While real-time reverse transcriptase–polymerase chain reaction (RT-PCR) on nasopharyngeal swabs is the current standard for SARS-CoV-2 detection, saliva is an attractive alternative for diagnosis and screening due to ease of collection and minimal supply requirements. Studies on the sensitivity of saliva-based SARS-CoV-2 molecular testing have shown considerable variability.We conducted a prospective, longitudinal study to investigate the testing time frame that optimizes saliva sensitivity for SARS-CoV-2 detection.Methods
Between June 17, 2020, and February 15, 2021, a convenience sample of individuals exposed to a household member with RT-PCR–confirmed SARS-CoV-2 within 2 weeks were recruited from Children’s Hospital Los Angeles and nearby community testing sites into the Household Exposure and Respiratory Virus Transmission and Immunity Study (HEARTS). Paired nasopharyngeal and saliva samples were collected every 3 to 7 days for up to 4 weeks or until 2 negative nasopharyngeal test results. RT-PCR for SARS-CoV-2 N1 and N2 genes was performed; cycle threshold less than 40 defined a positive result. A nasopharyngeal N1 cycle threshold of 34 or less was defined as high viral load. Detailed specimen collection and RT-PCR methods are reported in the eMethods in the Supplement.
Saliva sensitivity was calculated using nasopharyngeal-positive RT-PCR as the reference standard. COVID-19 onset was defined as the earlier date between first symptom (collected by questionnaire daily) or first RT-PCR positivity. Pre- and postsymptomatic were defined as asymptomatic time points before and after a symptomatic interval, respectively.
Saliva sensitivity by week of collection and between symptomatic and asymptomatic individuals were compared using the χ2 test or the Fisher exact test. Generalized estimating equations were used to determine clinical characteristics (Table) associated with saliva sensitivity in nasopharyngeal-positive pairs while accounting for repeated samples from the same individuals. Analyses were performed using SPSS version 27.0 (IBM Corp) with a 2-sided P < .05 considered significant. Written informed consent was obtained from participants. The study was approved by the institutional review board of Children’s Hospital Los Angeles.Results
We tested 889 paired nasopharyngeal swab-saliva samples from 404 participants, of which SARS-CoV-2 was detected in 524 nasopharyngeal (58.9%) and 318 saliva (35.7%) specimens. SARS-CoV-2 was detected in both specimens in 258 pairs (29.0%). Of the 256 nasopharyngeal SARS-CoV-2–positive participants (63.4%), the mean age was 28.2 years (range, 3.0-84.5 years); 108 (42.2%) were male. Participants returned for a median of 3 visits (interquartile range, 2-4). Ninety-three participants (36.3%) were asymptomatic throughout their infection; 126 (77.3%) of 163 symptomatic individuals reported mild severity.
Saliva sensitivity was highest in samples collected during the first week of infection at 71.2% (95% CI, 62.6%-78.8%) but decreased each subsequent week . Participants who presented with COVID-19–associated symptoms on the specimen collection day during week 1 of infection had significantly higher saliva sensitivity compared with asymptomatic participants (88.2% [95% CI, 77.6%-95.1%] vs 58.2% [95% CI, 46.3%-69.5%]; P < .001).
Saliva sensitivity remained significantly higher in symptomatic participants in week 2 (83.0% [95% CI, 70.6%-91.8%] vs 52.6% [95% CI, 42.6%-62.5%]; P < .001). No difference was observed more than 2 weeks after COVID-19 onset . Sensitivities did not significantly differ for never-symptomatic (34.7% [95% CI, 27.3%-42.7%]), presymptomatic (57.1% [95% CI, 31.7%-80.2%]), and postsymptomatic (42.9% [95% CI, 36.8%-49.1%]) time points (P = .26).
For each day after COVID-19 onset, the odds ratio for saliva detection was 0.94 (95% CI, 0.91-0.96) compared with the previous day (P < .001) . Participants presenting with COVID-19–associated symptoms at the time of specimen collection or with high nasopharyngeal viral loads had 2.8 (95% CI, 1.6-5.1; P < .001) and 5.2 (95% CI, 2.9-9.3; P < .001) higher odds of having a saliva-positive RT-PCR result compared with those with asymptomatic presentation or low nasopharyngeal viral loads, respectively.
Saliva was sensitive for detecting SARS-CoV-2 in symptomatic individuals during initial weeks of infection, but sensitivity in asymptomatic SARS-CoV-2 carriers was less than 60% at all time points. As COVID-19 testing strategies in workplaces, schools, and other shared spaces are optimized, low saliva sensitivity in asymptomatic infections must be considered. This study suggests saliva-based RT-PCR should not be used for asymptomatic COVID-19 screening.
This study has limitations. Samples were collected following household exposure; therefore, pretest probability was high. Nasopharyngeal swab testing was the reference standard, but this is not a perfect test for SARS-CoV-2 infection, and a positive RT-PCR result from any sample past 10 days of infection may not be predictive of viral replication or infectivity.
Our study is concordant with multiple published works supporting saliva as an alternative sample for COVID-19 screening and diagnosis, and one of a minority where saliva was shown to be more sensitive than the corresponding NP swab8,9,13, although the results by Leung et al. (53.7% saliva vs. 47.4% NP swab, 95 subjects) were not statistically different8. Several reasons may account for this difference in the studies, including enrichment from nasal and oropharyngeal secretions, where the viral load is potentially higher8,9, or a higher volume of samples collection, where approximately 10 mL of saliva was collected for testing13. Steps were taken to minimize biases and errors—NP swabs performed by trained healthcare staff, environmental testing of CAP-accredited laboratory (no evidence of contamination), conduction of tests for most of the samples in the same laboratory, and pre-processing of saliva samples with dithiothreitol before RNA extraction to resolve the issues of saliva specimen viscosity, which can lead to false negatives.
Interestingly but perhaps unsurprisingly, the use of different RT-PCR kits in the present study resulted in different test-positive rates in saliva, suggesting that this can potentially be an important consideration for clinical laboratories, where more sensitive laboratory protocols should be deployed for clinical diagnosis as opposed to mass screening for low-prevalence populations. More validation would be required to confirm this finding.
SN swabs, however, appeared less sensitive compared to both saliva and NP swabs for the diagnosis of COVID-19. Although it was convenient, less time-consuming to perform relative to saliva collection, and caused less discomfort compared to NP swabs, the markedly lower sensitivity should preclude its use where other sample types can be collected.
In our study, NGS provided efficient whole-genome profiling of SARS-CoV-2 for phylogenetic analysis directly from the clinical samples without culture. NGS detection sensitivity was excellent with a threshold of 1.7% genome coverage or 5 amplicons targets, confirming all CDC-LDT positives tested. Other groups have reported highly sensitive performance for NGS with limits of detection ranging between a threshold of 5% genome coverage or 84 genome-equivalents per mL21, or at least 5 SARS-CoV-2 targets for detection22. The phylogeny results were consistent with the virus belonging to a viral type (Clade O, lineage B.6) known to be circulating in the geographical regions of Singapore and India.
There are several limitations to our work. Firstly, the study population was confined to young and middle-aged men who were either asymptomatic or had mild disease. The results cannot be extrapolated to other populations (e.g., paediatric), where there is a clear need for alternate sample types to NP swabs. Secondly, we did not extend the follow-up testing sufficiently to determine when saliva viral shedding stopped for the majority of subjects, although this has been explored in other studies. Thirdly, we did not test for the difference, if any, between saliva obtained from naso-oropharyngeal or the mouth alone, although it is biologically plausible that the latter would result in lower sensitivity for COVID-19 diagnosis.
