Pub Date : 2022-12-21DOI: 10.3389/frans.2022.1020111
R. Lenz, Him K. Shrestha, A. Carrell, J. Labbé, R. Hettich, P. Abraham, J. LeBoldus
A major threat to forest ecosystems and plantation forestry is the introduction of a non-native pathogen. Among non-domesticated populations with relatively high levels of genetic diversity, a measurable range of susceptibility to resistance can be expected. Identifying genetic determinants of resistant and susceptible individuals can inform the development of new strategies to engineer disease resistance. Here we describe pathogen-induced changes in the proteome of Populus trichocarpa stem tissue in response to Sphaerulia musiva (Septoria canker). This hemibiotrophic fungal pathogen causes stem canker disease in susceptible poplar genotypes. Proteomics analyses were performed on stem tissue harvested across 0-, 12-, 24- and 48-h post-inoculation with Septoria from three genotypes including one resistant (BESC-22) and two susceptible [BESC-801; Nisqually-1 (NQ-1)]. In total, 11,897 Populus proteins at FDR <0.01 were identified across all time points and genotypes. Analysis of protein abundances between genotypes revealed that the resistant poplar genotype (BESC-22) mounts a rapid and sustained defense response involving pattern recognition receptors, calcium signaling proteins, SAR inducers, transcriptional regulators, resistance proteins, and proteins involved with the hypersensitive response. One susceptible genotype (BESC-801) had a downregulated and delayed defense response whereas the second susceptible genotype (NQ-1) lacked a distinct pattern. Overall, the proteome-wide and protein-specific trends suggest that responses to the Septoria canker infection are genotype-specific for the naïve host, Populus trichocarpa.
{"title":"Proteomics reveals pathways linked to septoria canker resistance and susceptibility in Populus trichocarpa","authors":"R. Lenz, Him K. Shrestha, A. Carrell, J. Labbé, R. Hettich, P. Abraham, J. LeBoldus","doi":"10.3389/frans.2022.1020111","DOIUrl":"https://doi.org/10.3389/frans.2022.1020111","url":null,"abstract":"A major threat to forest ecosystems and plantation forestry is the introduction of a non-native pathogen. Among non-domesticated populations with relatively high levels of genetic diversity, a measurable range of susceptibility to resistance can be expected. Identifying genetic determinants of resistant and susceptible individuals can inform the development of new strategies to engineer disease resistance. Here we describe pathogen-induced changes in the proteome of Populus trichocarpa stem tissue in response to Sphaerulia musiva (Septoria canker). This hemibiotrophic fungal pathogen causes stem canker disease in susceptible poplar genotypes. Proteomics analyses were performed on stem tissue harvested across 0-, 12-, 24- and 48-h post-inoculation with Septoria from three genotypes including one resistant (BESC-22) and two susceptible [BESC-801; Nisqually-1 (NQ-1)]. In total, 11,897 Populus proteins at FDR <0.01 were identified across all time points and genotypes. Analysis of protein abundances between genotypes revealed that the resistant poplar genotype (BESC-22) mounts a rapid and sustained defense response involving pattern recognition receptors, calcium signaling proteins, SAR inducers, transcriptional regulators, resistance proteins, and proteins involved with the hypersensitive response. One susceptible genotype (BESC-801) had a downregulated and delayed defense response whereas the second susceptible genotype (NQ-1) lacked a distinct pattern. Overall, the proteome-wide and protein-specific trends suggest that responses to the Septoria canker infection are genotype-specific for the naïve host, Populus trichocarpa.","PeriodicalId":73063,"journal":{"name":"Frontiers in analytical science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47828347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-12-16DOI: 10.3389/frans.2022.1091869
David Geilfuss, R. Boukherroub, J. Dostálek, W. Knoll, J. Masson, A. Baeumner, S. Szunerits
For nearly 40 years, surface plasmon resonance (SPR) analysis has been used to better understand the binding interaction strength between surface immobilized bioreceptors and the analytes of interest. The advantage of surface plasmon resonance, over other affinity sensing approaches such as Western blots and ELISAs approaches, resides in its possibility to reveal binding kinetics in a label-free manner. The concept of surface plasmon resonance has in addition been widely employed for the development of biosensors capitalizing on its direct assay format, short response times, simple sample treatments along with multiplexed sensing possibilities. To this must be added the possibility to reach high sensitivity due to the capability of surface plasmon resonance to detect very small changes in refractive index at the sensing interfaces in particular for analytes of larger size such as cells (e.g., bacteria), proteins, peptides and oligonucleotides. Challenges inherent to all affinity approaches call for further research and include non-specific surface binding events, mass transportation restrictions, steric hindrance, and the risk of data misinterpretation in case of lack of selective analyte binding. This opinion article is devoted to outlining the different approaches proposed to address these challenges by e.g., coupling with fluorescence read out, electrochemical sensing, mass spectroscopy analysis and more recently to integrate lateral flow concepts into surface plasmon resonance. Other plasmonic methods such as localized surface plasmon resonance (LSPR), surface enhanced Raman spectroscopy (SERS) will not be considered in detail, as such techniques have nowadays their own standing.
