Expression vectors for quatitating in vivo translational ambiguity: Their potential use to analyse frameshifting at the HIV gag-pol junction

Essential oils are gaining interest as environmentally friendly alternatives to synthetic fungicides for management of seedborne pathogens. Here, seven essential oils were initially tested in vivo for disinfection of squash seeds (Cucurbita maxima) naturally contaminated by Stagonosporopsis cucurbitacearum, Alternaria alternata, Fusarium fujikuro, Fusarium solani, Paramyrothecium roridum, Albifimbria verrucaria, Curvularia spicifera, and Rhizopus stolonifer. The seeds were treated with essential oils from Cymbopogon citratus, Lavandula dentata, Lavandula hybrida, Melaleuca alternifolia, Laurus nobilis, and Origanum majorana (#1 and #2). Incidence of S. cucurbitacearum was reduced, representing a range between 67.0% in L. nobilis to 84.4% in O. majorana #2. Treatments at 0.5 mg/mL essential oils did not affect seed germination, although radicles were shorter than controls, except with C. citratus and O. majorana #1 essential oils. Four days after seeding, seedling emergence was 20%, 30%, and 10% for control seeds and seeds treated with C. citratus essential oil (0.5 mg/mL) and fungicides (25 g/L difenoconazole plus 25 g/L fludioxonil). S. cucurbitacearum incidence was reduced by ~40% for plantlets from seeds treated with C. citratus essential oil. These data show the effectiveness of this essential oil to control the transmission of S. cucurbitacearum from seeds to plantlets, and thus define their potential use for seed decontamination in integrated pest management and organic agriculture.

Download Full-text

Ultramicronized Palmitoylethanolamide (um-PEA): A New Possible Adjuvant Treatment in COVID-19 patients

Pharmaceuticals ◽

10.3390/ph14040336 ◽

2021 ◽

Vol 14 (4) ◽

pp. 336

Author(s):

Annalisa Noce ◽

Maria Albanese ◽

Giulia Marrone ◽

Manuela Di Lauro ◽

Anna Pietroboni Zaitseva ◽

...

Keyword(s):

Adjuvant Treatment ◽

Pulmonary Involvement ◽

In Vivo Studies ◽

Pharmacological Treatments ◽

Beneficial Effects ◽

Coronavirus Infection ◽

Ultramicronized Palmitoylethanolamide ◽

Potential Use

The Coronavirus Disease-19 (COVID-19) pandemic has caused more than 100,000,000 cases of coronavirus infection in the world in just a year, of which there were 2 million deaths. Its clinical picture is characterized by pulmonary involvement that culminates, in the most severe cases, in acute respiratory distress syndrome (ARDS). However, COVID-19 affects other organs and systems, including cardiovascular, urinary, gastrointestinal, and nervous systems. Currently, unique-drug therapy is not supported by international guidelines. In this context, it is important to resort to adjuvant therapies in combination with traditional pharmacological treatments. Among natural bioactive compounds, palmitoylethanolamide (PEA) seems to have potentially beneficial effects. In fact, the Food and Drug Administration (FDA) authorized an ongoing clinical trial with ultramicronized (um)-PEA as an add-on therapy in the treatment of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection. In support of this hypothesis, in vitro and in vivo studies have highlighted the immunomodulatory, anti-inflammatory, neuroprotective and pain-relieving effects of PEA, especially in its um form. The purpose of this review is to highlight the potential use of um-PEA as an adjuvant treatment in SARS-CoV-2 infection.

Download Full-text

The Novel Arylamidine T-2307 MaintainsIn VitroandIn VivoActivity against Echinocandin-Resistant Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04228-14 ◽

2014 ◽

Vol 59 (2) ◽

pp. 1341-1343 ◽

Cited By ~ 14

Author(s):

Nathan P. Wiederhold ◽

Laura K. Najvar ◽

Annette W. Fothergill ◽

Rosie Bocanegra ◽

Marcos Olivo ◽

...

