scholarly journals Molecular sexing of degraded DNA from elephants and mammoths: a genotyping assay relevant both to conservation biology and to paleogenetics

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Laetitia Aznar-Cormano ◽  
Julie Bonnald ◽  
Sabrina Krief ◽  
Nelson Guma ◽  
Régis Debruyne
Keyword(s):  
A Priori ◽  
Cost Effective ◽  
Degraded Dna ◽  
Molecular Sexing ◽  
African Elephants ◽  
Actual Case ◽  
The Right ◽  
High Level

AbstractIt is important to determine the sex of elephants from their samples—faeces from the field or seized ivory—for forensic reasons or to understand population demography and genetic structure. Molecular sexing methods developed in the last two decades have often shown limited efficiency, particularly in terms of sensitivity and specificity, due to the degradation of DNA in these samples. These limitations have also prevented their use with ancient DNA samples of elephants or mammoths. Here we propose a novel TaqMan-MGB qPCR assay to address these difficulties. We designed it specifically to allow the characterization of the genetic sex for highly degraded samples of all elephantine taxa (elephants and mammoths). In vitro experiments demonstrated a high level of sensitivity and low contamination risks. We applied this assay in two actual case studies where it consistently recovered the right genotype for specimens of known sex a priori. In the context of a modern conservation survey of African elephants, it allowed determining the sex for over 99% of fecal samples. In a paleogenetic analysis of woolly mammoths, it produced a robust hypothesis of the sex for over 65% of the specimens out of three PCR replicates. This simple, rapid, and cost-effective procedure makes it readily applicable to large sample sizes.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Rapid and Scalable Production of Functional Anti-Coronavirus Monoclonal Antibody CR3022 in Plants

2020 ◽  
Author(s):  
Kaewta Rattanapisit ◽  
Balamurugan Shanmugaraj ◽  
Suwimon Manopwisedjaroen ◽  
Priyo Budi Purwono ◽  
Konlavat Siriwattananon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Developing Economies ◽  
Functional Characterization ◽  
Cost Effective ◽  
Technology Platforms ◽  
Effective Manner ◽  
Rapid Spread ◽  
Significant Public Health

Abstract Severe acute respiratory syndrome coronavirus-2 is responsible for an ongoing global outbreak of coronavirus disease (COVID-19) and represents a significant public health threat. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of diagnostic reagents/biopharmaceuticals for COVID-19. We explored the possibility of producing an anti-SARS-CoV monoclonal antibody (mAb) CR3022 and the receptor binding domain (RBD) of SARS-CoV-2 in Nicotiana benthamiana. Both RBD and the mAb were transiently expressed with the expression of 8μg/g and 130μg/g leaf fresh weight respectively. The plant-purified mAb binds to SARS-CoV-2, but fails to neutralize it in vitro. This is the first report showing the functional characterization of an anti- SARS-CoV mAb CR3022 in plants. Overall these findings showed that plants are a promising platform to produce anti-SARS-CoV mAb to use as a research reagent or a biotherapeutic in a cost-effective manner, which is especially important to developing economies during epidemics.

scholarly journals Characterization of Vancomycin-Resistant Enterococcus faecium Isolates from the United States and Their Susceptibility In Vitro to Dalfopristin-Quinupristin

1998 ◽  
Vol 42 (5) ◽  
pp. 1088-1092 ◽  
Author(s):  
G. M. Eliopoulos ◽  
C. B. Wennersten ◽  
H. S. Gold ◽  
T. Schülin ◽  
M. Souli ◽  
...  
Keyword(s):  
United States ◽  
Enterococcus Faecium ◽  
Multiple Drug Resistance ◽  
The United States ◽  
Multiple Drug ◽  
Data Set ◽  
Vancomycin Resistant ◽  
High Level