{"title":"Can classical surface plasmon resonance advance via the coupling to other analytical approaches?","authors":"David Geilfuss, R. Boukherroub, J. Dostálek, W. Knoll, J. Masson, A. Baeumner, S. Szunerits","doi":"10.3389/frans.2022.1091869","DOIUrl":"https://doi.org/10.3389/frans.2022.1091869","url":null,"abstract":"For nearly 40 years, surface plasmon resonance (SPR) analysis has been used to better understand the binding interaction strength between surface immobilized bioreceptors and the analytes of interest. The advantage of surface plasmon resonance, over other affinity sensing approaches such as Western blots and ELISAs approaches, resides in its possibility to reveal binding kinetics in a label-free manner. The concept of surface plasmon resonance has in addition been widely employed for the development of biosensors capitalizing on its direct assay format, short response times, simple sample treatments along with multiplexed sensing possibilities. To this must be added the possibility to reach high sensitivity due to the capability of surface plasmon resonance to detect very small changes in refractive index at the sensing interfaces in particular for analytes of larger size such as cells (e.g., bacteria), proteins, peptides and oligonucleotides. Challenges inherent to all affinity approaches call for further research and include non-specific surface binding events, mass transportation restrictions, steric hindrance, and the risk of data misinterpretation in case of lack of selective analyte binding. This opinion article is devoted to outlining the different approaches proposed to address these challenges by e.g., coupling with fluorescence read out, electrochemical sensing, mass spectroscopy analysis and more recently to integrate lateral flow concepts into surface plasmon resonance. Other plasmonic methods such as localized surface plasmon resonance (LSPR), surface enhanced Raman spectroscopy (SERS) will not be considered in detail, as such techniques have nowadays their own standing.","PeriodicalId":73063,"journal":{"name":"Frontiers in analytical science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45622122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-12-08DOI: 10.3389/frans.2022.1039606
D. Kumar, Ramiz M. R. Azad, D. Oulkar, H. Oberoi, S. Jacob, B. C. Koner, Subash Chandra S
Backgrounds: Oxytocin is nowadays used to increase the agricultural products besides its use during the milking of cattle leading to the contamination of agricultural produce and milk with oxytocin. Monitoring of accurate oxytocin contaminations from foodstuffs is sometimes required to maintain the quality standard. The commonly used oxytocin assays in this study were interfered with by the food matrix. There is a need to develop an accurate and confirmed method for monitoring oxytocin contaminations in foodstuffs. Objective: An attempt is made to develop an accurate assay method of oxytocin from milk and agricultural produces. Methods: The acidified methanol was used for the extraction of oxytocin from target food stuff/matrices (agricultural produce and Milk). LC-MS/MS was used for its detection and quantification. In the chromatographic separation, Oxytocin concentration was optimized using selective reaction monitoring (SRM) with heated electrospray ionization (HESI) in positive polarity. The chromatographic separation was performed using a reversed-phase C18 column with gradient elution at a flow rate of 0.4 ml/min. The acidified methanol was used for the extraction of oxytocin in all target food matrices. The method performance was verified as per the SANTE 2021 guideline. After method validation, the method was applied in real food samples analysis for assessing the presence/absence of oxytocin. Results: The calibration curve offered excellent linearity (R 2 = 0.999) with less than 15% residuals. The matrix effect was <20% observed for all target matrices. The mean recoveries were within 70%–115% with <11% RSD at four different levels in milk and 0.01 mg/kg in fruits and vegetables. The optimized method was applied to 50 random samples of milk, fruits, and vegetables from the market for the purposes of an established quality control approach. Based on the results, we did observe a signal of oxytocin in the random samples Therefore, this method has shown its practical suitability for the detection of oxytocin in milk, fruits, and vegetables. Conclusion: Extraction of oxytocin using acidified methanol followed by assays using LC-MS/MS is a simple, sensitive, accurate, reproducible, and practically suitable method for detection and quantification of oxytocin from milk, fruits, and vegetables.