In conclusion, our study adds to the body of evidence supporting saliva as a sensitive and less intrusive sample for COVID-19 diagnosis and further defines the role of naso-oropharyngeal secretions and the impact of different RT-PCR kits in increasing the sensitivity of testing. In our study, SN swabs were inferior to both saliva and NP swabs. Our study also provides evidence to support NGS in challenging samples for sensitive COVID-19 molecular diagnosis. Such an NGS workflow can also provide direct-from-sample phylogenetic analysis for public health decision-making, such as contact tracing.
While the causes of bladder cancer remain largely unknown, smoking is considered to be the main risk factor for the disease. By recreating the DNA damage caused by smoking toxins, these scientists at York University will help to better understand the causes of bladder cancer.
The team, led by Dr. Simon Baker of the Department of Biology at the University of York, cultivated human bladder tissue in the laboratory and exposed it to a common toxin from cigarette smoke. After the tissues were damaged by the toxins from the smoke, the team analyzed the 2 billion letters of the genetic code (DNA) to identify a pattern of change or “mutational signature”.
Identification of a mutational signature of tobacco-related cell damage This mutational signature consists of a unique pattern of DNA damage that could be found in the event of the development of bladder cancer in connection with smoking: “Mutational signatures can be used as fingerprints on a stage. crime. When we analyze DNA in cancer, we can identify these fingerprints and therefore find the criminals involved in the damage that led to cancer. ”
Weigh the importance of the different risk factors: the study reveals that the toxins from tobacco smoke do leave a specific imprint on the DNA of bladder tissues grown in the laboratory. However, this mutational signature linked to smoke toxins appears, after an in-depth analysis of the tissues, only responsible for a small part of the damage. This is the second lesson of this study: the technique of biopsy analysis could, for a given tumor, make it possible to balance the incidence of different risk factors. Here, the researchers conclude that although smoking is the main risk factor for bladder cancer, direct damage to DNA by smoke toxins does not appear to be the main reason for the formation of these cancers.
The toxins from tobacco smoke could be triggers that accelerate other DNA damaging events. The enzymes called “APOBEC” could thus be involved in this cascade of events:
recent studies suggest that they may indeed mistakenly target our own DNA in a number of types of cancer and, in particular, in bladder cells.
Cheese rind microbiomes are helpful mannequin techniques for figuring out the mechanisms that management microbiome variety. Here, we describe the strategies we now have optimized to first deconstruct in situ cheese rind microbiome variety and then reconstruct that variety in laboratory environments to conduct managed microbiome manipulations.
Most cheese rind microbial species, together with micro organism, yeasts, and filamentous fungi, could be simply cultured utilizing commonplace lab media. Colony morphologies of taxa are numerous and can usually be used to tell apart taxa on the phylum and generally even genus degree. Through the usage of cheese curd agar medium, hundreds of distinctive neighborhood mixtures or microbial interactions could be assessed. Transcriptomic experiments and transposon mutagenesis screens can pinpoint mechanisms of interactions between microbial species.
Preparation of lyophilized and powdered cheese curd Support Protocol 3: Preparation of 10% cheese curd agar plates and tubes Basic Protocol 6: Interaction screens utilizing responding lawns Support Protocol 4: Preparation of liquid 2% cheese curd Basic Protocol 7: Experimental evolution Basic Protocol 8: Measuring neighborhood perform: pH/acidification Basic Protocol 9: Measuring neighborhood perform: Pigment manufacturing Basic Protocol 10: RNA sequencing of cheese rind biofilms.
Selection of Lactic Acid Bacteria (LAB) Antagonizing Vibrio Parahaemolyticus: The Pathogen of Acute Hepatopancreatic Necrosis Disease (AHPND) in Whiteleg Shrimp (Penaeus Vannamei)
Acute hepatopancreatic necrosis illness (AHPND) has just lately emerged as a critical illness of cultured shrimp. A complete of 19 lactic acid micro organism (LAB) strains remoted from shrimp samples have been characterised based mostly on morphological traits, biochemical assessments, sequencing evaluation, and their means to antagonize Vibrio parahaemolyticus, which causes AHPND in whiteleg shrimp.
Results from the agar nicely diffusion technique indicated that Three out of 19 remoted LAB strains confirmed the best antagonizing means in opposition to AHPND V. parahaemolyticus pressure with an inhibition zone diameter starting from 18 to 20 mm. Experiments the place shrimps got feed supplemented with these LAB strains and challenged with AHPND pressure confirmed excessive survival charges (roughly 80.0%), which weren’t considerably completely different as in comparison with these recorded in the destructive management remedy (86.6%), however considerably completely different to these recorded in the optimistic management remedy (40.6%) after 16 days of the experiment.
However, the histological pictures of shrimp hepatopancreas indicated that the an infection price considerably lowered from 60.0% to 11.1% in shrimps fed with LAB-supplemented feeds and challenged with AHPND V. parahaemolyticus pressure as in comparison with these in the optimistic management remedy. A polymerase chain response (PCR) and 16S rRNA gene sequencing confirmed the identification of LAB pressure. These outcomes could be utilized in additional experiments to analyze the power of L. plantarum in stopping AHPND in intensively cultured whiteleg shrimp.
Micropropagation of Rosaceous Species SAM Grown in Temperate Climate
The advantages of in vitro plant cultivation are primarily attributable to very excessive multiplication price. Cultivation of plant materials in vitro could be carried out throughout the entire yr whatever the time of the yr or climate circumstances.
We create synthetic circumstances in the lab (warmth, gentle, humidity), and we will regulate these circumstances at any time. For the preservation of cultivar id, we advocate establishing in vitro cultures from shoot suggestions often bigger than 0.2 mm.
In follow, in vitro cultivation of crops makes use of these development regulators to attain organogenesis, for instance, root formation, extended development, or multiplication. During every subculture, these cultures are then transferred on a strong agar medium in the type of actively rising a number of shoots with a well-differentiated shoot tip containing meristematic space. Cytokinins are vital for cell division and causes branching of crops.
Auxins, each endogenous and exogenous, act at as a set off for the differentiation and formation of root primordia. Morphological traits (formation of leaves or callus) and shoot improvement must be noticed throughout in vitro multiplication and after switch to ex vitro circumstances.
Campylobacter has been acknowledged as a big foodborne pathogen worldwide. An rising frequency of antibiotic-resistant pathogens along with Campylobacter is acknowledged to transmit from meals merchandise to folks and causes excessive threats to public effectively being.
To greater mitigate antibiotic resistance catastrophe, quick detection methods are required to provide effectively timed antimicrobial resistance surveillance info for the agri-food packages. Herein, we developed a polymer-based microfluidic machine for the identification and antimicrobial susceptibility testing (AST) of Campylobacter An array of bacterial incubation chambers was created throughout the microfluidic machine, the place chromogenic medium and antibiotics had been loaded.
The progress of Campylobacter was visualized by color change because of this of chromogenic response. This platform achieved 100% specificity of Campylobacter identification. Sensitive detection of a quantity of Campylobacter species (i.e., C. jejuni, C. coli, and C. lari) was obtained in artificially contaminated milk and poultry meat with detection limit proper right down to 1×102 CFU/mL and 1×104 CFU/25 g, respectively. On-chip AST determined Campylobacter antibiotic susceptibilities by the underside focus of antibiotics that will inhibit bacterial progress (i.e., no color change).
High coincidences (91-100%) of on-chip AST and customary agar dilution method had been achieved in opposition to a quantity of clinically important antibiotics. For a presumptive colony, on-chip identification and AST had been achieved in parallel inside 24 h, whereas commonplace methods take a quantity of days for a quantity of sequential steps along with biochemical assays and standard culture-based AST.
