>We utilized hemoglobin values obtained for clinical purposes, classifying subjects into categories consisting of no anemia (hemoglobin >13 g/dL), at least borderline (≤ 13 g/dL), at least mild (≤ 12 g/dL), at least moderate (≤ 10 g/dL), and severe (≤ 8 g/dL) anemia. We stratified our results by gender, comorbidity, ICU admission, and development of severe sepsis. We used multivariable logistic regression to determine factors independently associated with the development of moderate to severe anemia and to examine the relationship between anemia and 90d mortality.</p> <p>Results</p> <p>A total of 8240 daily hemoglobin values were measured in 1893 subjects. Mean (SD) number of hemoglobin values per patient was 4.4 (4.0). One in three subjects (33.9%) had at least mild anemia at presentation, 3 in 5 (62.1%) were anemic at some point during their hospital stay, and 1 in 2 (54.5%) survivors were discharged from the hospital anemic. Anemia increased with illness severity and was more common in those with comorbid illnesses, female gender, and poor outcomes. Yet, even among men and in those with no comorbidity or only mild illness, anemia during hospitalization was common (~55% of subjects). When anemia was moderate to severe (≤ 10 g/dL), its development was independently associated with increased 90d mortality, even among hospital survivors.</p> <p>Conclusions</p> <p>Anemia was common in hospitalized CAP and independently associated with 90d mortality when hemoglobin values were 10 g/dL or less. Whether prevention or treatment of CAP-associated anemia would improve clinical outcomes remains to be seen.</p

Early to late sparing of radiation damage to the parotid gland by adrenergic and muscarinic receptor agonists

Damage to salivary glands after radiotherapeutic treatment of head and neck tumours can severely impair the quality of life of the patients. In the current study we have investigated the early-to-late pathogenesis of the parotid gland after radiation. Also the ability to ameliorate the damage using pretreatment with adrenergic or muscarinic receptor agonists is studied. Rats were locally irradiated with or without i.p. pretreatment with phenylephrine (α-adrenoceptor agonist, 5 mg kg−1), isoproterenol (β-adrenoceptor agonist, 5 mg kg−1), pilocarpine (4 mg kg−1), methacholine (3.75 mg kg−1) (muscarinic receptor agonists) or methacholine plus phenylephrine. Parotid salivary flow rate, amylase secretion, the number of cells and gland histology were monitored sequentially up to 240 days postirradiation. The effects were described in 4 distinct phases. The first phase (0–10 days) was characterised by a rapid decline in flow rate without changes in amylase secretion or acinar cell number. The second phase (10–60 days) consists of a decrease in amylase secretion and is paralleled by acinar cell loss. Flow rate, amylase secretion and acinar cell numbers do not change in the third phase (60–120 days). The fourth phase (120–240 days) is determined by a further deterioration of gland function but an increase in acinar cell number, albeit with poor tissue morphology. All drug pretreatments used could reduce radiation effects in phase I and II. The protective effects were lost during phase IV, with the exception of methacholine plus phenylephrine pretreatment. The latter combination of drugs ameliorated radiation-damage throughout the entire follow-up time. The data show that combined pre-irradiation stimulation of muscarinic acetylcholine receptors with methacholine plus α-adrenoceptors with phenylephrine can reduce both early and late damage, possibly involving the PLC/PIP2 second messenger pathways. This opens perspectives for the development of clinical applicable methods for long-term sparing of parotid glands subjected to radiotherapy of head and neck cancer patients. © 2001 Cancer Research Campaignhttp://www.bjcancer.co

Factors associated with diversity, quantity and zoonotic potential of ectoparasites on urban mice and voles

Wild rodents are important hosts for tick larvae but co-infestations with other mites and insects are largely neglected. Small rodents were trapped at four study sites in Berlin, Germany, to quantify their ectoparasite diversity. Host-specific, spatial and temporal occurrence of ectoparasites was determined to assess their influence on direct and indirect zoonotic risk due to mice and voles in an urban agglomeration. Rodent-associated arthropods were diverse, including 63 species observed on six host species with an overall prevalence of 99%. The tick Ixodes ricinus was the most prevalent species, found on 56% of the rodents. The trapping location clearly affected the presence of different rodent species and, therefore, the occurrence of particular host-specific parasites. In Berlin, fewer temporary and periodic parasite species as well as non-parasitic species (fleas, chiggers and nidicolous Gamasina) were detected than reported from rural areas. In addition, abundance of parasites with low host-specificity (ticks, fleas and chiggers) apparently decreased with increasing landscape fragmentation associated with a gradient of urbanisation. In contrast, stationary ectoparasites, closely adapted to the rodent host, such as the fur mites Myobiidae and Listrophoridae, were most abundant at the two urban sites. A direct zoonotic risk of infection for people may only be posed by Nosopsyllus fasciatus fleas, which were prevalent even in the city centre. More importantly, peridomestic rodents clearly supported the life cycle of ticks in the city as hosts for their subadult stages. In addition to trapping location, season, host species, body condition and host sex, infestation with fleas, gamasid Laelapidae mites and prostigmatic Myobiidae mites were associated with significantly altered abundance of I. ricinus larvae on mice and voles. Whether this is caused by predation, grooming behaviour or interaction with the host immune system is unclear. The present study constitutes a basis to identify interactions and vector function of rodent-associated arthropods and their potential impact on zoonotic diseases