{"title":"A quantification method for trace level of oxytocin in food matrices using LC-MS/MS","authors":"D. Kumar, Ramiz M. R. Azad, D. Oulkar, H. Oberoi, S. Jacob, B. C. Koner, Subash Chandra S","doi":"10.3389/frans.2022.1039606","DOIUrl":"https://doi.org/10.3389/frans.2022.1039606","url":null,"abstract":"Backgrounds: Oxytocin is nowadays used to increase the agricultural products besides its use during the milking of cattle leading to the contamination of agricultural produce and milk with oxytocin. Monitoring of accurate oxytocin contaminations from foodstuffs is sometimes required to maintain the quality standard. The commonly used oxytocin assays in this study were interfered with by the food matrix. There is a need to develop an accurate and confirmed method for monitoring oxytocin contaminations in foodstuffs. Objective: An attempt is made to develop an accurate assay method of oxytocin from milk and agricultural produces. Methods: The acidified methanol was used for the extraction of oxytocin from target food stuff/matrices (agricultural produce and Milk). LC-MS/MS was used for its detection and quantification. In the chromatographic separation, Oxytocin concentration was optimized using selective reaction monitoring (SRM) with heated electrospray ionization (HESI) in positive polarity. The chromatographic separation was performed using a reversed-phase C18 column with gradient elution at a flow rate of 0.4 ml/min. The acidified methanol was used for the extraction of oxytocin in all target food matrices. The method performance was verified as per the SANTE 2021 guideline. After method validation, the method was applied in real food samples analysis for assessing the presence/absence of oxytocin. Results: The calibration curve offered excellent linearity (R 2 = 0.999) with less than 15% residuals. The matrix effect was <20% observed for all target matrices. The mean recoveries were within 70%–115% with <11% RSD at four different levels in milk and 0.01 mg/kg in fruits and vegetables. The optimized method was applied to 50 random samples of milk, fruits, and vegetables from the market for the purposes of an established quality control approach. Based on the results, we did observe a signal of oxytocin in the random samples Therefore, this method has shown its practical suitability for the detection of oxytocin in milk, fruits, and vegetables. Conclusion: Extraction of oxytocin using acidified methanol followed by assays using LC-MS/MS is a simple, sensitive, accurate, reproducible, and practically suitable method for detection and quantification of oxytocin from milk, fruits, and vegetables.","PeriodicalId":73063,"journal":{"name":"Frontiers in analytical science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48279800","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-11-29DOI: 10.3389/frans.2022.1010317
D. Vos, S. Meijer, R. Pouw, S. Ellis, R. Heeren, B. Balluff
Glycosylation plays an important role in the progression of esophageal adenocarcinoma (EAC). Being able to image these glycosylation changes directly in endoscopic resection specimens could provide useful insights into the molecular mechanisms of the disease progression and potential markers for EAC staging. For this purpose, both 3D and 2.5D matrix-assisted laser/desorption ionization (MALDI) mass spectrometry imaging (MSI) have been employed in this study to investigate glycosidase-cleaved N-glycans in a total of 24 formalin-fixed paraffin-embedded esophageal local excision specimens spanning all stages of disease progression, namely from non-dysplastic Barrett’s esophagus to metastatic EAC. 3D-MSI was first used to estimate the number of sections needed to sufficiently cover the molecular heterogeneity of each stage of progression. This analysis showed that a total of four sections out of 20 were sufficient. This subset of four sections was measured for all remaining specimens and is called 2.5D-MSI. Subsequent analyses of the 2.5D-MSI datasets revealed significant elevations of five high-mannose N-glycans (Man3, Man4, Man6, Man7, and Man8) in EAC and three complex (Hex6HexNAc5, Hex6HexNAc5NeuAc1, Hex7HexNAc6) N-glycans in metastatic EAC as compared to previous stages of the disease. The augmented levels of these glycans in EAC could be explained by publically available gene expression data of enzymes involved in glycan synthesis and processing. As the role of glycosylation is gaining more interest in MSI and cancer research, our results show the added value of combining localized N-glycan levels, as provided by MSI, with gene expression to gain a deeper understanding of the mechanisms behind N-glycan changes. This gives evidence at multiple levels that specific N-glycosylation plays an important role during progression of dysplasia to EAC and could play a role in patient surveillance.