In conclusion, this lab-on-a-chip machine can get hold of a quick and reliable detection of antibiotic-resistant Campylobacter
Increasing points of antibiotic-resistant Campylobacter to public effectively being have emphasised the importance of setting pleasant and fast detection. This analysis described the effectively timed identification and antimicrobial susceptibility testing of Campylobacter via the use of a microfluidic machine. Our developed method not solely lowered complete analysis time, however moreover simplified meals sample preparation and chip operation for end prospects.
Due to the miniaturized dimension of lab-on-a-chip platform, the detection was achieved via the use of as a lot as 1000-time a lot much less reagents than commonplace reference methods, making it a aggressive technique for quick screening and surveillance analysis in meals industries. In addition, a quantity of clinically important Campylobacter species (C. jejuni, C. coli, C. lari) might presumably be examined by our machine. This machine has potential for big utility in meals safety administration and medical diagnostics significantly throughout the resource-limited areas.
Evaluation of EUCAST quick antimicrobial susceptibility testing (RAST) for optimistic blood cultures in medical comply with using an entire lab automation
Our purpose was to guage EUCAST’s ‘quick antimicrobial susceptibility testing’ (RAST) immediately from optimistic blood custom that delivers antimicrobial outcomes inside 6 h for Staphylococcus aureus, Enterococcus spp., Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, using complete lab automation. Zone diameters from RAST had been in distinction with MIC outcomes. Furthermore, its have an effect on on time to report was investigated.
RAST was carried out to all optimistic cardio and anaerobic blood custom bottles by subculturing them, i.e. onto Mueller-Hinton agar and together with six antibiotic discs defending Gram-negative and Gram-positive treatment (cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin). RAST was routinely imaged after 6 h. Zone sizes had been measured using a TLA software program program software program and interpreted in accordance with EUCAST medical breakpoints.
Bacteria had been acknowledged using MALDI-TOF MS and MIC outcomes had been determined using Vitek2 panels. Categorial settlement between agar diffusion and MIC outcomes was investigated. Additionally, time to RAST and time to Vitek had been in distinction for 100 isolates (20 per species). Between November 2018 and April 2019, 3313 optimistic mono-bacterial blood custom bottles had been collected of which 894 bottles with RAST-validated species had been investigated.
Among these bottles, 2029 specific particular person antibiotic measurements had been in distinction with MIC outcomes from Vitek2 and 14 very foremost, 28 foremost and 12 minor errors had been found. A median low cost of 17:30 h in time to report was observed. Introduction of RAST with automated TLA imaging function would possibly cut back time to report by 17:30 h. Excellent accordance between zone diameter and MIC outcomes, considerably for cefoxitin, vancomycin and meropenem, was observed, nonetheless drawbacks because of this of ATU had been seen.
Characterization of lactic acid micro organism isolated from a standard Ivoirian beer course of to develop starter cultures for protected sorghum-based drinks
The present analysis aimed to characterize lactic acid micro organism involved throughout the fully totally different processing steps of tchapalo, a standard Ivoirian beverage, for his or her potential utility as starter cultures in meals and drinks. Lactic acid micro organism (LAB) had been as a result of this reality isolated and enumerated at fully totally different steps of the strategy on MRS and BEA agars.
Of the 465 isolates, 27 produced bacteriocins that inhibit Lactobacillus delbrueckii F/31 stress. Of these, two moreover inhibited Listeria innocua ATCC 33090, whereas two others displayed inhibitory train in opposition to L.innocua ATCC 33090, E. faecalis CIP 105042, E. faecalis ATCC 29212, Streptococcus sp. medical LNSP, E. faecalis CIP 105042 and E. faecium ATCC 51558.
The dominant species involved in tchapalo LAB fermentation, as determined by 16S rRNA gene sequencing, had been Lactobacillus fermentum (64%), adopted by Pediococcus acidilactici (14%). Two strains representing the two dominant species, L. fermentum S6 and P. acidilactici S7, and two potential bacteriocin producers, Weissella confusa AB3E41 and Enterococcus faecium AT1E22, had been chosen for added characterization.
First, genome analysis confirmed that these strains do not present potential harmful genes resembling pathogenic parts or transmissible antibiotic resistance genes. Furthermore, phylogenetic analyses had been carried out to judge proof of eventual hyperlinks to groups of strains with express properties. They revealed that (i) L. fermentum S6 and P.
acidilactici S7 are rigorously related to strains that ferment crops, (ii) E. faecium AT1E22 belongs to the environmental clade B of E. faecium, whereas W. confusa is variety of very like totally different strains moreover isolated from plant fermentations. Further genome analysis confirmed that E. faecium AT1E22 incorporates the Enterocin P gene almost definitely carried by a megaplasmid, whereas no proof of a bacteriocin gene was current in W.
confusa AB3E41. The metabolic and the 1st step of the probiotic potentials of the fully totally different strains had been analyzed. Lactobacillus fermentum S6 and P. acidilactici S7 are good candidates to develop starter cultures, and E. faecium AT1E22 should be further examined to substantiate its potential as a probiotic stress throughout the manufacturing of sorghum wort.
Using a field to lab method, mature deep-rooting traits in wheat had been correlated to root phenotypes measured on young plants from controlled situations. Mature deep-rooting root traits of twenty wheat genotypes at maturity had been established by way of coring in three field trials throughout two years. Field traits had been correlated to phenotypes expressed by the 20 genotypes after progress in 4 commonly-used lab screens:
i. soil tubes for root emergence, elongation, size and branching at 4 ages to 34 days after sowing (DAS); ii. paper pouches 7 DAS and iii. agar chambers for major root (PR) quantity and angles at Eight DAS; and iv. soil baskets for PR and nodal root (NR) quantity and angle at 42 DAS.
Correlations between lab and field root traits (r2 = 0.45 – 0.73) had been extremely inconsistent, with many traits uncorrelated and nobody lab phenotype correlating equally throughout three field experiments. Phenotypes most positively related with deep field roots had been: longest PR and NR axiles from soil tube display at 20 DAS; and slim PR angle and extensive NR angle from soil baskets at 42 DAS. Paper and agar PR angles had been positively and considerably correlated to one another, however solely extensive outer PRs in paper display correlated positively to shallower field root traits.
NR phenotypes in soil baskets weren’t predicted by PR phenotypes in any display, suggesting impartial developmental controls and worth in measuring each root varieties in lab screens.Strong temporal and edaphic results on mature root traits, and an absence of understanding of root trait adjustments over plant improvement, are main challenges in creating controlled surroundings root screens for mature root traits in the field.
Genetic Diversity and Virulence Characteristics of Staphylococcus aureus Isolates from Cases of Bovine Mastitis
Staphylococcus aureus is one of the main bacterial mastitis pathogens with important results on animal and human well being. Some research confirmed that S. aureus strains that infect completely different host species are genetically distinct, though most strains can infect a variety of host species. However, there aren’t any clearly outlined clonal patterns of S. aureus strains which can be identified to contaminate a selected host.
The aims of this research had been to guage the clonal variety and virulence traits of S. aureus isolates from instances of bovine mastitis. Bacteriological exams had been carried out on milk samples from instances of bovine mastitis from 11 dairy farms together with some milk samples from unknow farms in Eastern Tennessee. Overall, a complete of 111 S.
aureus had been remoted and recognized, and additional evaluated for his or her genetic variety by pulsed-field gel electrophoresis (PFGE) and virulence traits by PCR. Genotypic virulence components together with staphylococcal enterotoxins, and poisonous shock syndrome toxin 1 (tsst-1) had been examined by PCR. In addition, the affiliation amongst a number of identified virulence components of these isolates primarily based on our present and former research in our lab had been evaluated.