ABSTRACT In the course of clinical studies with the investigational streptogramin antimicrobial dalfopristin-quinupristin, isolates of vancomycin-resistant Enterococcus faecium were referred to our laboratory from across the United States. Seventy-two percent of the strains were of the VanA type, phenotypically and genotypically, while 28% were of the VanB type. High-level resistance to streptomycin or gentamicin was observed in 86 and 81%, respectively, of the VanA strains but in only 69 and 66%, respectively, of the VanB strains. These enterococci were resistant to ampicillin (MIC for 50% of the isolates tested [MIC50] and MIC90, 128 and 256 μg/ml, respectively) and to the other approved agents tested, with the exception of chloramphenicol (MIC90, 8 μg/ml) and novobiocin (MIC90, 1 μg/ml). Considering all of the isolates submitted, dalfopristin-quinupristin inhibited 86.4% of them at concentrations of ≤1 μg/ml and 95.1% of them at ≤2 μg/ml. However, for the data set comprised of only the first isolate submitted for each patient, 94.3% of the strains were inhibited at concentrations of ≤1 μg/ml and 98.9% were inhibited at concentrations of ≤2 μg/ml. Multiple drug resistance was very common among these isolates of vancomycin-resistant E. faecium, while dalfopristin-quinupristin inhibited the majority at concentrations that are likely to be clinically relevant.

SEMI-QUANTITATIVE RADIONUCLIDE PHLEBOGRAPHIC (RNP) ASSESSMENT OF DEEP VEIN THROMBOSIS (DVT) AND CHRONIC VENOUS INSUFFICIENCY (CVI)

1987 ◽  
Author(s):  
J Zahavi ◽  
S Zaltzman ◽  
E Firsteter ◽  
E Avrahami
Keyword(s):  
Venous Insufficiency ◽  
Cost Effective ◽  
Vein Thrombosis ◽  
Below The Knee ◽  
High Incidence ◽  
Complete Thrombosis ◽  
The Right ◽  
High Level ◽  
Deep Vein

A semi-quantitative RNP using 99Technetium macroaggregated albumin for the evaluation and follow-up of DVT and CVI has been developed. Values were assigned to the deep veins of the calf, knee, tigh and pelvis based upon the localization and the characteristics of the images obtained: stasis, hot spots and collateral circulation. A maximum score of 18 reflected complete thrombosis of all 4 segments. 208 patients (mean age 53.7 years, range 18-92), 161 of whom had a proven risk factor for DVT were studied. 99Technetium was injected into the dorsal foot vein of 407 limbs with appropriate tourniquets and early and late imaging of the limbs, pelvis and lungs was performed. In 48 patients, 83 limbs, X-ray contrast phlebography (CP) was also done. The mean RNP score was 4.1 units (range 0.4-18) and higher in the left than the right lower limb. It was mostly high in patients with proximal recurrent DVT or in DVT superimposed on CVI. The score was easy to follow and helpful in the assessment of the extent of DVT. It was particularly helpful in 3 instances. 1) Assessment of venous patency following anticoagulant therapy. 2) Estimation of recurrent DVT. 3) Differentiation of recent DVT from venous insufficiency. Overall RNP method had a sensitivity of 87.6%, a specificity of 54% and an accuracy of 64.8%. The sensitivity was similar in above & below-knee thrombi. Yet the specificity was higher in above-knee thrombi. The highest accuracy (87.3%) was observed in pelvic and groin thrombi. The distribution of thrombi on CP was 19% below the knee, 31% above it and 50% both above and below the knee. Pulmonary embolism (PE) was initially observed in 54 patients (26%) with no clinical evidence of DVT and therefore untreated. This high level is most probably related to the high incidence of proximal DVT in the patients. 181 patients were treated with heparin & coumadin and the RNP score was decreased to 3.6 units (range 0.4-8.8). PE occurred during treatment in 11 (6.1%) and recurrent DVT in 16 (8.8%) patients. CVI was observed in 23 patients before treatment and in another 24 patients (13.2%) after treatment. These results indicate that the RNP method is a simple, semi-quantitative and useful technique for the evaluation and follow-up of DVT and CVI. It is most helpful in the assessment of the extent of DVT. It is also a rapid, noninvasive and cost effective techniaue.

