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Journal of Proteomics Oct 2023Here we introduce hyperthermoacidic archaeal proteases (HTA-Proteases©) isolated from organisms that thrive in nearly boiling acidic volcanic springs and investigate...
Here we introduce hyperthermoacidic archaeal proteases (HTA-Proteases©) isolated from organisms that thrive in nearly boiling acidic volcanic springs and investigate their use for bottom-up proteomic experiments. We find that HTA-Proteases have novel cleavage specificities, show no autolysis, function in dilute formic acid, and store at ambient temperature for years. HTA-Proteases function optimally at 70-90 °C and pH of 2-4 with rapid digestion kinetics. The extreme HTA-Protease reaction conditions actively denature sample proteins, obviate the use of chaotropes, are largely independent of reduction and alkylation, and allow for a one-step/five-minute sample preparation protocol without sample manipulation, dilution, or additional cleanup. We find that brief one-step HTA-Protease protocols significantly increase proteome and protein sequence coverage with datasets orthogonal to trypsin. Importantly, HTA-Protease digests markedly increase coverage and identifications for ribonucleoproteins, histones, and mitochondrial membrane proteins as compared to tryptic digests alone. In addition to increased coverage in these classes, HTA-Proteases and simplified one-step protocols are expected to reduce technical variability and advance the fields of clinical and high-throughput proteomics. This work reveals significant utility of heretofore unavailable HTA-Proteases for proteomic workflows. We discuss some of the potential for these remarkable enzymes to empower new proteomics methods, approaches, and biological insights. SIGNIFICANCE: Here we introduce new capabilities for bottom-up proteomics applications with hyperthermoacidic archaeal proteases (HTA-Proteases©). HTA-Proteases have novel cleavage specificity, require no chaotropes, and allow simple one-step/five-minute sample preparations that promise to reduce variability between samples and laboratories. HTA-Proteases generate unique sets of observable peptides that are non-overlapping with tryptic peptides and significantly increase sequence coverage and available peptide targets relative to trypsin alone. HTA-Proteases show some bias for the detection and coverage of nucleic acid-binding proteins and membrane proteins relative to trypsin. These new ultra-stable enzymes function optimally in nearly boiling acidic conditions, show no autolysis, and do not require aliquoting as they are stable for years at ambient temperatures. Used independently or in conjunction with tryptic digests, HTA-Proteases offer increased proteome coverage, unique peptide targets, and brief one-step protocols amenable to automation, rapid turnaround, and high-throughput approaches.
Topics: Peptide Hydrolases; Trypsin; Proteome; Proteomics; Workflow; Peptides; Membrane Proteins
PubMed: 37634627
DOI: 10.1016/j.jprot.2023.104992 -
Journal of Dairy Science Jul 2023The objectives of this study were to evaluate the abundance and viability of leukocytes, the abundance of microRNA, and the activity of the complement pathway in (1)...
The objectives of this study were to evaluate the abundance and viability of leukocytes, the abundance of microRNA, and the activity of the complement pathway in (1) colostrum following heat-treatment or freezing, and (2) colostrum, transition milk, and mature milk. In experiment 1, composite colostrum samples were harvested from individual cows (n = 14) on a commercial dairy farm in NY and split into 3 aliquots using single-use colostrum bags. One aliquot was immediately cooled on ice following harvest (RAW) and stored at 4°C overnight, one was heat-treated for 60 min at 60°C (HT) before being cooled on ice and stored at 4°C overnight, and one was frozen at -20°C overnight (FR). The following morning, all samples were warmed to 40°C before further processing. In experiment 2, cows were sampled in a longitudinal study where composite samples were collected from colostrum (first milking, n = 23), transition milk (3 to 4 d postpartum, n = 13), and mature milk (6 to 7 d postpartum, n = 13). In both experiments colostrum was harvested from the first milking within 8 h of calving and samples were processed within 14 h of collection. Colostral leukocytes were isolated before viability was determined by trypan blue exclusion and manual differential cell counts were performed. Extracellular vesicles were isolated from whey by ultracentrifugation to isolate and quantify microRNA. Activity of the alternative complement pathway was determined in casein-depleted whey by semi-solid phase hemolysis assay. Somatic cell counts were determined for all raw samples. Macrophages and neutrophils made up the greatest proportion of leukocytes in colostrum followed by lymphocytes. Lymphocyte proportion increased as colostrum transitioned to mature milk, but overall somatic cell numbers declined concurrently. Viable cells were not isolated from HT or FR samples. Abundance of microRNA isolated from transition and mature milk was decreased compared with colostrum, did not differ between HT and RAW, but was increased in FR compared with RAW. Alternative complement pathway activity was decreased in HT, but not FR compared with RAW, and was not measurable in transition or mature milk. Postharvest heat-treatment and freezing of colostrum eliminated viable colostral leukocytes and affected microRNA abundance and complement activity. Leukocyte proportions, microRNA abundance, and complement activity changed as colostrum transitioned to mature milk. Although there were clear changes in the colostral components under study in relation to treatment and transition to mature milk, the biological significance of the described treatment effects and temporal changes were not investigated here.