Previously generated information that had been included in the evaluation of affiliation amongst virulence components had been the presence of biofilm manufacturing related genes in the ica operon resembling icaA, icaD and icaAB, and phenotypic virulence traits resembling hemolysis on blood agar, slime manufacturing and resistance or susceptibility to 10 generally used antimicrobials in dairy farms.
The PFGE outcomes confirmed the presence of 16 PFGE varieties (designated A – P) all through farms, of which three pulsotypes, I, M and O had been the most often remoted PFGE varieties from most farms. The PFGE sort M was the most prevalent of all 16 PFGE varieties, with 64 isolates being current amongst 9 farms.
The PCR outcomes of enterotoxin genes confirmed that out of the complete 111 examined 84 (75.7%) had been destructive whereas 13 (11.7%), 2 (1.8%), 3 (2.7%), 1 (0.9%) and eight (7.2%) had been optimistic for seb, seb and sec, sec, see, and tsst-1, respectively. All 111 isolates had been destructive for sea and sej. Results of the analysis of I, M and O strains adhesion to and invasion into mammary epithelial cells confirmed that the complete rely of every pressure of micro organism adhered to and invaded into mammary epithelial cell line (MAC-T cells) was not considerably completely different (P>0.05).
This could also be a sign that there is no such thing as a important distinction in their capability to ascertain early host-pathogen interplay and colonization of the host.outcomes. There had been no statistically important associations amongst PFGE varieties and different identified virulence components of these strains.
However, PFGE varieties O and M are likely to cluster with β-hemolysin, absence of enterotoxins and susceptibility to antimicrobials. In conclusion, we didn’t discover any affiliation of pulsotype with genotypic and phenotypic virulence components and S. aureus isolates from instances of mastitis included in this research had numerous genotypes that possessed variable virulence components.
Quinoa, a dietary grain, can be utilized as an ingredient in gluten-free sourdoughs. This research characterizes quinoa flour spontaneous fermentation with emphasis in the isolation of exopolysaccharide (EPS) producer micro organism. Real, pink and black grains had been studied.
Dough yield, microbiota composition and fermentation biochemistry had been decided for a complete of 36 quinoa flour fermentations. The fermentation biochemistry was monitored by high-performance liquid chromatography (HPLC) evaluation, pH measurement and titratable acidity. Changes in the microbiota had been monitored by plating on deMann Rogosa and Sharp 5 agar (MRS5) and yeast and mildew agar (YMA) plates and with metagenetic evaluation.
The capability to supply exopolysaccharides was screened in chosen lactic acid micro organism (LAB) isolates. Production of natural acids in the spontaneous fermentation dropped the pH to 4.0 ± 0.3. The group of presumptive LAB reached 8.37 ± 0.01 log colony forming models (CFU)/mL by day 8 of back-slopped fermentations.
The microbiota was composed of Lactobacillus, Enterococcus, Leuconostoc, Lactococcus, Pediococcus and Weissella. P. pentosaceous,L. citreum and W. cibaria had been capable of produce EPS in a starch-rich medium. P. pentosaceous confirmed larger exopolysaccharide yield, speedy acidifying kinetics and was capable of drop the dough broth pH to values under 4.Zero and a optimistic fermentation quotient after 24 h of incubation. Therefore, the bacterium could be a possible candidate for quinoa sourdough manufacturing.
Vaccine Plasmid Topology Monitoring by Capillary Gel Electrophoresis.
Plasmid DNA has been widely used in vaccination as well as in cell and gene therapy. It exists in multiple isoforms including supercoiled, nicked or open circular and linear forms. Regulatory agencies recommend having more than 80% of the supercoiled isoform for bulk release of plasmid products; thus it should be analyzed accordingly.The traditional analysis method for plasmid DNA is agarose gel electrophoresis. However, due to time-consuming manual sample loading, visualization, and data analysis, it has limitations in obtaining consistently quantitative results. In this short communication, we introduce a fast, sensitive, and robust plasmid analysis method using capillary electrophoresis with laserinduced fluorescence detection (CE-LIF). CE-LIF analysis of the supercoiled isoform and its open circular counterpart was completed in 20 minutes with excellent sensitivity by using a common fluorescent groove binding dye. The advantage of the method was demonstrated by the purity analysis of two large plasmids (7 kb and 10 kb). The fully automated sample loading, separation and data analysis featured enhanced assay repeatability and ease of quantitation over agarose gel electrophoresis.As a worked example, analysis of plasmid samples treated at elevated temperature during an accelerated stability test also demonstrated the applicability of CELIF to monitor plasmid degradation.
Preparation of Multiwall Carbon Nanotubes Embedded Electroconductive Multi-Microchannel Scaffolds for Neuron Growth under Electrical Stimulation.
To prepare the conductive MWCNT (multiwall carbon nanotube)-agarose scaffolds with multi-microchannel for neuron growth under electrical stimulation.The scaffolds were produced by gradient freeze and lyophilization methods. The synthesized materials were characterized by SEM and near-infrared spectroscopy, and their microstructure, swelling-deswelling, conductivity, biocompatibility, and shape memory behavior were measured. A three-dimensional culture model by implanting cells into scaffolds was built, and the behaviors of RSC96 cells on scaffolds under electrical stimulation were evaluated.The addition of MWCNT did not affect the pore composition ratio and shape memory of agarose scaffolds, but 0.025% wt MWCNT in scaffolds improved the swelling ratio and water retention at the swelling equilibrium state. Though MWCNTs in high concentration had slight effect on proliferation of RSC96 cells and PC12 cells, there was no difference that the expressions of neurofilament of RSC96 cells on scaffolds with MWCNTs of different concentration. RSC96 cells arranged better along the longitudinal axis of scaffolds and showed better adhesion on both 0.025% MWCNT-agarose scaffolds and 0.05% MWCNT-agarose scaffolds compared to other scaffolds.Agarose scaffolds with MWCNTs possessed promising applicable prospect in peripheral nerve defects.
Serum Protein Gel Agarose Electrophoresis in Captive Tigers.
Given the endangered status of tigers (Panthera tigris), the health of each individual is important and any data on blood chemistry values can provide valuable information alongside the assessment of physical condition. The nature of tigers in the wild makes it is extremely difficult to obtain biological samples from free-living subjects, therefore the values obtained from captive tigers provide very useful data. Serum protein electrophoresis is a useful tool in the diagnosis and monitoring of a number of diseases. In this study, we evaluated agarose gel serum protein electrophoresis on samples from 11 healthy captive tigers. Serum electrophoresis on all 11 tiger samples successfully separated proteins into albumin, α1, α2, β1, β2 and γ globulin fractions as in other mammals. Electrophoretic patterns were comparable in all tigers. Mean± standard deviation or median and range values obtained for each protein fraction in healthy tigers were, respectively: 3.6 ± 0.2, 0.21 (0.2-0.23), 1.2 ± 0.2, 10.7 ± 0.2, 0.4 (0.3-0.6), 1.2 (1-1.8) gr/dL. The results of this preliminary study provide the first data on serum electrophoretic patterns in tigers and may be a useful diagnostic tool in the health assessment of this endangered species.
Human Keratinocyte UVB-Protective Effects of a Low Molecular Weight Fucoidan from Sargassum horneri Purified by Step Gradient Ethanol Precipitation.