Characterization of sulfidopeptide leukotriene responses in sheep tracheal smooth muscle in vitro

1991 ◽  
Vol 69 (6) ◽  
pp. 805-811 ◽  
Author(s):  
K. Tomioka ◽  
J. T. Jackowski ◽  
W. M. Abraham
Keyword(s):  
Smooth Muscle ◽  
Dose Response ◽  
Tracheal Smooth Muscle ◽  
Response Curves ◽  
Leukotriene Antagonist ◽  
Dose Response Curves ◽  
The Right ◽  
Glutamyl Transpeptidase

We have investigated the effects of leukotrienes (LTs) on isolated tracheal smooth muscle from sheep sensitive to Ascaris suum antigen. LTC4 and LTD4 produced dose-dependent contractions of sheep trachea, but LTE4 was virtually inactive. YM-17690, a non-analogous LT agonist, produced no contractile response up to 100 μM. Indomethacin (5 μM) had no effect on LTC4- and LTD4-induced contractions. L-Serine borate (45 mM), an inhibitor of γ-glutamyl transpeptidase, shifted the dose–response curve of LTC4 to the left by 161-fold, and L-cysteine (6 mM), an inhibitor of aminopeptidase, shifted the dose–response curves of LTC4 and LTD4 to the left by 67- and 23-fold, respectively. YM-16638 (1 μM), an LT antagonist, shifted the dose–response curves of LTC4 and LTD4 to the right with pKB values of 6.57 and 7.13, respectively. YM-16638 did not affect LTC4-induced contractions of L-serine borate-treated tissues, indicating that the compound acts only on LTD4 receptors in sheep trachea. LTE4 (1 μM) shifted the dose–response curves of LTC4 and LTD4 to the right with pKB values of 6.87 and 7.31, respectively. YM-17690 (10 μM) showed effects similar to LTE4, suggesting that the compound acts as an LTE4 agonist in sheep trachea. These results suggest that in sheep tracheal smooth muscle (a) LTC4 and LTD4 produce contractions, (b) these LT-induced contractions are not mediated by cyclooxygenase products, (c) LTC4 is converted to LTD4 and then to LTE4, and (d) the potency of the LTC4- and LTD4-induced contractions is increased when their conversion to LTE4 is inhibited. This potentiation may result from the inability of LTE4 to contract sheep trachea and (or) its antagonist actions.Key words: leukotriene antagonist, receptors, asthma.

scholarly journals THE GENETICS OF DOPA DECARBOXYLASE IN DROSOPHILA MELANOGASTER. II. ISOLATION AND CHARACTERIZATION OF DOPA-DECARBOXYLASE-DEFICIENT MUTANTS AND THEIR RELATIONSHIP TO THE α-METHYL-DOPA-HYPERSENSITIVE MUTANTS

Genetics ◽  
1976 ◽  
Vol 84 (2) ◽  
pp. 287-310
Author(s):  
Theodore R F Wright ◽  
Glenn C Bewley ◽  
Allen F Sherald
Keyword(s):  
Drosophila Melanogaster ◽  
Recombination Frequency ◽  
Dopa Decarboxylase ◽  
Isolation And Characterization ◽  
Methyl Dopa ◽  
Level Of Activity ◽  
The Right ◽  
Balancer Chromosome

ABSTRACT Of 84 lethals isolated over the dopa decarboxylase (DDC) deficiency Df(2L)50, 8 have been identified as DDC-deficient alleles on the basis of their effect on DDC activity when heterozygous over the CyO balancer chromosome with activities ranging from 28% to 53% of controls. Some of the Ddc-deficient alleles exhibit intracistronic complementation. Most of the complementing pairs of alleles are much reduced in viability, e.g. < 5% of expected, and express a common syndrome of mutant phenes which can reasonably be inferred to derive from inadequately sclerotinized cuticle. Individuals heterozygous for the noncomplementing allele, Ddcn7, over the 12-band DDC deficiency, Df(2L)130, die at the end of embryogenesis as unhatched larvae with unpigmented mouth parts. The Ddc alleles and the l(2)amd α-methyl dopa (αMD) hypersensitive alleles are both located within the 11 band region 37B10-C7. The l(2)amd locus is immediately to the right of hk(2-53.9).Ddc has been mapped within 0.004 Map Units to the right of l(2)amd with a maximum estimated recombination frequency of 0.01%. None of the Ddc/CyO strains are sensitive to the dietary administration of α-methyl dopa (αMD), and complementation occurs between the Ddc deficient alleles and the l(2)amd alleles both on the basis of viability and DDC activity. No effect on DDC by the amd alleles has been found to date. Even in the complementing heterozygote, amdH1/amdH89, the level of activity, thermostability, and in vitro αMD inhibition of DDC remains unaffected. Although no biochemical phene has yet been established for the αMD hypersensitive amd alleles, it seems likely that the two groups of mutants are functionally related.