Topics: Pregnancy; Female; Cattle; Animals; Milk; Colostrum; Hot Temperature; MicroRNAs; Freezing; Ice; Longitudinal Studies; Leukocytes; Lactation
PubMed: 37164855
DOI: 10.3168/jds.2022-22876 -
BMJ Open Sep 2020The ChicagO Multiethnic Prevention and Surveillance Study or 'COMPASS' is a population-based cohort study with a goal to examine the risk and determinants of cancer and...
PURPOSE
The ChicagO Multiethnic Prevention and Surveillance Study or 'COMPASS' is a population-based cohort study with a goal to examine the risk and determinants of cancer and chronic disease. COMPASS aims to address factors causing and/or exacerbating health disparities using a precision health approach by recruiting diverse participants in Chicago, with an emphasis on those historically underrepresented in biomedical research.
PARTICIPANTS
Nearly 8000 participants have been recruited from 72 of the 77 Chicago community areas. Enrolment entails the completion of a 1-hour long survey, consenting for past and future medical records from all sources, the collection of clinical and physical measurement data and the on-site collection of biological samples including blood, urine and saliva. Indoor air monitoring data and stool samples are being collected from a subset of participants. On collection, all biological samples are processed and aliquoted within 24 hours before long-term storage and subsequent analysis.
FINDINGS TO DATE
The cohort reported an average age of 53.7 years, while 80.5% identified as African-American, 5.7% as Hispanic and 47.8% as men. Over 50% reported earning less than US$15 000 yearly, 35% were obese and 47.8% were current smokers. Moreover, 38% self-reported having had a diagnosis of hypertension, while 66.4% were measured as hypertensive at enrolment.
FUTURE PLANS
We plan to expand recruitment up to 100 000 participants from the Chicago metropolitan area in the next decade using a hybrid community and clinic-based recruitment framework that incorporates data collection through mobile medical units. Follow-up data collection from current cohort members will include serial samples, as well as longitudinal health, lifestyle and behavioural assessment. We will supplement self-reported data with electronic medical records, expand the collection of biometrics and biosamples to facilitate increasing digital epidemiological study designs and link to state and/or national level databases to ascertain outcomes. The results and findings will inform potential opportunities for precision disease prevention and mitigation in Chicago and other urban areas with a diverse population.
REGISTRATION
NA.
Topics: Black or African American; Chicago; Chronic Disease; Cohort Studies; Hispanic or Latino; Humans; Male; Middle Aged
PubMed: 32938600
DOI: 10.1136/bmjopen-2020-038481 -
Prostaglandins & Other Lipid Mediators Apr 2021Two experiments were conducted to determine whether mifepristone (RU486) and PGFα activate the phosphatidylinositol hydrolysis pathway during the midluteal phase of the...
Two experiments were conducted to determine whether mifepristone (RU486) and PGFα activate the phosphatidylinositol hydrolysis pathway during the midluteal phase of the ovine estrous cycle. In experiment 1, ewes on day 8 of the cycle were given 10 μg RU486 or vehicle into the ovarian artery with removal of the corpus luteum (CL) after 10 min. Blood collected prior to and after treatment was analyzed for progesterone. Aliquots of CL were incubated with 10 μCi of H-inositol and in the presence and absence of PGFα (10 nM) for 15 min. Exposure of CL to RU486 and PGFα increased phosphatidylinositol hydrolysis (p < 0.05). Serum progesterone was reduced in both control and RU486-treated ewes (p < 0.05) compared to concentrations before treatments. In experiment 2, aliquots of CL collected from ewes on day 8 of the cycle were incubated with H-inositol and exposed to RU486 (2 μM) in the presence and absence of PGFα (1 μM) for 15 min. Treatments stimulated phosphatidylinositol hydrolysis as in Exp 1 (p < 0.05). Progesterone concentrations in incubation medium were increased in response to RU486 and PGFα (p < 0.05). Collectively, these data suggest that RU486 and PGFα act to stimulate phosphatidylinositol hydrolysis in the mature ovine CL.
