The simple and rapid detection of specific PCR products from bacterial genomes using Zn finger proteins

A novel method of rapid and specific detection of polymerase chain reaction (PCR) products from bacterial genomes using Zn finger proteins was developed. Zn finger proteins are DNA-binding proteins that can sequence specifically recognize PCR products. Since Zn finger proteins can directly detect PCR products without undergoing dehybridization, unlike probe DNA, and can double check the specific PCR amplification and sequence specificity of the PCR products, this novel method would be quick and highly accurate. In this study, we tried to detect Legionella pneumophila using Sp1. It was found that a 49 bp L. pneumophila-specific region containing the Sp1 recognition site is located on the flhA gene of the L. pneumophila genome. We succeeded in specifically detecting PCR products amplified from L. pneumophila in the presence of other bacterial genomes by ELISA, and demonstrated that Sp1 enables the discrimination of L. pneumophila-specific PCR products from others. By fluorescence depolarization measurement, these specific PCR products could be detected within 1 min. These results indicate that the rapid and simple detection of PCR products specific to L. pneumophila using a Zn finger protein was achieved. This methodology can be applied to the detection of other bacteria using various Zn finger proteins that have already been reported

New light on plant ash glass found in Africa: evidence for Indian Ocean Silk Road trade using major, minor, trace element and lead isotope analysis of glass from the 15th—16th century AD from Malindi and Mambrui, Kenya

Seventeen glass vessels and twenty glass beads recovered from the excavations at the ancient city of Malindi and the archaeological site of Mambrui in Kenya, east Africa were analysed using electron probe microanalysis (EPMA) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The results show that all of the glass samples are soda-lime-silica glass. They belong to the high alumina -plant ash glass type, characterised by high alumina and relatively low calcium contents, widely distributed in eastern (10th- 16th centuries AD) and southern Africa (13th - 15th centuries AD), Central Asia (9th- 14th centuries AD) and southeast Asia (12th- 13th centuries AD), made with plant ashes and sands. This is an understudied glass type for which previous research has indicated there were three types. When compared with published research on such glasses using Zr, Ti, Ba, Cr, La, Li, Cs, Na2O, MgO and CaO we have identified at least four different compositional groups of v-Na-Al glass: Types A, B, C and D. By comparing the results with contemporary v-Na-Al glass vessels and beads from Central Asia, Africa, and southeast Asia we show that most of the Malindi and Mambrui glass share similar characteristics to the compositions of Mapungubwe Oblate and some of the Madagascar glass beads from southern Africa. They belong to Type A v-Na-Al glass which is characterised by an elevated level of Ti and Ba and a relatively high ratios of Cr/La, relatively low Zr concentrations and low ratios of Zr/ Ti. Differences in Zr, Li, MgO and Na2O concentrations in Type A glass indicates that there are subgroups which might derive from different glass workshop(s) specialising in Type A v- Na-Al glass production. Comparison with the chemical compositions of glass from Ghazni, Afghanistan and Termez, Uzbekistan, and by using lead isotope analysis, we suggest v-Na- Al glass was manufactured in Central Asia and possibly worked into vessels and beads there. Copyright: © 2020 Siu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone

Pyrroloquinoline quinone is a prominent redox cofactor in many prokaryotes, produced from a ribosomally synthesized and post-translationally modified peptide PqqA via a pathway comprising four conserved proteins PqqB?E. These four proteins are now fairly well-characterized and span radical SAM activity (PqqE), aided by a peptide chaperone (PqqD), a dual hydroxylase (PqqB), and an eight-electron, eight-proton oxidase (PqqC). A full description of this pathway has been hampered by a lack of information regarding a protease/peptidase required for the excision of an early, cross-linked di-amino acid precursor to pyrroloquinoline quinone. Herein, we isolated and characterized a two-component heterodimer protein from the ?-proteobacterium Methylobacterium (Methylorubrum) extorquens that can rapidly catalyze cleavage of PqqA into smaller peptides. Using pulldown assays, surface plasmon resonance, and isothermal calorimetry, we demonstrated the formation of a complex PqqF/PqqG, with a K-D of 300 nm. We created a molecular model of the heterodimer by comparison with the Sphingomonas sp. A1 M16B Sph2681/Sph2682 protease. Analysis of time-dependent patterns for the appearance of proteolysis products indicates high specificity of PqqF/PqqG for serine side chains. We hypothesize that PqqF/PqqG initially cleaves between the PqqE/PqqD-generated cross-linked form of PqqA, with nonspecific cellular proteases completing the release of a suitable substrate for the downstream enzyme PqqB. The finding of a protease that specifically targets serine side chains is rare, and we propose that this activity may be useful in proteomic analyses of the large family of proteins that have undergone post-translational phosphorylation at serine.National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [GM118117, GM124002, 1S10OD020062-01