Keyword(s):

Body Weight ◽

Candida Albicans ◽

Invasive Candidiasis ◽

Placebo Control ◽

The Novel ◽

Content Type ◽

Fungal Burden ◽

Potential Use

ABSTRACTWe evaluated thein vitroandin vivoactivities of the investigational arylamidine T-2307 against echinocandin-resistantCandida albicans. T-2307 demonstrated potentin vitroactivity, and daily subcutaneous doses between 0.75 and 6 mg/kg of body weight significantly improved survival and reduced fungal burden compared to placebo control and caspofungin (10 mg/kg/day) in mice with invasive candidiasis caused by an echinocandin-resistant strain. Thus, T-2307 may have potential use in the treatment of echinocandin-resistantC. albicansinfections.

Download Full-text

The nuclear orphan receptors COUP-TFII and Ear-2 act as silencers of the human oxytocin gene promoter

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0190163 ◽

1997 ◽

Vol 19 (2) ◽

pp. 163-172 ◽

Cited By ~ 27

Author(s):

K Chu ◽

HH Zingg

Keyword(s):

Receptor Binding ◽

Promoter Activity ◽

Transcriptional Activity ◽

Gene Promoter ◽

Fine Tuning ◽

Orphan Receptor ◽

Expression Vectors ◽

Mobility Shift ◽

Interaction Sites

We have previously shown that COUP-TFII and Ear-2, two members of the nuclear orphan receptor family, are able to repress oestrogen-stimulated transcriptional activity of the human oxytocin (OT) gene promoter by binding to a site that overlaps with the oestrogen response element (ERE) present in the 5' flanking region of the gene. Although most nuclear receptor-mediated transcriptional repression conforms with the paradigm of passive repression and involves competitive binding to an activator site, active repression, i.e. silencing of basal promoter activity, has been observed in a limited number of cases. Here we show by co-transfection experiments using COUP-TFII and Ear-2 expression vectors and reporter constructs containing OT gene promoter fragments linked to the chloramphenicol acetyltransferase gene that both COUP-TFII and Ear-2 are capable of silencing basal OT gene promoter activity by 54 and 75% respectively. 5' Deletion and footprint analyses revealed two areas of functionally important interaction sites: (1) a direct TGACC(T/C) repeat overlapping the ERE and (2) a more promoter-proximal area centred at - 90 containing three imperfect direct repeats (R1-R3) spaced by four nucleotides each. Mutagenesis of reporter constructs as well as electrophoretic mobility-shift assays demonstrated that each of the three proximal repeats R1-R3 contributed to orphan receptor binding and the silencing effect. Inasmuch as the orphan receptor-binding sites are not involved in mediating basal transcriptional activity of the OT gene promoter, the observed effects are best interpreted as active repression or promoter silencing. Moreover, since COUP-TFII and Ear-2 are both co-expressed in OT-expressing uterine epithelial cells, the novel transcriptional effects described here are likely to be of functional importance in the fine-tuning of uterine OT gene expression in vivo.

Download Full-text

Mutation-specific hemostatic variability in mice expressing common type 2B von Willebrand disease substitutions

Blood ◽

10.1182/blood-2009-11-253120 ◽

2010 ◽

Vol 115 (23) ◽

pp. 4862-4869 ◽

Cited By ~ 25

Author(s):

Mia Golder ◽

Cynthia M. Pruss ◽

Carol Hegadorn ◽

Jeffrey Mewburn ◽

Kimberly Laverty ◽

...

Keyword(s):

Ferric Chloride ◽

Von Willebrand Disease ◽

Expression Vectors ◽

Severe Thrombocytopenia ◽

Normal Platelet ◽

Injury Model ◽

Von Willebrand ◽

Willebrand Factor

Abstract Type 2B von Willebrand disease (2B VWD) results from von Willebrand factor (VWF) A1 mutations that enhance VWF-GPIbα binding. These “gain of function” mutations lead to an increased affinity of the mutant VWF for platelets and the binding of mutant high-molecular-weight VWF multimers to platelets in vivo, resulting in an increase in clearance of both platelets and VWF. Three common 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf cDNA sequence and the expression vectors delivered to 8- to 10-week-old C57Bl6 VWF−/− mice, using hydrodynamic injection. The resultant phenotype was examined, and a ferric chloride–induced injury model was used to examine the thrombogenic effect of the 2B VWD variants in mice. Reconstitution of only the plasma component of VWF resulted in the generation of the 2B VWD phenotype in mice. Variable thrombocytopenia was observed in mice expressing 2B VWF, mimicking the severity seen in 2B VWD patients: mice expressing the V1316M mutation showed the most severe thrombocytopenia. Ferric chloride–induced injury to cremaster arterioles showed a marked reduction in thrombus development and platelet adhesion in the presence of circulating 2B VWF. These defects were only partially rescued by normal platelet transfusions, thus emphasizing the key role of the abnormal plasma VWF environment in 2B VWD.