Ultraviolet B (UVB) radiation-induced oxidative skin cell damage is a major cause of photoaging. In the present study, a low molecular weight fucoidan fraction (SHC4) was obtained from Sargassum horneri by Celluclast-assisted extraction, followed by step gradient ethanol precipitation. The protective effect of SHC4 was investigated in human keratinocytes against UVB-induced oxidative stress. The purified fucoidan was characterized by Fourier-transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (NMR), agarose gel-based molecular weight analysis and monosaccharide composition analysis. SHC4 had a mean molecular weight of 60 kDa, with 37.43% fucose and 28.01 ± 0.50% sulfate content. The structure was mainly composed of α-L-Fucp-(1→4) linked fucose units. SHC4 treatment dose-dependently reduced intracellular reactive oxygen species (ROS) levels and increased the cell viability of UVB exposed HaCaT keratinocytes. Moreover, SHC4 dose-dependently inhibited UVB-induced apoptotic body formation, sub-G1 accumulation of cells and DNA damage. Inhibition of apoptosis was mediated via the mitochondria-mediated pathway, re-establishing the loss of mitochondrial membrane potential. The UVB protective effect of SHC4 was facilitated by enhancing intracellular antioxidant defense via nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling. Further studies may promote the use of SHC4 as an active ingredient in cosmetics and nutricosmetics.
IKZF1 Deletions as a Prognostic Factor in Costa Rican Patients With Pediatric B-Cell Acute Lymphoblastic Leukemia.
The IKZF1 gene encodes for Ikaros, a transcriptional factor in B-cell development. Deletions in this gene have been associated with a worse prognosis in B-cell acute lymphoblastic leukemia (B-ALL). We evaluated the presence of these alterations in all Costa Rican pediatric patients diagnosed with B-ALL between 2011 and 2014, treated with a modified Berlin-Frankfurt-Münster therapeutic protocol. Multiplex polymerase chain reaction with 2 detection methods (agarose gel and gene scanning) was used to detect intragenic deletions and multiplex ligation-dependent probe amplification for whole-gene deletions. Differences between groups (normal vs. deleted IKZF1) were analyzed by the χ test, the Kaplan-Meier test was used to calculate relapse-free survival and overall survival, and Cox regression was performed for multivariant analysis. Minimum follow-up was 4.5 years. Incidence of IKZF1 deletions was 12.9% (n=20), with an equal amount of intragenic and complete gene deletions. Adverse karyotype (P=0.048), high-risk category (P=0.030), occurrence of relapse (P=0.021), and medullar relapse (P=0.011) were statistically associated with the presence of deletions in IKZF1. Relapse-free survival at 54 months was lower in patients harboring an IKZF1 deletion than that in patients with IKZF1-wt (40.0% vs. 66.7%; P=0.014). Patients with B-ALL and IKZF1 deletions, showed a poorer relapse-free survival, in comparison with patients with IKZF1-wt, suggesting that IKZF1 status is an independent prognostic factor for pediatric patients with B-ALL.
An enzyme inhibition-based lab-in-a-syringe device for point-of-need determination of pesticides.
An enzyme inhibition-based lab-in-a-syringe (EI-LIS) device was developed by integrating a 1-naphthol-linked bi-enzymatic reaction (sensor core) into the LIS (sensor device) for point-of-need monitoring of pesticide residues. The integration relies on the rational design of two reaction pads. The conjugate pad is a polyester fiber membrane loaded with plant-esterase, an alternative to acetylcholinesterase. Besides pesticide capture, plant-esterase also mediates the hydrolysis of 1-naphthyl acetate, generating 1-naphthol. The detection pad is an agarose gel entrapping oxidized 3,3′,5,5′-tetramethylbenzidine (oxTMB) from Fe(iii) meso-tetra(N-methyl-4-pyridyl) porphyrin (FeTMPyP4)-catalyzed TMB oxidation. Both pads were embedded into their cartridges and then connected to a syringe. Under syringe pumping, 1-naphthol vertically flowed from the conjugate to the detection cartridge, linking the two pads. If plant-esterase was intact, 1-naphthol would reduce oxTMB, causing a color change of the detection pad from blue to colorless. If the plant-esterase activity was inhibited by pesticides, less 1-naphthol was produced, and the blue color of the detection pad would be partially or wholly retained. The deeper the blue color, the greater the pesticide concentration. This chromogenic pattern is responsible for a highly sensitive readout (detection limits of dichlorvos: 0.1 nM with the naked eye and 0.07 nM with a spectrometer).
CircSAMD4A regulates cell progression and epithelial‑mesenchymal transition by sponging miR‑342‑3p via the regulation of FZD7 expression in osteosarcoma.
Osteosarcoma (OS) is a primary malignant tumor with a complex etiology. Therefore, research into the pathogenesis of osteosarcoma is considered a priority. Circular RNAs play important roles in cell metabolism and in the immune response and are closely associated with cancer treatment. However, research into the association of circular RNAs with osteosarcoma is limited. In the present study, CircSAMD4A was validated by RT‑qPCR and agarose gel electrophoresis. CircSAMD4A and miR‑342‑3p expression was detected by RT‑qPCR. The relative protein expression levels were measured by western blot analysis. MTT assay and flow cytometry were used to detect cell cytotoxicity and apoptosis, respectively. Transwell assay was applied to assess cell migration and invasion. Dual‑luciferase reporter assay was used to determine the association among CircSAMD4A, Frizzled‑7 (FZD7) and miR‑342‑3p. In vivo, subcutaneous tumor formation assay was performed in an experiment with nude mice. The results revealed that the expression levels of CircSAMD4A and FZD7 were upregulated, while those of miR‑342‑3p were downregulated in OS tissues and cells. The inhibition of CircSAMD4A suppressed cell progression and epithelial‑mesenchymal transition (EMT), and promoted cell apoptosis in OS. The reduction of miR‑342‑3p reversed the effects of CircSAMD4A downregulation on cell cytotoxicity, migration, invasion, apoptosis and EMT in OS, while FZD7 overexpression blocked the effect of miR‑342‑3p upregulation on OS progression. The suppressive effect of sh‑CircSAMD4A on tumor growth was thus verified in OS. Overall, the present study demonstrated that CircSAMD4A affected cell cytotoxicity, invasion, apoptosis, migration and EMT by regulating the miR‑342‑3p/FDZ7 axis in OS, thereby providing a novel regulatory mechanism and a potential therapeutic target for OS.
Injectable Hydrogel for NIR-II Photo-Thermal Tumor Therapy and Dihydroartemisinin-Mediated Chemodynamic Therapy.
In traditional Chinese medicine, dihydroartemisinin (DHA) is the focus of extensive attention because of its unique activity with Fe2+ to produce reactive oxygen species (ROS) and promote apoptosis. In this work, we designed a newfangled ink@hydrogel containing FeCl3, traditional Chinese ink (Hu Kaiwen ink), and agarose hydrogel to create a synergistic activity with DHA in the treatment of cancer. When the system is irradiated under 1,064 nm for a few minutes, the ink in the ink@hydrogel converts the light to heat and hyperthermia causes the reversible hydrolysis of hydrogel. Then, Fe3+ quickly diffuses from the hydrogel to the tumor microenvironment and is reduced to Fe2+ to break the endoperoxide bridge in pre-injected DHA, which results in the release of free radicals for a potent anticancer action. To our knowledge, this is the first report of a hydrogel tumor therapy system that induces a photo-thermal response in the second near infrared window (NIR-II). in vivo experiments also showed a significant effect of DHA-Fe2+ in chemodynamic therapy (CDT) and in photo-thermal therapy. This hydrogel platform provided an encouraging idea for synergistic tumor therapy.
A dual-stimulation strategy in a micro-chip for the investigation of mechanical associative learning behavior of C. elegans.