Topics: Animals; Corpus Luteum; Female; Hydrolysis; Mifepristone; Phosphatidylinositols; Progesterone; Sheep
PubMed: 33545368
DOI: 10.1016/j.prostaglandins.2021.106538 -
Applied Radiation and Isotopes :... Mar 2022Thermoluminescence (TL) dating is one of the most significant chronological tools used in Quaternary research. However, for changes in the characteristics of quartz, the... (Review)
Review
Thermoluminescence (TL) dating is one of the most significant chronological tools used in Quaternary research. However, for changes in the characteristics of quartz, the larger deviation is still a problem in TL dating, especially with the single-aliquot regeneration-does (SAR) procedure. In the SAR-TL protocol, changes in the characteristics of quartz inevitably cause a shift in the TL peak position and a reduction in the sensitivity of the TL peak during repetitive thermal treatment. In this paper, we studied the optimal TL parameters to minimize the effect of the above problems for TL dating. Based on the optimization experiment combining OSL and TL measurements, the optimal preheat temperature was found to be 300 °C for both silt-sized grains and sand-sized grains, which eliminates the remainder of the 325 °C TL signals and inhibits the 375 °C TL peak position shift. Referring to the test does in SAR-OSL dating protocol, the optimal test doses, 200 Gy and 250 Gy for the silt-sized grains and sand-sized grains respectively, were determined to correct the reduction in TL sensitivity, and they were added to improve the SAR-TL protocol. The improved SAR-TL protocol with the optimal measurement parameters, which we called the accurate-parametric SAR-TL protocol, improves the accuracy of quartz TL dating and expands the range of accurate TL dating. For the experimental doses of 400 Gy and 700 Gy, the relative error of D obtained by the accurate-parametric SAR-TL protocol was less than ±5.5% for both silt-sized grains and sand-sized grains. In addition, we discussed the application conditions of the accurate-parametric SAR-TL protocol and the method that obtains the same level of thermal lag for different luminescence measurement equipment.
PubMed: 34968882
DOI: 10.1016/j.apradiso.2021.110072 -
Journal of Oral and Maxillofacial... 2022The micro-flora of oral cavity is a myriad of micro-organism. Any infection of oral cavity leads to diseased condition which is a transitional transformation of the...
Detection and comparison of prevalence of through culture and Real Time-polymerase chain reaction in subgingival plaque samples of chronic periodontitis and healthy individuals.
INTRODUCTION
The micro-flora of oral cavity is a myriad of micro-organism. Any infection of oral cavity leads to diseased condition which is a transitional transformation of the micro-organism in a specific paradigm depending upon the diseased condition. Periodontitis is one of the predominant chronic diseases which is a multifactorial infection. is a key etiological agent in causing periodontitis. To study the predominance of these bacteria in the diseased condition is important to detect, quantify and to find its efficacy by comparing different methods for identification.
AIM AND OBJECTIVES
The aim of the study is to determine the prevalence of by anerobic culture and by real-time polymerase chain reaction (PCR) from subgingival plaque samples of chronic periodontitis and healthy individual and to compare efficacy of two methods.
MATERIALS AND METHODS
A total of 400 subjects were considered, and subgingival plaque was collected using paper points. Individual were equally divided into two groups: chronic periodontitis (200) and healthy individuals (200). Each plaque sample collected was divided into two aliquots of which the first aliquot was subjected for anerobic culture to isolate . Phenotypical identification was done morphologically and biochemically further quantification of was done by colony-forming unit. The second aliquot was subjected for DNA extraction and real-time PCR was conducted to detect and quantify using specific primer.