Download Full-text

Is There Potential for Repurposing Statins as Novel Antimicrobials?

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00192-16 ◽

2016 ◽

Vol 60 (9) ◽

pp. 5111-5121 ◽

Cited By ~ 52

Author(s):

Emma Hennessy ◽

Claire Adams ◽

F. Jerry Reen ◽

Fergal O'Gara

Keyword(s):

Serum Cholesterol ◽

Bacterial Growth ◽

Bacterial Infections ◽

Statin Treatment ◽

Therapeutic Agents ◽

Pleiotropic Effects ◽

Current Information ◽

Potential Use

ABSTRACTStatins are members of a class of pharmaceutical widely used to reduce high levels of serum cholesterol. In addition, statins have so-called “pleiotropic effects,” which include inflammation reduction, immunomodulation, and antimicrobial effects. An increasing number of studies are emerging which detail the attenuation of bacterial growth andin vitroandin vivovirulence by statin treatment. In this review, we describe the current information available concerning the effects of statins on bacterial infections and provide insight regarding the potential use of these compounds as antimicrobial therapeutic agents.

Download Full-text

A Single Pitx1 Binding Site Is Essential for Activity of the LHβ Promoter in Transgenic Mice

Molecular Endocrinology ◽

10.1210/mend.15.5.0628 ◽

2001 ◽

Vol 15 (5) ◽

pp. 734-746 ◽

Cited By ~ 3

Author(s):

Christine C. Quirk ◽

Kristen L. Lozada ◽

Ruth A. Keri ◽

John H. Nilson

Keyword(s):

Transgenic Mice ◽

Binding Site ◽

Cell Lines ◽

Regulatory Element ◽

Regulatory Elements ◽

Vital Role ◽

Proximal Region ◽

Expression Vectors ◽

Homeodomain Protein

Abstract Reproduction depends on regulated expression of the LHβ gene. Tandem copies of regulatory elements that bind early growth response protein 1 (Egr-1) and steroidogenic factor 1 (SF-1) are located in the proximal region of the LHβ promoter and make essential contributions to its activity as well as mediate responsiveness to GnRH. Located between these tandem elements is a single site capable of binding the homeodomain protein Pitx1. From studies that employ overexpression paradigms performed in heterologous cell lines, it appears that Egr-1, SF-1, and Pitx1 interact cooperatively through a mechanism that does not require the binding of Pitx1 to its site. Since the physiological ramifications of these overexpression studies remain unclear, we reassessed the requirement for a Pitx1 element in the promoter of the LHβ gene using homologous cell lines and transgenic mice, both of which obviate the need for overexpression of transcription factors. Our analysis indicated a striking requirement for the Pitx1 regulatory element. When assayed by transient transfection using a gonadotrope-derived cell line (LβT2), an LHβ promoter construct harboring a mutant Pitx1 element displayed attenuated transcriptional activity but retained responsiveness to GnRH. In contrast, analysis of wild-type and mutant expression vectors in transgenic mice indicated that LHβ promoter activity is completely dependent on the presence of a functional Pitx1 binding site. Indeed, the dependence on an intact Pitx1 binding site in transgenic mice is so strict that responsiveness to GnRH is also lost, suggesting that the mutant promoter is inactive. Collectively, our data reinforce the concept that activity of the LHβ promoter is determined, in part, through highly cooperative interactions between SF-1, Egr-1, and Pitx1. While Egr-1 can be regarded as a key downstream effector of GnRH, and Pitx1 as a critical partner that activates SF-1, our data firmly establish that the Pitx1 element plays a vital role in permitting these functions to occur in vivo.