During the past decades, few micro-devices for analysis of associative learning behavior have been reported. In this work, an agarose-PDMS hybridized micro-chip was developed to establish a new associative learning model between mechanosensation and food reward in C. elegans. The micro-chip consisted of column arrays which mimicked mechanical stimulation to C. elegans. After trained by pairing bacterial food and mechanical stimuli in the chip, the worms exhibited associative learning behavior and gathered in the regions where there was food during training. The key research findings include: (1) Associative learning behavior of C. elegans could be generated and quantitatively analyzed by this developed micro-chip. (2) Associative learning behavior could be enhanced by extending the training time and developmental stage. (3) Mechanosensation-related genes and neurotransmitters signals had effects on the learning behavior. (4) The associative learning ability could be strengthened by exogenous dopamine in both wild type and mutants. We validated that the design of the micro-chip was useful and convenient for the study of learning behavior based on mechanosensation.
High SOX8 expression promotes tumor growth and predicts poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma.
According to our previous study, GOLPH3 is markedly up-expressed in tongue squamous cell carcinoma (TSCC), which is also associated with poor survival. However, it remains unclear about key upstream and downstream mechanisms of GOLPH3. This study aimed to illuminate new mechanisms modulating GOLPH3 upregulation and promoting TSCC development at the molecular level. Using mass spectrometry and agarose-streptavidin-biotin pull-down analyses, SOX8 (SRY-Box 8) was identified to be the new protein to bind the GOLPH3 promoter.
Agarose LE to meet their electrophoretic needs for a variety of applications. Now the same agarose is available in a convenient, no mess tablet. Agarose LE is refined in an advanced process that excludes the use of organic solvents. The result is a cleaner end product with significantly reduced environmental impact. The agarose can be used for analyses of nucleic acids from 150bp to 6kbp, protein electrophoresis and various blotting protocols.
The low EEO of the agarose promotes increased electrophoretic mobility, yielding improved resolution and shorter run times. This also allows macromolecules and larger particles (subcellular fragments, viruses, etc.) to migrate more freely through the gel matrix. The consistently low EEO reduces band distortion (caused by counterflow) that can result from the presence of excessive sulfate-rich negative ions.
The quick dissolving EZ Pack tablet contains 0.5g (500mg) of Agarose LE, eliminating the hands-on time and inaccuracies normally associated with weighing. Use of the tablet is not only convenient and cleaner, it also provides better consistency and reproducbilty gel to gel. Gel preparation is simple – add the desired number of tablets to electrophoresis buffer, alow to sit for two minutes and then heat and pour as usual. The resulting gels are highly transparent, have exceptional thermal stability (ensuring safe and easy handling) and exhibit exceptionally low absorption of chemical staining agents.
Agarose Tablets are supplied in convenient blister packs for safe, clean dispensing.
Agarose LE is a highly purified agarose, suitable for a variety of molecular biology applications. It is refined using an advanced process that excludes the use of organic solvents, yielding a cleaner end-product with a significantly reduced environmental impact. Agarose LE can be used for analyses of proteins and nucleic acids of various sizes (150 bp to 25 kbp).
It’s low EEO (≤ 0.13-mr ) promotes increased electrophoretic mobility, yielding improved resolution and shorter run times. This also allows macromolecules and larger particles (subcellular fragments, viruses, etc.) to migrate more freely through the gel matrix. The consistently low EEO also provides a reduction in band distortion (caused by counterflow) that can result from the presence of excessive sulfate-rich negative ions. Agarose LE is widely used for nucleic acid electrophoresis (analytical and preparative), protein electrophoresis (including radial immunodiffusion) and various blotting protocols. It is easily soluble, free of nucleases, and easy to use. It is highly transparent (forms a clear, colorless solution at 1g:100ml H2O), and exhibits exceptionally low absorption of chemical staining agents. Pore size can be adjusted by simple modifications to the concentration ratio. Formulated for high gel strength and integrity, Agarose LE exhibits exceptional thermal stability and mechanical resistance, ensuring safe, easy handling, regardless of whether a denaturing agent has been added. Benchmark Scientific A1705
The beauty of Molecular Biology lies in capturing a number of key processes which are truly exciting – like the DNA replication itself, studied are the mechanism of transcription into RNA and the following protein actualization which happens through translation.
All these interesting processes are subject to a very delicate regulation which takes place with the purpose of proceeding with their cellular functions accurately. In this category you can find a number of specialised CRISPR products, Nucleic Acid Kits, of course, and related reagents.
Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3- to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches. …
RNA-Seq-enabled proteomics of a weakly electric fish
The brown ghost knifefish (Apteronotus leptorhynchus) is a weakly electric teleost fish of particular interest as a versatile model system for a variety of research areas in neuroscience and biology. The comprehensive information available on the neurophysiology and neuroanatomy of this organism has enabled significant advances in such areas as the study of the neural network.
Fine structure HDX-MS software
Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is confounded by deuterium exchange-associated peaks overlapping with peaks of heavy, natural abundance isotopes, such as carbon-13. Recent studies demonstrated that high-performance mass spectrometers could resolve isotopic fine structure and eliminate this peak overlap.
Bottom-up MS studies typically employ a reduction and alkylation step that eliminates a class of PTM, S-thiolation. Given that molecular oxygen can mediate S-thiolation from reduced thiols, which are abundant in the reducing intracellular milieu, we investigated the possibility that some S-thiolation modifications are artifacts of protein preparation. Cu/Zn-superoxide dismutase (SOD1), Gentaur Bvba, Kampenhout.
BACKGROUND: Neuropeptides are a diverse category of signaling molecules in the nervous system regulating a variety of processes including food intake, social behavior, circadian rhythms, learning, and memory. Both the identification and functional characterization of specific neuropeptides are ongoing fields of research. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of nervous tissues.
Isotopic Fine Structure HDX MS
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is used for analyzing protein dynamics, protein folding/unfolding, and molecular interactions. Until this study, HDX MS experiments employed mass spectral resolving powers that afforded only one peak per nominal mass in a given peptide’s isotope distribution, and HDX MS data analysis methods were developed accordingly.
Structural impact of Cys mod of SOD1
The metalloenzyme Cu/Zn-superoxide dismutase (SOD1) catalyzes the reduction of superoxide anions into molecular oxygen and hydrogen peroxide. Hydrogen peroxide can oxidize SOD1, resulting in aberrant protein conformational changes, disruption of SOD1 function, and DNA damage. Cells may have evolved mechanisms of regulation that prevent such oxidation. We observed that cysteinylation of cysteine 111 (Cys111).
Cysteine modification blocks oxidative damage
Reactive oxygen species (ROS) are cytotoxic. To remove ROS, cells have developed ROS-specific defense mechanisms, including the enzyme Cu/Zn superoxide dismutase (SOD1), which catalyzes the disproportionation of superoxide anions into molecular oxygen and hydrogen peroxide. Although hydrogen peroxide is less reactive than superoxide, it is still capable of oxidizing, unfolding, and inactivating SOD1.
LC-MS performance evaluation
Selecting a suitable nano-liquid chromatography system (LC), ionization source and mass spectrometer for LC-tandem mass spectrometry (MS-MS) studies is complicated by numerous competing technologies. This study compares four popular nano-LC systems, four ionization sources and three MS facilities that use completely different LC-MS-MS systems. Statistically significant differences in LC performance.
X-linking MS analysis
The small quantities of protein required for mass spectrometry (MS) make it a powerful tool to detect binding (protein-protein, protein-small molecule, etc.) of proteins that are difficult to express in large quantities, as is the case for many intrinsically disordered proteins. Chemical cross-linking, proteolysis, and MS analysis, combined, are a powerful tool for the identification.
Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3- to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches.