RESULTS
Out of 400 samples, 73% showed detection of by culture method and through reverse transcription-PCR (RT-PCR), the detection was 75%. Individual detection of by culture in chronic periodontitis was 89.5% and 54.4% in healthy individuals, while detection by RT-PCR was found to be 91.5% in chronic periodontitis and 58% in healthy individuals. However, comparison between two techniques in detection of was statistically insignificant.
CONCLUSION
When we compared RT-PCR with culture RT-PCR showed higher positivity. RT-PCR is more sensitive and requires less time to detect. However, in the present study, culture also showed good positivity, suggesting proper dilution and with extended incubation, the specificity of culture can be improved to a great extent.
PubMed: 35968159
DOI: 10.4103/jomfp.jomfp_163_21 -
Transfusion Aug 2022Preparing small-dose red cell concentrates (RCCs) is a common practice for pediatric and neonatal transfusions. However, there is a lack of quality monitoring data to...
BACKGROUND
Preparing small-dose red cell concentrates (RCCs) is a common practice for pediatric and neonatal transfusions. However, there is a lack of quality monitoring data to indicate that both the preparation and storage of small-dose RCCs does not alter in vitro red cell quality. The present study seeks to provide data to support this practice.
MATERIALS AND METHODS
To evaluate quality of stored small aliquots, six ABO/Rh matched leukoreduced citrate phosphate-dextrose/saline-adenine-glucose-mannitol (LR CPD/SAGM) RCCs were pooled and split into 30 ml aliquots, 80 ml aliquots, and a standard 290 ml unit, with testing performed for up to 43 days post-collection. To evaluate the impact of irradiation on small-dose RCC preparation, a total of 48 independent LR CPD/SAGM RCCs were used (non-irradiated: n = 24; irradiated: n = 24). Aliquoting with/without irradiation was performed within 7 days of collection and baseline testing was performed within 24 h of aliquot production.
RESULTS
Limited variability in hemolysis, mean cell volume, and extracellular potassium concentrations were seen between the different aliquot sizes throughout the 43-day storage period. Aliquot production did not accentuate damage based on any of these tested parameters in both the non-irradiated and irradiated subsets. A significant increase was seen in the potassium concentrations in the irradiated parent and aliquot samples relative to their non-irradiated counterparts.
CONCLUSIONS
Non-irradiated small-aliquot dose RCCs meet in vitro quality criteria required for safe transfusion throughout the 42-day storage period. The same can be said for aliquots derived from irradiated units and tested within 24 h of aliquot production.
Topics: Blood Preservation; Carcinoma, Renal Cell; Child; Erythrocytes; Gamma Rays; Hemolysis; Humans; Infant, Newborn; Kidney Neoplasms; Potassium; Time Factors
PubMed: 35869790
DOI: 10.1111/trf.17027 -
Cryobiology Dec 2022Leptospires are preserved by frequent sub-culturing in semisolid media due to the challenge of low recovery by freezing or liquid nitrogen methods. The present study...
Leptospires are preserved by frequent sub-culturing in semisolid media due to the challenge of low recovery by freezing or liquid nitrogen methods. The present study evaluated three liquid EMJH medium compositions (Medium A: Leptospira medium base EMJH, Leptospira enrichment EMJH, 5-fluorouracil (3%), rabbit serum (1%) and calf serum (1%); Medium B: same as Medium A but without 5-fluorouracil; Medium C: same as Medium B but with the addition of sodium pyruvate) for the revival of leptospires after storage at -80 °C. A total of 18 Leptospira serovars cultured in Medium A was aliquoted into cryogenic vials and directly stored at -80 °C. A hundred microlitre from each serovar culture stored at -80 °C was sub-cultured on a selected time over a period of 30 months into Media A, B and C. Regrowth on Media B and C showed a better and faster recovery (89-100%) (p-value <0.05) compared to Medium A (67-100%). Leptospires can be stored longer at -80 °C and a good recovery could be obtained when sub-cultured on EMJH medium without 5-fluorouracil.
Topics: Animals; Rabbits; Cryopreservation; Leptospira; Freezing; Fluorouracil; Nitrogen; Culture Media
PubMed: 36179819
DOI: 10.1016/j.cryobiol.2022.09.007 -
Analytical Chemistry Feb 2021Isolation and analysis of circulating rare cells is a promising approach for early detection of cancer and other diseases and for prenatal diagnosis. Isolation of rare...