Download Full-text

MicroRNAs in Valvular Heart Diseases: Biological Regulators, Prognostic Markers and Therapeutical Targets

International Journal of Molecular Sciences ◽

10.3390/ijms222212132 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12132

Author(s):

Francesco Nappi ◽

Adelaide Iervolino ◽

Sanjeet Singh Avtaar Singh ◽

Massimo Chello

Keyword(s):

Mitral Valve ◽

Mitral Valve Prolapse ◽

Heart Diseases ◽

Osteoblastic Differentiation ◽

New Drugs ◽

Expression Vectors ◽

Chordae Tendineae ◽

Valvular Heart Diseases

miRNAs have recently attracted investigators’ interest as regulators of valvular diseases pathogenesis, diagnostic biomarkers, and therapeutical targets. Evidence from in-vivo and in-vitro studies demonstrated stimulatory or inhibitory roles in mitral valve prolapse development, aortic leaflet fusion, and calcification pathways, specifically osteoblastic differentiation and transcription factors modulation. Tissue expression assessment and comparison between physiological and pathological phenotypes of different disease entities, including mitral valve prolapse and mitral chordae tendineae rupture, emerged as the best strategies to address miRNAs over or under-representation and thus, their impact on pathogeneses. In this review, we discuss the fundamental intra- and intercellular signals regulated by miRNAs leading to defects in mitral and aortic valves, congenital heart diseases, and the possible therapeutic strategies targeting them. These miRNAs inhibitors are comprised of antisense oligonucleotides and sponge vectors. The miRNA mimics, miRNA expression vectors, and small molecules are instead possible practical strategies to increase specific miRNA activity. Advantages and technical limitations of these new drugs, including instability and complex pharmacokinetics, are also presented. Novel delivery strategies, such as nanoparticles and liposomes, are described to improve knowledge on future personalized treatment directions.

Download Full-text

Different expression levels of two KgmB-His fusion proteins

Journal of the Serbian Chemical Society ◽

10.2298/jsc0512401m ◽

2005 ◽

Vol 70 (12) ◽

pp. 1401-1407 ◽

Cited By ~ 3

Author(s):

Sandra Markovic ◽

Sandra Vojnovic ◽

Milija Jovanovic ◽

Branka Vasiljevic

Keyword(s):

Recombinant Proteins ◽

Fusion Proteins ◽

Kanamycin Resistance ◽

Expression Vectors ◽

E Coli ◽

C Terminus ◽

N Terminus ◽

Different Levels ◽

Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

Download Full-text

Is Phasmarhabditis papillosa (Nematoda: Rhabditidae) a possible biological control agent against the Spanish slug, Arion vulgaris (Gastropoda: Arionidae)?

Nematology ◽

10.1163/15685411-bja10063 ◽

2020 ◽

pp. 1-9

Author(s):

Žiga Laznik ◽

Ivana Majić ◽

Stanislav Trdan ◽

Antoinette P. Malan ◽

Annika Pieterse ◽

...

Keyword(s):

Biological Control ◽

Laboratory Experiments ◽

Biological Control Agent ◽

First Record ◽

Control Agent ◽

Molecular Techniques ◽

Parasitic Nematodes ◽

Arion Vulgaris ◽

Potential Use

Summary In the period from August to October 2018, 140 specimens of the Spanish slug, Arion vulgaris, were collected from Podbrezje, Slovenia. Slugs were dissected and examined for the presence of parasitic nematodes within the cadavers. Identification of the nematodes was conducted using morphological and molecular techniques and confirmed the presence of Phasmarhabditis papillosa. This is the first record of P. papillosa from the mollusc host, A. vulgaris. Laboratory experiments aimed at testing the efficacy of P. papillosa against A. vulgaris were conducted using nematodes grown in vivo. Nematodes were applied at concentration rates of 50, 100 and 200 nematodes slug−1, respectively. Three weeks following treatment, the mortality of slugs was confirmed in all treatments (50 nematodes slug−1, 37.4 ± 2.7%; 100 nematodes slug−1, 48.4 ± 2.7%; 200 nematodes slug−1, 50.6 ± 2.7%). However, the pathogenesis of P. papillosa was observed first in the treatments with the lowest nematode dose at 4 days after treatments, while a decrease in the feeding behaviour of slugs was noted first in the treatments with the highest nematode dose. Future opportunities for the potential use of P. papillosa as a biological control agent against slugs are discussed. This is the first report of P. papillosa from Slovenia, and of its virulence against A. vulgaris.

Download Full-text