{"title":"2.5D mass spectrometry imaging of N-glycans in esophageal adenocarcinoma and precursor lesions","authors":"D. Vos, S. Meijer, R. Pouw, S. Ellis, R. Heeren, B. Balluff","doi":"10.3389/frans.2022.1010317","DOIUrl":"https://doi.org/10.3389/frans.2022.1010317","url":null,"abstract":"Glycosylation plays an important role in the progression of esophageal adenocarcinoma (EAC). Being able to image these glycosylation changes directly in endoscopic resection specimens could provide useful insights into the molecular mechanisms of the disease progression and potential markers for EAC staging. For this purpose, both 3D and 2.5D matrix-assisted laser/desorption ionization (MALDI) mass spectrometry imaging (MSI) have been employed in this study to investigate glycosidase-cleaved N-glycans in a total of 24 formalin-fixed paraffin-embedded esophageal local excision specimens spanning all stages of disease progression, namely from non-dysplastic Barrett’s esophagus to metastatic EAC. 3D-MSI was first used to estimate the number of sections needed to sufficiently cover the molecular heterogeneity of each stage of progression. This analysis showed that a total of four sections out of 20 were sufficient. This subset of four sections was measured for all remaining specimens and is called 2.5D-MSI. Subsequent analyses of the 2.5D-MSI datasets revealed significant elevations of five high-mannose N-glycans (Man3, Man4, Man6, Man7, and Man8) in EAC and three complex (Hex6HexNAc5, Hex6HexNAc5NeuAc1, Hex7HexNAc6) N-glycans in metastatic EAC as compared to previous stages of the disease. The augmented levels of these glycans in EAC could be explained by publically available gene expression data of enzymes involved in glycan synthesis and processing. As the role of glycosylation is gaining more interest in MSI and cancer research, our results show the added value of combining localized N-glycan levels, as provided by MSI, with gene expression to gain a deeper understanding of the mechanisms behind N-glycan changes. This gives evidence at multiple levels that specific N-glycosylation plays an important role during progression of dysplasia to EAC and could play a role in patient surveillance.","PeriodicalId":73063,"journal":{"name":"Frontiers in analytical science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49103256","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-11-22DOI: 10.3389/frans.2022.1080042
Entesar Al‐Hetlani
In this inspiring issue, three interesting and diverse peer-reviewed articles were published: two full research articles and one review article. The topics of these articles covered luminescence and electrochemical methods for the analysis of physical evidence. The three articles discussed the detection of fentanyl and its analogs via in situ electrochemical-surface enhanced Raman spectroscopy (EC-SERS), the utilization of electrochemistry to probe the degradation and time since deposition of blood and, finally, the role of fluorescence spectroscopy in the analysis of blood. These articles highlighted some of the main challenges currently facing forensic practitioners working with different types of physical evidence and provided suggestions for some of the fundamental unanswered questions. Can we rapidly differentiate between fentanyl and its analogs? Can we predict the degradation behavior of bloodstains at different temperatures using electrochemistry? How can fluorescence spectroscopy be beneficial for blood analysis? Due to the increase in casework related to fentanyl and its analogs, there is a genuine need for simple, rapid and sensitive methods for their identification and differentiation. In this respect, the in situ electrochemical-surface enhanced Raman spectroscopy (EC-SERS) method was developed by Ott et al. for the rapid detection of fentanyl and six of its analogs. In this detailed study, the authors initially optimized the experimental conditions for the SERS substrate and amperometric detection. The synthesis and roughening of the SERS substrate (Ag nanoparticles) were performed in situ utilizing cyclic voltammetry (CV) and multipulse amperometric detection (MPD). Consequently, MPD parameters were optimized to obtain the maximum response and improved enhancement due to SERS hot spot generation. Major functional groups in fentanyl were identified, and acetyl fentanyl, methoxyacetyl fentanyl, furanyl fentanyl, acryl fentanyl, valeryl fentanyl and despropionyl fentanyl (4-ANPP) analogs were successfully analyzed due to their difference in the amide portion. This is significant for crime laboratories when minor differences in the drug molecule structure can make identification challenging. Sensitivity was evaluated by determining the limit of detection of each drug by measuring the signal OPEN ACCESS