Biologics registers in RA: methodological aspects, current role and future applications

The beginning of the 21st century saw a biopharmaceutical revolution in the treatment of inflammatory rheumatic diseases, particularly rheumatoid arthritis. The fast-evolving use of biologic therapies highlighted the need to develop registers at national and international levels with the aim of collecting long-term data on patient outcomes. Over the past 15 years, many biologics registers have contributed a wealth of data and provided robust and reliable evidence on the use, effectiveness and safety of these therapies. The unavoidable challenges posed by the continuous introduction of new therapies, particularly with regard to understanding their long-term safety, highlights the importance of learning from experience with established biologic therapies. In this Perspectives article, the role of biologics registers in bridging the evidence gap between efficacy in clinical trials and real-world effectiveness is discussed, with a focus on methodological aspects of registers, their unique features and challenges and their role going forward

Characterization of different FAD-dependent glucose dehydrogenases for possible use in glucose-based biosensors and biofuel cells

In this study, different flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) were characterized electrochemically after “wiring” them with an osmium redox polymer [Os(4,4′-dimethyl-2,2′-bipyridine)2(PVI)10Cl]+ on graphite electrodes. One tested FADGDH was that recently discovered in Glomerella cingulata (GcGDH), another was the recombinant form expressed in Pichia pastoris (rGcGDH), and the third was a commercially available glycosylated enzyme from Aspergillus sp. (AspGDH). The performance of the Os-polymer “wired” GDHs on graphite electrodes was tested with glucose as the substrate. Optimal operational conditions and analytical characteristics like sensitivity, linear ranges and current density of the different FADGDHs were determined. The performance of all three types of FADGDHs was studied at physiological conditions (pH 7.4). The current densities measured at a 20 mM glucose concentration were 494 ± 17, 370 ± 24, and 389 ± 19 μA cm−2 for GcGDH, rGcGDH, and AspGDH, respectively. The sensitivities towards glucose were 2.16, 1.90, and 1.42 μA mM−1 for GcGDH, rGcGDH, and AspGDH, respectively. Additionally, deglycosylated rGcGDH (dgrGcGDH) was investigated to see whether the reduced glycosylation would have an effect, e.g., a higher current density, which was indeed found. GcGDH/Os-polymer modified electrodes were also used and investigated for their selectivity for a number of different sugars

Prader-Willi syndrome: A primer for clinicians

The advent of sensitive genetic testing modalities for the diagnosis of Prader-Willi syndrome has helped to define not only the phenotypic features of the syndrome associated with the various genotypes but also to anticipate clinical and psychological problems that occur at each stage during the life span. With advances in hormone replacement therapy, particularly growth hormone children born in circumstances where therapy is available are expected to have an improved quality of life as compared to those born prior to growth hormone

A Glycemia Risk Index (GRI) of Hypoglycemia and Hyperglycemia for Continuous Glucose Monitoring Validated by Clinician Ratings

BackgroundA composite metric for the quality of glycemia from continuous glucose monitor (CGM) tracings could be useful for assisting with basic clinical interpretation of CGM data.MethodsWe assembled a data set of 14-day CGM tracings from 225 insulin-treated adults with diabetes. Using a balanced incomplete block design, 330 clinicians who were highly experienced with CGM analysis and interpretation ranked the CGM tracings from best to worst quality of glycemia. We used principal component analysis and multiple regressions to develop a model to predict the clinician ranking based on seven standard metrics in an Ambulatory Glucose Profile: very low-glucose and low-glucose hypoglycemia; very high-glucose and high-glucose hyperglycemia; time in range; mean glucose; and coefficient of variation.ResultsThe analysis showed that clinician rankings depend on two components, one related to hypoglycemia that gives more weight to very low-glucose than to low-glucose and the other related to hyperglycemia that likewise gives greater weight to very high-glucose than to high-glucose. These two components should be calculated and displayed separately, but they can also be combined into a single Glycemia Risk Index (GRI) that corresponds closely to the clinician rankings of the overall quality of glycemia (r = 0.95). The GRI can be displayed graphically on a GRI Grid with the hypoglycemia component on the horizontal axis and the hyperglycemia component on the vertical axis. Diagonal lines divide the graph into five zones (quintiles) corresponding to the best (0th to 20th percentile) to worst (81st to 100th percentile) overall quality of glycemia. The GRI Grid enables users to track sequential changes within an individual over time and compare groups of individuals.ConclusionThe GRI is a single-number summary of the quality of glycemia. Its hypoglycemia and hyperglycemia components provide actionable scores and a graphical display (the GRI Grid) that can be used by clinicians and researchers to determine the glycemic effects of prescribed and investigational treatments