RNA-Seq-enabled proteomics of a weakly electric fish
The brown ghost knifefish (Apteronotus leptorhynchus) is a weakly electric teleost fish of particular interest as a versatile model system for a variety of research areas in neuroscience and biology. The comprehensive information available on the neurophysiology and neuroanatomy of this organism has enabled significant advances.
RNA-Seq-enabled proteomics of a weakly electric fishFine structure HDX-MS softwareAnaerobic top-downDifferential neuropeptidomicsIsotopic Fine Structure HDX MSStructural impact of Cys mod of SOD1Cysteine modification blocks oxidative damageLC-MS performance evaluationX-linking MS analysisStabilizing SOD1 Mar 20
The brown ghost knifefish (Apteronotus leptorhynchus) is a weakly electric teleost fish of particular interest as a versatile model system for a variety of research areas in neuroscience and biology. The comprehensive information available on the neurophysiology and neuroanatomy of this organism has enabled significant advances in such areas as the study of the neural basis of behavior, the development of adult-born neurons in the central nervous system and their involvement in the regeneration of nervous tissue, as well as brain aging and senescence. Despite substantial scientific interest in this species, no genomic resources are currently available. Here, we report de novo assembly and annotation of the A. leptorhynchus transcriptome. After evaluating several trimming and transcript reconstruction strategies, de novo assembly using Trinity uncovered 42,459 unique contigs containing at least a partial protein-coding sequence based on alignment to a reference set of known Actinopterygii sequences. As many as 11,847 of these contigs contained full or near-full length protein sequences, providing broad coverage of the proteome. A variety of non-coding RNA sequences were also identified and annotated, including conserved long intergenic non-coding RNA and other long non-coding RNA observed previously to be expressed in adult zebrafish (Danio rerio) brain, as well as a variety of miRNA, snRNA, and snoRNA. Shotgun proteomics confirmed translation of open reading frames from over 2,000 transcripts, including alternative splice variants. Assignment of tandem mass spectra was greatly improved by use of the assembly compared to databases of sequences from closely related organisms. The assembly and raw reads have been deposited at DDBJ/EMBL/GenBank under the accession number GBKR00000000. Tandem mass spectrometry data is available via ProteomeXchange with identifier PXD001285. Presented here is the first release of an annotated de novo transcriptome assembly from Apteronotus leptorhynchus, providing a broad overview of RNA expressed in central nervous system tissue. The assembly, which includes substantial coverage of a wide variety of both protein coding and non-coding transcripts, will allow the development of better tools to understand the mechanisms underlying unique characteristics of the knifefish model system, such as their tremendous regenerative capacity and negligible brain senescence.
Background: Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is confounded by deuterium exchange-associated peaks overlapping with peaks of heavy, natural abundance isotopes, such as carbon-13. Recent studies demonstrated that high-performance mass spectrometers could resolve isotopic fine structure and eliminate this peak overlap, allowing direct detection and quantification of deuterium incorporation. Results: Here, we present a graphical tool that allows for a rapid and automated estimation of deuterium incorporation from a spectrum with isotopic fine structure. Given a peptide sequence (or elemental formula) and charge state, the mass-to-charge ratios of deuterium-associated peaks of the specified ion is determined. Intensities of peaks in an experimental mass spectrum within bins corresponding to these values are used to determine the distribution of deuterium incorporated. A theoretical spectrum can then be calculated based on the estimated distribution of deuterium exchange to confirm interpretation of the spectrum. Deuterium incorporation can also be detected for ion signals without a priori specification of an elemental formula, permitting detection of exchange in complex samples of unidentified material such as natural organic matter. A tool is also incorporated into QUDeX-MS to help in assigning ion signals from peptides arising from enzymatic digestion of proteins. MATLAB-deployable and standalone versions are available for academic use at qudex-ms.sourceforge.net and agarlabs.com. Conclusion: Isotopic fine structure HDX-MS offers the potential to increase sequence coverage of proteins being analyzed through mass accuracy and deconvolution of overlapping ion signals. As previously demonstrated, however, the data analysis workflow for HDX-MS data with resolved isotopic fine structure is distinct. QUDeX-MS we hope will aid in the adoption of isotopic fine structure HDX-MS by providing an intuitive workflow and interface for data analysis. Click here for full text. This post has no tag
Bottom-up MS studies typically employ a reduction and alkylation step that eliminates a class of PTM, S-thiolation. Given that molecular oxygen can mediate S-thiolation from reduced thiols, which are abundant in the reducing intracellular milieu, we investigated the possibility that some S-thiolation modifications are artifacts of protein preparation. Cu/Zn-superoxide dismutase (SOD1) was chosen for this case study as it has a reactive surface cysteine residue, which is readily cysteinylated in vitro. The ability of oxygen to generate S-thiolation artifacts was tested by comparing purification of SOD1 from postmortem human cerebral cortex under aerobic and anaerobic conditions. S-thiolation was ∼50% higher in aerobically processed preparations, consistent with oxygen-dependent artifactual S-thiolation. The ability of endogenous small molecule disulfides (e.g. cystine) to participate in artifactual S-thiolation was tested by blocking reactive protein cysteine residues during anaerobic homogenization. A 50-fold reduction in S-thiolation occurred indicating that the majority of S-thiolation observed aerobically was artifact. Tissue-specific artifacts were explored by comparing brain- and blood-derived protein, with remarkably more artifacts observed in brain-derived SOD1. Given the potential for such artifacts, rules of thumb for sample preparation are provided. This study demonstrates that without taking extraordinary precaution, artifactual S-thiolation of highly reactive, surface-exposed, cysteine residues can result.
Read the full paper: Artifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation Tags: amyotrophic lateral sclerosis, anaerobic, cysteinylation, Fourier transform mass spectrometry, oxidative damage, PTMs, SOD1, top-down proteomics
Institute Core Mass Spectrometry Facility
Tags: electrospray ionization, Fourier transform mass spectrometry, isotopic fine structure, LC-MS, LC-MS-MS, nanoESI-LC-MS, nanoLC, performance, QqTOF
BACKGROUND: Neuropeptides are a diverse category of signaling molecules in the nervous system regulating a variety of processes including food intake, social behavior, circadian rhythms, learning, and memory. Both the identification and functional characterization of specific neuropeptides are ongoing fields of research. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of nervous tissues from a variety of organisms allows direct detection and identification of neuropeptides. Here, we demonstrate an analysis workflow that allows for the detection of differences in specific neuropeptides amongst a variety of neuropeptides being simultaneously measured. For sample preparation, we describe a straight-forward and rapid (minutes) method where individual adult Drosophila melanogaster brains are analyzed. Using a MATLAB-based data analysis workflow, also compatible with MALDI-TOF mass spectra obtained from other sample preparations and instrumentation, we demonstrate how changes in neuropeptides levels can be detected with this method.
RESULTS: Over fifty isotopically resolved ion signals in the peptide mass range are reproducibly observed across experiments. MALDI-TOF MS profile spectra were used to statistically identify distinct relative differences in organ-wide endogenous levels of detected neuropeptides between biological conditions. In particular, three distinct levels of a particular neuropeptide, pigment dispersing factor, were detected by comparing groups of preprocessed spectra obtained from individual brains across three different D. melanogaster strains, each of which express different amounts of this neuropeptide. Using the same sample preparation, MALDI-TOF/TOF tandem mass spectrometry confirmed that at least 14 ion signals observed across experiments are indeed neuropeptides. Among the identified neuropeptides were three products of the neuropeptide-like precursor 1 gene previously not identified in the literature.