{"title":"Editorial: Luminescence and electrochemical methods: Analysis of physical evidence","authors":"Entesar Al‐Hetlani","doi":"10.3389/frans.2022.1080042","DOIUrl":"https://doi.org/10.3389/frans.2022.1080042","url":null,"abstract":"In this inspiring issue, three interesting and diverse peer-reviewed articles were published: two full research articles and one review article. The topics of these articles covered luminescence and electrochemical methods for the analysis of physical evidence. The three articles discussed the detection of fentanyl and its analogs via in situ electrochemical-surface enhanced Raman spectroscopy (EC-SERS), the utilization of electrochemistry to probe the degradation and time since deposition of blood and, finally, the role of fluorescence spectroscopy in the analysis of blood. These articles highlighted some of the main challenges currently facing forensic practitioners working with different types of physical evidence and provided suggestions for some of the fundamental unanswered questions. Can we rapidly differentiate between fentanyl and its analogs? Can we predict the degradation behavior of bloodstains at different temperatures using electrochemistry? How can fluorescence spectroscopy be beneficial for blood analysis? Due to the increase in casework related to fentanyl and its analogs, there is a genuine need for simple, rapid and sensitive methods for their identification and differentiation. In this respect, the in situ electrochemical-surface enhanced Raman spectroscopy (EC-SERS) method was developed by Ott et al. for the rapid detection of fentanyl and six of its analogs. In this detailed study, the authors initially optimized the experimental conditions for the SERS substrate and amperometric detection. The synthesis and roughening of the SERS substrate (Ag nanoparticles) were performed in situ utilizing cyclic voltammetry (CV) and multipulse amperometric detection (MPD). Consequently, MPD parameters were optimized to obtain the maximum response and improved enhancement due to SERS hot spot generation. Major functional groups in fentanyl were identified, and acetyl fentanyl, methoxyacetyl fentanyl, furanyl fentanyl, acryl fentanyl, valeryl fentanyl and despropionyl fentanyl (4-ANPP) analogs were successfully analyzed due to their difference in the amide portion. This is significant for crime laboratories when minor differences in the drug molecule structure can make identification challenging. Sensitivity was evaluated by determining the limit of detection of each drug by measuring the signal OPEN ACCESS","PeriodicalId":73063,"journal":{"name":"Frontiers in analytical science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46625692","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-11-01DOI: 10.3389/frans.2022.1005558
L. Stockdale, N. de Haan, J. Hill, M. Johnson, A. Tomic, M. Wuhrer, E. Jones, C. Jin, J. Nouta, C. Koeleman, M. Verheul, B. Basnyat, M. Shakya, D. Pant, S. Provstgaard-Morys, A. Pollard
Vaccines against typhoid fever have been shown to be safe and effective in field trials. The mechanism through which the vaccines protect remains elusive. Recent data have implicated antibody glycosylation, and specifically afucosylated antibodies, as an important factor in vaccine-induced effector function for a range of viral infections, however this has not been evaluated for vaccines against bacterial infections such as Salmonella typhi. Here, we studied antibody glycosylation after either Vi-conjugate or Vi-polysaccharide vaccine in a UK cohort who were then challenged with virulent S. typhi, and compared findings to antibody glycosylation after Vi-conjugate vaccine in Nepalese children living in a typhoid endemic region. We compared vaccine-induced responses and correlated these measures with antibody-dependent function. Robust antigen-specific antibody galactosylation and sialylation modifications were induced by both vaccines in UK adults, with Vi-conjugate vaccine inducing Vi-specific glycan changes of higher magnitude than Vi-polysaccharide. Among those individuals diagnosed with typhoid fever after challenge, a distinct glycan profile was correlated with disease severity. Elevated galactosylation and sialylation was correlated with increased antibody-dependent phagocytosis by macrophages and neutrophils among UK adults. While bulk IgG glycosylation differed between Nepalese children and UK adults, vaccination with the Vi-conjugate vaccine overcame these differences to result in similar Vi-specific antibody glycosylation profiles 28 days after vaccination in both cohorts.