scholarly journals Molecular Viral Diagnosis and Sanitation of Yam Genetic Resources: Implications for Safe Yam Germplasm Exchange

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1101
Author(s):  
Marie Umber ◽  
Denis Filloux ◽  
Suzia Gélabale ◽  
Rose-Marie Gomez ◽  
Armelle Marais ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Cost Effective ◽  
Meristem Culture ◽  
Germplasm Exchange ◽  
Tropical Plants ◽  
High Prevalence ◽  
Clean Seed ◽  
High Level ◽  
Viral Diagnosis

Yam (Dioscorea spp.) is an important crop in tropical and subtropical regions. Many viruses have been recently identified in yam, hampering genetic conservation and safe international exchanges of yam germplasm. We report on the implementation of reliable and cost-effective PCR-based detection tools targeting eight different yam-infecting viruses. Viral indexing of the in vitro yam collection maintained by the Biological Resources Center for Tropical Plants (BRC-TP) in Guadeloupe (French West Indies) unveiled a high prevalence of potyviruses, badnaviruses, Dioscorea mosaic associated virus (DMaV) and yam asymptomatic virus 1 (YaV1) and a high level of coinfections. Infected yam accessions were subjected to a combination of thermotherapy and meristem culture. Sanitation levels were monitored using PCR-based and high-throughput sequencing-based diagnosis, confirming the efficacy and reliability of PCR-based detection tools. Sanitation rates were highly variable depending on viruses. Sixteen accessions were successfully sanitized, paving the way to safe yam germplasm exchanges and the implementation of clean seed production programs worldwide.

scholarly journals Quasi-spectral characterization of intracellular regions in bright-field light microscopy images

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kirill Lonhus ◽  
Renata Rychtáriková ◽  
Ganna Platonova ◽  
Dalibor Štys
Keyword(s):  
Cell Structure ◽  
A Priori ◽  
Semantic Segmentation ◽  
Bright Field ◽  
Model Free ◽  
Bright Field Microscopy ◽  
Priori Information ◽  
The Right ◽  
Microscopy Images

Abstract Investigation of cell structure is hardly imaginable without bright-field microscopy. Numerous modifications such as depth-wise scanning or videoenhancement make this method being state-of-the-art. This raises a question what maximal information can be extracted from ordinary (but well acquired) bright-field images in a model-free way. Here we introduce a method of a physically correct extraction of features for each pixel when these features resemble a transparency spectrum. The method is compatible with existent ordinary bright-field microscopes and requires mathematically sophisticated data processing. Unsupervised clustering of the spectra yields reasonable semantic segmentation of unstained living cells without any a priori information about their structures. Despite the lack of reference data (to prove strictly that the proposed feature vectors coincide with transparency), we believe that this method is the right approach to an intracellular (semi)quantitative and qualitative chemical analysis.

scholarly journals Characterization of Dihydrofolate Reductase Genes from Trimethoprim-Susceptible and Trimethoprim-Resistant Strains ofEnterococcus faecalis

1999 ◽  
Vol 43 (1) ◽  
pp. 141-147 ◽  
Author(s):  
Teresa M. Coque ◽  
Kavindra V. Singh ◽  
George M. Weinstock ◽  
Barbara E. Murray
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
E Coli ◽  
Integrative Vector ◽  
Dihydrofolate Reductases ◽  
Multiple Copies ◽  
Gram Negative Organisms ◽  
High Level