CONCLUSIONS: Using MALDI-TOF MS and preprocessing/statistical analysis, changes in relative levels of a particular neuropeptide in D. melanogaster tissue can be statistically detected amongst a variety of neuropeptides. While the data analysis methods should be compatible with other sample preparations, the presented sample preparation method was sufficient to identify previously unconfirmed D. melanogaster neuropeptides.
Read the full paper: A rapid MALDI-TOF mass spectrometry workflow for Drosophila melanogaster differential neuropeptidomics Tags: drosophila melanogaster, isotopic resolution, MALDI-TOF, mass spectrometry, neuropeptidomics, neuroscience, NPLP1
Isotopic Fine Structure HDX MS
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is used for analyzing protein dynamics, protein folding/unfolding, and molecular interactions. Until this study, HDX MS experiments employed mass spectral resolving powers that afforded only one peak per nominal mass in a given peptide’s isotope distribution, and HDX MS data analysis methods were developed accordingly. A level of complexity that is inherent to HDX MS remained unaddressed, namely, various combinations of natural abundance heavy isotopes and exchanged deuterium shared the same nominal mass and overlapped at previous resolving powers. For example, an A + 2 peak is comprised of (among other isotopomers) a two-2H-exchanged/zero-13C isotopomer, a one-2H-exchanged/one-13C isotopomer, and a zero-2H-exchanged/two-13C isotopomer. Notably, such isotopomers differ slightly in mass as a result of the ~3 mDa mass defect between 2H and 13C atoms. Previous HDX MS methods did not resolve these isotopomers, requiring a natural-abundance-only (before HDX or “time zero”) spectrum and data processing to remove its contribution. It is demonstrated here that high-resolution mass spectrometry can be used to detect isotopic fine structure, such as in the A + 2 profile example above, deconvolving the isotopomer species resulting from deuterium incorporation. Resolving isotopic fine structure during HDX MS therefore permits direct monitoring of HDX, which can be calculated as the sum of the fractional peak magnitudes of the deuterium-exchanged isotopomers. This obviates both the need for a time zero spectrum as well as data processing to account for natural abundance heavy isotopes, saving instrument and analysis time.
Read the full paper: Resolving Isotopic Fine Structure to Detect and Quantify Natural Abundance- and Hydrogen/Deuterium Exchange-Derived Isotopomers
Tags: Fourier transform mass spectrometry, hydrogen-deuterium exchange, isotopic fine structure, isotopic resolution
Structural impact of Cys mod of SOD1
The metalloenzyme Cu/Zn-superoxide dismutase (SOD1) catalyzes the reduction of superoxide anions into molecular oxygen and hydrogen peroxide. Hydrogen peroxide can oxidize SOD1, resulting in aberrant protein conformational changes, disruption of SOD1 function, and DNA damage. Cells may have evolved mechanisms of regulation that prevent such oxidation. We observed that cysteinylation of cysteine 111 (Cys111) of SOD1 prevents oxidation by peroxide (DOI 10.1021/bi4006122 ). In this article, we characterize cysteinylated SOD1 using differential scanning fluorometry and X-ray crystallography. The stoichiometry of binding was one cysteine per SOD1 dimer, and there does not appear to be free volume for a second cysteine without disrupting the dimer interface. Much of the three-dimensional structure of SOD1 is unaffected by cysteinylation. However, local conformational changes are observed in the cysteinylated monomer that include changes in conformation of the electrostatic loop (loop VII; residues 133-144) and the dimer interface (loop VI; residues 102-115). In addition, our data shows how cysteinylation precludes oxidation of cysteine 111 and suggests possible cross-talk between the dimer interface and the electrostatic loop.
Read the full paper: Structural Consequences of Cysteinylation of Cu/Zn-Superoxide Dismutase
Tags: crystallography, cysteinylation, dimer stability, protein structure, PTMs, SOD1
Cysteine modification blocks oxidative damage
Reactive oxygen species (ROS) are cytotoxic. To remove ROS, cells have developed ROS-specific defense mechanisms, including the enzyme Cu/Zn superoxide dismutase (SOD1), which catalyzes the disproportionation of superoxide anions into molecular oxygen and hydrogen peroxide. Although hydrogen peroxide is less reactive than superoxide, it is still capable of oxidizing, unfolding, and inactivating SOD1, at least in vitro. To explore the relevance of post-translational modification (PTM) of SOD1, including peroxide-related modifications, SOD1 was purified from postmortem human nervous tissue. As much as half of all purified SOD1 protein contained non-native post-translational modifications (PTMs), the most prevalent modifications being cysteinylation and peroxide-related oxidations. Many PTMs targeted a single reactive SOD1 cysteine, Cys111. An intriguing observation was that unlike native SOD1, cysteinylated SOD1 was not oxidized. To further characterize how cysteinylation may protect SOD1 from oxidation, cysteine-modified SOD1 was prepared in vitro and exposed to peroxide. Cysteinylation conferred nearly complete protection from peroxide-induced oxidation of SOD1. Moreover, SOD1 that has been cysteinylated and peroxide oxidized in vitro comprised a set of PTMs that bear a striking resemblance to the myriad of PTMs observed in SOD1 purified from human tissue.
Read the full paper: Post-Translational Modification by Cysteine Protects Cu/Zn-Superoxide Dismutase from Oxidative Damage
Tags: amyotrophic lateral sclerosis, cysteinylation, Fourier transform mass spectrometry, mass spectrometry, oxidative damage, protein stability, PTMs
LC-MS performance evaluation
Selecting a suitable nano-liquid chromatography system (LC), ionization source and mass spectrometer for LC-tandem mass spectrometry (MS-MS) studies is complicated by numerous competing technologies. This study compares four popular nano-LC systems, four ionization sources and three MS facilities that use completely different LC-MS-MS systems. Statistically significant differences in LC performance were identified with similarly performing Proxeon, Waters and Eksigent nanoLC-Ultra systems [retention time routinely at 0.7-0.9% relative standard deviation (RSD)], and all outperformed the Eksigent nanoLC-2D (RSD ∼2%). In addition, compatibility issues were identified between the Bruker HCT ion trap mass spectrometer and both the Eksigent nanoLC-2D and the Bruker nanoelectrospray source. The electrospray source itself had an unexpected and striking effect on chromatographic reproducibility on the Bruker HCT ion trap. The New Objective nanospray source significantly outperformed the Bruker nanospray source in retention time RSD (1% RSD versus 14% RSD, respectively); and the Bruker nebulized nanospray source outperformed both of these traditional, non-nebulized sources (0.5% RSD in retention time). Finally, to provide useful benchmarks for overall proteomics sensitivity, different LC-MS-MS platforms were compared by analyzing a range of concentrations of tryptic digests of bovine serum albumin at three MS facilities. The results indicate that similar sensitivity can be realized with a Bruker HCT-Ultra ion trap, a Thermo LTQ-Velos Linear ion trap and a Thermo LTQ-Orbitrap XL-ETD.
Read the full paper: Performance Comparisons of Nano-LC Systems, Electrospray Sources and LC–MS-MS Platforms
Tags: electrospray ionization, LC-MS, LC-MS-MS, mass spectrometry, nanoESI-LC-MS, nanoLC, performance
X-linking MS analysis
The small quantities of protein required for mass spectrometry (MS) make it a powerful tool to detect binding (protein-protein, protein-small molecule, etc.) of proteins that are difficult to express in large quantities, as is the case for many intrinsically disordered proteins. Chemical cross-linking, proteolysis, and MS analysis, combined, are a powerful tool for the identification of binding domains. Here, we present a traditional approach to determine protein-protein interaction binding sites using heavy water ((18)O) as a label. This technique is relatively inexpensive and can be performed on any mass spectrometer without specialized software.