{"title":"Distinct glycosylation and functional profile of typhoid vaccine-induced antibodies in a UK challenge study and Nepalese children","authors":"L. Stockdale, N. de Haan, J. Hill, M. Johnson, A. Tomic, M. Wuhrer, E. Jones, C. Jin, J. Nouta, C. Koeleman, M. Verheul, B. Basnyat, M. Shakya, D. Pant, S. Provstgaard-Morys, A. Pollard","doi":"10.3389/frans.2022.1005558","DOIUrl":"https://doi.org/10.3389/frans.2022.1005558","url":null,"abstract":"Vaccines against typhoid fever have been shown to be safe and effective in field trials. The mechanism through which the vaccines protect remains elusive. Recent data have implicated antibody glycosylation, and specifically afucosylated antibodies, as an important factor in vaccine-induced effector function for a range of viral infections, however this has not been evaluated for vaccines against bacterial infections such as Salmonella typhi. Here, we studied antibody glycosylation after either Vi-conjugate or Vi-polysaccharide vaccine in a UK cohort who were then challenged with virulent S. typhi, and compared findings to antibody glycosylation after Vi-conjugate vaccine in Nepalese children living in a typhoid endemic region. We compared vaccine-induced responses and correlated these measures with antibody-dependent function. Robust antigen-specific antibody galactosylation and sialylation modifications were induced by both vaccines in UK adults, with Vi-conjugate vaccine inducing Vi-specific glycan changes of higher magnitude than Vi-polysaccharide. Among those individuals diagnosed with typhoid fever after challenge, a distinct glycan profile was correlated with disease severity. Elevated galactosylation and sialylation was correlated with increased antibody-dependent phagocytosis by macrophages and neutrophils among UK adults. While bulk IgG glycosylation differed between Nepalese children and UK adults, vaccination with the Vi-conjugate vaccine overcame these differences to result in similar Vi-specific antibody glycosylation profiles 28 days after vaccination in both cohorts.","PeriodicalId":73063,"journal":{"name":"Frontiers in analytical science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44409707","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-11-01DOI: 10.3389/frans.2022.1030701
S. Mahmoud, S. Ganesan, P. Raheja, F. Cantarutti, Hagar Ateia, W. Zaher
Introduction: The COVID-19 pandemic has led to the rapid development and launch of several commercial RT-PCR-based assays for identification of SARS-CoV-2. However, there is need for peer-reviewed evaluation of these assays that can support their clinical performance. In this study, we, therefore, conduct an in-house evaluation of the automated Cobas 68000 RT-PCR assay in detecting SARS-CoV-2 infections using different pooling techniques. Methods: An observational study is conducted to evaluate the clinical performance of the Cobas 6800 SARS-CoV-2 assay in comparison with the Labgun Exofast RT-PCR kit, using both pooled and non-pooled sample techniques. A total of 300 nasopharyngeal swab samples, 40 known positive samples and 260 negative samples, are used for pooling, while the performance is evaluated in three different sample pool sizes of 4, 5, and 6. Results: The sensitivity and specificity of the Cobas 6,800 was 100% when compared to the comparable assay. The sample pooling technique showed that specificity was 100% in all pool sizes and the sensitivity varied from 95% in the 6-pooled sample to 100% in both the 5- and 4-pooled samples. The lower limit of detection was verified as 25 copies/ml for un-pooled samples, and, therefore, the limit of detection was 100, 125, and 150 copies/ml for the 4, 5, and 6 sample pools, respectively. Strong correlation was observed between the Ct values of the target genes of both assays. Conclusion: Cobas 6800 RT-PCR assay is a reliable platform for qualitative and rapid detection of SARS-CoV-2 and can be effectively utilized for pooling of samples with highly efficient performance when disease prevalence is lower.
{"title":"Diagnostic accuracy of the Cobas 6800 RT-PCR assay for detection of SARS-CoV-2 RNA","authors":"S. Mahmoud, S. Ganesan, P. Raheja, F. Cantarutti, Hagar Ateia, W. Zaher","doi":"10.3389/frans.2022.1030701","DOIUrl":"https://doi.org/10.3389/frans.2022.1030701","url":null,"abstract":"Introduction: The COVID-19 pandemic has led to the rapid development and launch of several commercial RT-PCR-based assays for identification of SARS-CoV-2. However, there is need for peer-reviewed evaluation of these assays that can support their clinical performance. In this study, we, therefore, conduct an in-house evaluation of the automated Cobas 68000 RT-PCR assay in detecting SARS-CoV-2 infections using different pooling techniques. Methods: An observational study is conducted to evaluate the clinical performance of the Cobas 6800 SARS-CoV-2 assay in comparison with the Labgun Exofast RT-PCR kit, using both pooled and non-pooled sample techniques. A total of 300 nasopharyngeal swab samples, 40 known positive samples and 260 negative samples, are used for pooling, while the performance is evaluated in three different sample pool sizes of 4, 5, and 6. Results: The sensitivity and specificity of the Cobas 6,800 was 100% when compared to the comparable assay. The sample pooling technique showed that specificity was 100% in all pool sizes and the sensitivity varied from 95% in the 6-pooled sample to 100% in both the 5- and 4-pooled samples. The lower limit of detection was verified as 25 copies/ml for un-pooled samples, and, therefore, the limit of detection was 100, 125, and 150 copies/ml for the 4, 5, and 6 sample pools, respectively. Strong correlation was observed between the Ct values of the target genes of both assays. Conclusion: Cobas 6800 RT-PCR assay is a reliable platform for qualitative and rapid detection of SARS-CoV-2 and can be effectively utilized for pooling of samples with highly efficient performance when disease prevalence is lower.","PeriodicalId":73063,"journal":{"name":"Frontiers in analytical science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44644209","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-10-26DOI: 10.3389/frans.2022.1021008