Enterococci are usually susceptible in vitro to trimethoprim; however, high-level resistance (HLR) (MICs, >1,024 μg/ml) has been reported. We studied Enterococcus faecalis DEL, for which the trimethoprim MIC was >1,024 μg/ml. No transfer of resistance was achieved by broth or filter matings. Two different genes that conferred trimethoprim resistance when they were cloned in Escherichia coli (MICs, 128 and >1,024 μg/ml) were studied. One gene that coded for a polypeptide of 165 amino acids (MIC, 128 μg/ml forE. coli) was identical to dfr homologs that we cloned from a trimethoprim-susceptible E. faecalis strain, and it is presumed to be the intrinsic E. faecalis dfr gene (which causes resistance in E. coli when cloned in multiple copies); this gene was designated dfrE. The nucleotide sequence 5′ to this dfr gene showed similarity to thymidylate synthetase genes, suggesting that the dfr andthy genes from E. faecalis are located in tandem. The E. faecalis gene that conferred HLR to trimethoprim in E. coli, designated dfrF, codes for a predicted polypeptide of 165 amino acids with 38 to 64% similarity with other dihydrofolate reductases from gram-positive and gram-negative organisms. The nucleotide sequence 5′ to dfrFdid not show similarity to the thy sequences. A DNA probe for dfrF hybridized under high-stringency conditions only to colony lysates of enterococci for which the trimethoprim MIC was >1,024 μg/ml; there was no hybridization to plasmid DNA from the strain of origin. To confirm that this gene causes trimethoprim resistance in enterococci, we cloned it into the integrative vector pAT113 and electroporated it into RH110 (E. faecalisOG1RF::Tn916ΔEm) (trimethoprim MIC, 0.5 μg/ml), which resulted in RH110 derivatives for which the trimethoprim MIC was >1,024 μg/ml. These results indicate thatdfrF is an acquired but probably chromosomally located gene which is responsible for in vitro HLR to trimethoprim in E. faecalis.

Assessment of blackberry varieties suitable for growing in the Right-Bank Forest-Steppe part of Ukraine

2020 ◽  
pp. 201-206
Author(s):  
L. Shubenko ◽  
S. Shokh ◽  
Kumanska Yu.
Keyword(s):  
Crop Yields ◽  
Average Weight ◽  
Forest Steppe ◽  
Maximum Weight ◽  
Yield Level ◽  
High Yields ◽  
The Right ◽  
High Level ◽  
The University

The research aimed to study the productivity of blackberry foreign varieties (Rubus subgenus Rubus Watson) in the Right-Bank Forest-Steppe part of Ukraine as they are attractive for production due to their fruit taste and appearance. The task was to establish the beginning and duration of fruiting of blackberry varieties, to determine the yield level and the quality of the berries. A fi eld experiment was conducted on the experimental fi eld of the Scientific and Research Center of BNAU to assess the productivity of blackberry varieties. The studies were started in the spring of 2017 on blackberry seedlings with growing the culture in vitro in the biotechnological laboratory of the university. The tests included 5 varieties with climbing shoots: Smoothstem, Thornfree, Black Satin, Triple Crown, Arapaho. According to the results of observations, it was found that for two years of fruiting among the studied varieties, the earliest production was received in the Arapaho variety, in the first decade of July. Triple Crown, with the fi rst fruits ripened on August 2, was characterized by late fruiting. The Triple Crown variety diff ered by the longest period of berry coming – 35 days. The yield obtained in a year after planting is not an indicator of long-term crop yields. However, the Triple Crown variety diff ered by the highest yield in the fi rst year of fruiting, with an average of 3.7 kg of berries obtained from a bush. High yields were also observed in the Smoothstem variety. Black Satin variety was characterized by the high level of yield growth for over two years of fruiting, which amounted to 80 %. The largest in the fi rst years of fruiting were Triple Crown fruits with an average weight of 11.1 g. The maximum weight of individual berries of this variety reached 17.7 g. The Arapaho variety also stood out with its large fruits of 8.5 g on average. Some fruits reached a maximum weight of 12.1 g. Key words: blackberry, the beginning of fruiting, productivity, average weight of berries, duration of products supply.

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