Sequential Ensemble-Decision Aliquot Ranking Isolation and Fluorescence Hybridization Identification of Rare Cells from Blood by Using Concentrated Peripheral Blood Mononuclear Cells.
Isolation and analysis of circulating rare cells is a promising approach for early detection of cancer and other diseases and for prenatal diagnosis. Isolation of rare cells is usually difficult due to their heterogeneity as well as their low abundance in peripheral blood. We previously reported a two-stage ensemble-decision aliquot ranking platform (S-eDAR) for isolating circulating tumor cells from whole blood with high throughput, high recovery rate (>90%), and good purity (>70%), allowing detection of low surface antigen-expressing cancer cells linked to metastasis. However, due to the scarcity of these cells, large sample volumes and large quantities of antibodies were required to isolate sufficient cells for downstream analysis. Here, we drastically increased the number of nucleated cells analyzed by first concentrating peripheral blood mononuclear cells (PBMCs) from whole blood by density gradient centrifugation. The S-eDAR platform was capable of isolating rare cells from concentrated PBMCs (10/mL, equivalent to processing ∼20 mL of whole blood in the 1 mL sample volume used by our instrument) at a high recovery rate (>85%). We then applied the S-eDAR platform for isolating rare fetal nucleated red blood cells (fNRBCs) from concentrated PBMCs spiked with umbilical cord blood cells and confirmed fNRBC recovery by immunostaining and fluorescence hybridization, demonstrating the potential of the S-eDAR system for isolating rare fetal cells from maternal PBMCs to improve noninvasive prenatal diagnosis.
Topics: Cell Separation; Female; Fetal Blood; Humans; In Situ Hybridization, Fluorescence; Leukocytes; Leukocytes, Mononuclear; Neoplastic Cells, Circulating; Pregnancy
PubMed: 33528996
DOI: 10.1021/acs.analchem.0c04629 -
Scandinavian Journal of Clinical and... Jul 2022The body of literature varies significantly regarding serum and urine osmolality stability. Therefore, our aim was to investigate the stability of serum and urine...
OBJECTIVES
The body of literature varies significantly regarding serum and urine osmolality stability. Therefore, our aim was to investigate the stability of serum and urine osmolality at different temperatures (room temperature (RT) 4-8 °C, -20 °C) and time conditions (8 h, 24 h, 1 month).
METHODS
The stability study was conducted following the CRESS guidelines, including 40 serum and urine samples. Samples were aliquoted into three aliquots and stored as follows: primary tube stored at RT for 8 h; two capped aliquots stored at 4-8 °C for 8 h and 24 h; one aliquot stored at -20 °C for 1 month. To minimize imprecision error, serum and urine osmolality were measured by the freezing point depression method in triplicate on OSMOMAT 3000 (Gonotech, Germany) analyzer. Percentage difference (PD%) against baseline measurement was calculated. Deviations were assessed against a reference change value of 5.0%.
RESULTS
The PD% for serum and urine osmolality was below 2.0% for all time/temperature conditions. For serum samples: primary tube after 8 h at RT PD% (95% CI) = 0.0% (-0.3, 0.2%); 8 h at 4-8 °C PD% (95% CI) = -0.4% (-0.7, 0.0%); 24 h at 4-8 °C PD% (95% CI) = -0.7% (-0.7, -0.6%); 1 month at -20 °C PD% (95% CI) = -2.1% (-2.4, -1.5%). For urine samples: after 8 h at RT PD% (95% CI) =0.6% (0.2, 0.9%); 8 h at 4-8 °C PD% (95% CI) = -0.2% (-0.5, 0.1%); 24 h at 4-8 °C PD% (95% CI) = -0.2% (-0.5, 0.0%); 1 month at -20 °C PD% (95% CI) = -2.0% (-3.0, -1.0%).
CONCLUSIONS
Changes in osmolality for tested conditions for serum and urine samples, were within acceptance criteria. Reflex and add-on osmolality testing can be performed within the same day in samples kept at RT for 8 h in primary tube and within 24 h, in aliquoted refrigerated samples, without compromising the reliability of test results. For longer storage, samples should be kept at -20 °C.
Topics: Humans; Osmolar Concentration; Reproducibility of Results; Serum; Specimen Handling; Temperature; Time Factors; Urine
PubMed: 35654415
DOI: 10.1080/00365513.2022.2079094