E. Jones, R. Drake, James W Dressman, Vaunita Parihar, Rachel Stubler, E. Masters, K. Mercer
The current COVID-19 pandemic is characterized by a broad range of disease severity in patients. This diversity in clinical manifestations has complicated our understanding of the SARS-CoV-2 pathogenesis and highlights the significance of an individual’s ability to mount an effective viral immune response. Glycosylation is a common post-translational modification occurring in complex organisms and is imperative for immune cell function. In this study, a combination approach with immunohistochemistry (IHC) and matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was utilized to determine the spatial distribution of N-glycans and immune cell populations in COVID-19 lung tissues. Tissues from seven SARS-CoV-2, PCR + donors were analyzed. Tissues represented a spectrum of time spent on ventilators which was reflected in their respective viral infection status and lung pathologies. N-glycan distributions in the MALDI-IMS images were then correlated with H&E staining and IHC of SARS-CoV-2 spike protein, CD4, CD8, CD163 and CD11b. Distinct and shared N-glycan signatures were identified in association with specific immune cell types, and their co-localization with the viral spike protein. Additionally, we observed unique patterns of α2,3-linked and α2,6-linked sialic acid glycans that associated with both immune cell populations and fibrotic regions within the tissue architecture. N-glycan MALDI-IMS is an effective tool to further understand tissue-localized immune cell populations in response to emerging viral pathogens such as SARS-CoV-2.
{"title":"Applying imaging mass spectrometry to define the N-glycan profiles of co-localized virus and immune cell infiltrates in post-COVID-19 infected lung autopsy tissues","authors":"E. Jones, R. Drake, James W Dressman, Vaunita Parihar, Rachel Stubler, E. Masters, K. Mercer","doi":"10.3389/frans.2022.1021008","DOIUrl":"https://doi.org/10.3389/frans.2022.1021008","url":null,"abstract":"The current COVID-19 pandemic is characterized by a broad range of disease severity in patients. This diversity in clinical manifestations has complicated our understanding of the SARS-CoV-2 pathogenesis and highlights the significance of an individual’s ability to mount an effective viral immune response. Glycosylation is a common post-translational modification occurring in complex organisms and is imperative for immune cell function. In this study, a combination approach with immunohistochemistry (IHC) and matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was utilized to determine the spatial distribution of N-glycans and immune cell populations in COVID-19 lung tissues. Tissues from seven SARS-CoV-2, PCR + donors were analyzed. Tissues represented a spectrum of time spent on ventilators which was reflected in their respective viral infection status and lung pathologies. N-glycan distributions in the MALDI-IMS images were then correlated with H&E staining and IHC of SARS-CoV-2 spike protein, CD4, CD8, CD163 and CD11b. Distinct and shared N-glycan signatures were identified in association with specific immune cell types, and their co-localization with the viral spike protein. Additionally, we observed unique patterns of α2,3-linked and α2,6-linked sialic acid glycans that associated with both immune cell populations and fibrotic regions within the tissue architecture. N-glycan MALDI-IMS is an effective tool to further understand tissue-localized immune cell populations in response to emerging viral pathogens such as SARS-CoV-2.","PeriodicalId":73063,"journal":{"name":"Frontiers in analytical science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46857352","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-10-24DOI: 10.3389/frans.2022.928573
Colin I. Elliott, A. Shafer, T. Stotesbury
Estimating the time since deposition (TSD) of a bloodstain can provide important medico-legal information for crime scene investigation. Research in this area primarily investigates the degradation of either hemoglobin or genetic material over time. In this work, we present a comprehensive meta-analysis on bloodstain TSD research. Our results are interpreted from 25 quantitative studies used to probe the effect of biomolecule studied, analytical technique used, substrate porosity, environmental conditions, and blood source on TSD estimates. There was an overall strong effect of time across studies (Fisher’s Zr = 1.66, r = 0.93), and generally, we found that the type of biomolecule studied (e.g., hemoglobin, DNA) had equal effect sizes for TSD estimation. Differences in the mean TSD effect size were also observed between substrate porosity. Interestingly, the blood source does not significantly influence the magnitude of the effect sizes in TSD estimation. Despite the clear effect of time, forensically relevant prediction of bloodstain TSD remains complicated by inter-donor variability, type of substrate and environmental conditions. We recommend that future bloodstain TSD research increase sample size, include summary statistics and standardize experimental methodologies so that we can develop a quantitative understanding of the physicochemical processes involved in whole blood degradation in ex vivo conditions.
{"title":"The crux of time: A meta-analysis of ex vivo whole blood degradation","authors":"Colin I. Elliott, A. Shafer, T. Stotesbury","doi":"10.3389/frans.2022.928573","DOIUrl":"https://doi.org/10.3389/frans.2022.928573","url":null,"abstract":"Estimating the time since deposition (TSD) of a bloodstain can provide important medico-legal information for crime scene investigation. Research in this area primarily investigates the degradation of either hemoglobin or genetic material over time. In this work, we present a comprehensive meta-analysis on bloodstain TSD research. Our results are interpreted from 25 quantitative studies used to probe the effect of biomolecule studied, analytical technique used, substrate porosity, environmental conditions, and blood source on TSD estimates. There was an overall strong effect of time across studies (Fisher’s Zr = 1.66, r = 0.93), and generally, we found that the type of biomolecule studied (e.g., hemoglobin, DNA) had equal effect sizes for TSD estimation. Differences in the mean TSD effect size were also observed between substrate porosity. Interestingly, the blood source does not significantly influence the magnitude of the effect sizes in TSD estimation. Despite the clear effect of time, forensically relevant prediction of bloodstain TSD remains complicated by inter-donor variability, type of substrate and environmental conditions. We recommend that future bloodstain TSD research increase sample size, include summary statistics and standardize experimental methodologies so that we can develop a quantitative understanding of the physicochemical processes involved in whole blood degradation in ex vivo conditions.","PeriodicalId":73063,"journal":{"name":"Frontiers in analytical science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42619657","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-10-19DOI: 10.3389/frans.2022.1018115
Daniel Carvalho, Bruna Barbosa Laurentino, Camila Loreta Rocha, J. Kool, G. Somsen, Erika R Amstalden van Hove, C. Cardoso
Deregulated activity and expression of human kallikreins (KLKs) may be involved in various pathologies, so these enzymes are an attractive biological target for identifying molecules that can modulate KLK activity. This identification involves applying fast and efficient screening methods. This work describes an off-line assay with mass spectrometry (MS) detection that uses KLK immobilized on Sepharose-NHS as a micro-column configuration (IMER-KLK-Sepharose-NHS). The mass spectrometry used has an ion trap analyzer and electrospray ionization (EIS). The HPLC-MS method for quantifying KLK activity was developed. The enzymatic assay conditions were optimized, and the IMER-KLK-Sepharose-NHS kinetic parameter (KMapp = 15.48 ± 3 μmol L−1) was evaluated. Finally, the method was validated by using leupeptin as a reference inhibitor (IC50 = 0.85 ± 0.10 μmol L−1). The developed method was able to identify the reference inhibitor and can be an alternative for screening KLK inhibitors.
{"title":"Activity assay based on the immobilized enzyme kallikrein and mass spectrometry","authors":"Daniel Carvalho, Bruna Barbosa Laurentino, Camila Loreta Rocha, J. Kool, G. Somsen, Erika R Amstalden van Hove, C. Cardoso","doi":"10.3389/frans.2022.1018115","DOIUrl":"https://doi.org/10.3389/frans.2022.1018115","url":null,"abstract":"Deregulated activity and expression of human kallikreins (KLKs) may be involved in various pathologies, so these enzymes are an attractive biological target for identifying molecules that can modulate KLK activity. This identification involves applying fast and efficient screening methods. This work describes an off-line assay with mass spectrometry (MS) detection that uses KLK immobilized on Sepharose-NHS as a micro-column configuration (IMER-KLK-Sepharose-NHS). The mass spectrometry used has an ion trap analyzer and electrospray ionization (EIS). The HPLC-MS method for quantifying KLK activity was developed. The enzymatic assay conditions were optimized, and the IMER-KLK-Sepharose-NHS kinetic parameter (KMapp = 15.48 ± 3 μmol L−1) was evaluated. Finally, the method was validated by using leupeptin as a reference inhibitor (IC50 = 0.85 ± 0.10 μmol L−1). The developed method was able to identify the reference inhibitor and can be an alternative for screening KLK inhibitors.","PeriodicalId":73063,"journal":{"name":"Frontiers in analytical science","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45294919","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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