Reverse Protein Engineering Of Firefly Luciferase

Firefly luciferase is a bioluminescent protein commonly used as a bioluminescent tag in biological studies and applications. However, because the protein is fairly large in size, it is sometimes larger than the molecules it is intended to measure and is therefore not a sufficient tag in smaller applications. The active site of firely luciferase is also not well understood, making it difficult to engineer the protein without affecting its bioluminescent activity. In this paper, we discuss the experimental methods of Reverse Protein Engineering: a bioengineering technology that reduces the size of a protein while retaining its original function. This involves subcloning a core section of the protein, attaching a DNA library to the core to achieve a large pool of randomized peptide variants, and screening those variants for any bioluminescent properties. Successful conduction of this technique would achieve two goals: 1) create a peptide alternative to resolve the protein’s current size limitations and 2) confirm the importance of specific amino acids that might contribute to the active site’s activity. Our experiments show that Reverse Protein Engineering can be done to decrease the size of Firefly Luciferase (550 amino acids) to a much smaller peptidic version of the protein (less than 80 amino acids). However, to determine successful function of the peptide variants, more research in screening the peptides for bioluminescent activity needs to be done. In addition, Reverse Protein Engineering with a different range of amino acids within the core could further the chances of achieving a successful bioluminescent peptide variant of firefly luciferase

A füstölgő meddőhányóktól a globális közlekedésig : Beszélgetés Erdősi Ferenccel

Erdősi Ferenc 1934. április 19-én született Pécsett. Elsőként a Szegedi Tudományegyetem földrajz– geológia szakán diplomázott, majd az ELTE Bölcsészettudományi Karán szerzett történelemtanári oklevelet. 1976-ban kandidált, 1989-ben lett a földrajztudomány doktora, 1993-ban egyetemi tanári kinevezést kapott. Az utóbbi két évtizedben közlekedésföldrajzzal, valamint a telematika területi hatásainak vizsgálatával foglalkozott. 2004-ben Baross Gábor-díjjal és a Magyar Köztársasági Érdemrend lovagkeresztjével, 2010-ben tisztikereszttel tüntették ki. Az itt olvasható, utólagosan strukturált interjú 2021 szeptemberében, otthonában készült. A jelen beszélgetés célja, hogy jobban megismertesse a professzor életének fontos mozzanatait, azt a miliőt, amely szerepet játszott szakmai életútjának alakulásában. Célunk továbbá, hogy ezen keresztül érzékeltessük a hazai földrajz erényeit, kihívásait és feladatait

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Unexpected attraction of polarotactic water-leaving insects to matt black car surfaces: mattness of paintwork cannot eliminate the polarized light pollution of black cars.

The horizontally polarizing surface parts of shiny black cars (the reflection-polarization characteristics of which are similar to those of water surfaces) attract water-leaving polarotactic insects. Thus, shiny black cars are typical sources of polarized light pollution endangering water-leaving insects. A new fashion fad is to make car-bodies matt black or grey. Since rough (matt) surfaces depolarize the reflected light, one of the ways of reducing polarized light pollution is to make matt the concerned surface. Consequently, matt black/grey cars may not induce polarized light pollution, which would be an advantageous feature for environmental protection. To test this idea, we performed field experiments with horizontal shiny and matt black car-body surfaces laid on the ground. Using imaging polarimetry, in multiple-choice field experiments we investigated the attractiveness of these test surfaces to various water-leaving polarotactic insects and obtained the following results: (i) The attractiveness of black car-bodies to polarotactic insects depends in complex manner on the surface roughness (shiny, matt) and species (mayflies, dolichopodids, tabanids). (ii) Non-expectedly, the matt dark grey car finish is much more attractive to mayflies (being endangered and protected in many countries) than matt black finish. (iii) The polarized light pollution of shiny black cars usually cannot be reduced with the use of matt painting. On the basis of these, our two novel findings are that (a) matt car-paints are highly polarization reflecting, and (b) these matt paints are not suitable to repel polarotactic insects. Hence, the recent technology used to make matt the car-bodies cannot eliminate or even can enhance the attractiveness of black/grey cars to water-leaving insects. Thus, changing shiny black car painting to matt one is a disadvantageous fashion fad concerning the reduction of polarized light pollution of black vehicles

First Synthesis of DBU-Conjugated Cationic Carbohydrate Derivatives and Investigation of Their Antibacterial and Antifungal Activity

The emergence of drug-resistant bacteria and fungi represents a serious health problem worldwide. It has long been known that cationic compounds can inhibit the growth of bacteria and fungi by disrupting the cell membrane. The advantage of using such cationic compounds is that the microorganisms would not become resistant to cationic agents, since this type of adaptation would mean significantly altering the structure of their cell walls. We designed novel, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene)-derived amidinium salts of carbohydrates, which may be suitable for disturbing the cell walls of bacteria and fungi due to their quaternary ammonium moiety. A series of saccharide-DBU conjugates were prepared from 6-iodo derivatives of d-glucose, d-mannose, d-altrose and d-allose by nucleophilic substitution reactions. We optimized the synthesis of a d-glucose derivative, and studied the protecting group free synthesis of the glucose-DBU conjugates. The effect of the obtained quaternary amidinium salts against Escherichia coli and Staphylococcus aureus bacterial strains and Candida albicans yeast was investigated, and the impact of the used protecting groups and the sugar configuration on the antimicrobial activity was analyzed. Some of the novel sugar quaternary ammonium compounds with lipophilic aromatic groups (benzyl and 2-napthylmethyl) showed particularly good antifungal and antibacterial activity

First Synthesis of DBU-Conjugated Cationic Carbohydrate Derivatives and Investigation of Their Antibacterial and Antifungal Activity

The emergence of drug-resistant bacteria and fungi represents a serious health problem worldwide. It has long been known that cationic compounds can inhibit the growth of bacteria and fungi by disrupting the cell membrane. The advantage of using such cationic compounds is that the microorganisms would not become resistant to cationic agents, since this type of adaptation would mean significantly altering the structure of their cell walls. We designed novel, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene)-derived amidinium salts of carbohydrates, which may be suitable for disturbing the cell walls of bacteria and fungi due to their quaternary ammonium moiety. A series of saccharide-DBU conjugates were prepared from 6-iodo derivatives of d-glucose, d-mannose, d-altrose and d-allose by nucleophilic substitution reactions. We optimized the synthesis of a d-glucose derivative, and studied the protecting group free synthesis of the glucose-DBU conjugates. The effect of the obtained quaternary amidinium salts against Escherichia coli and Staphylococcus aureus bacterial strains and Candida albicans yeast was investigated, and the impact of the used protecting groups and the sugar configuration on the antimicrobial activity was analyzed. Some of the novel sugar quaternary ammonium compounds with lipophilic aromatic groups (benzyl and 2-napthylmethyl) showed particularly good antifungal and antibacterial activity

Total numbers of reactions (touching, landing and aerial looping) of tabanids to the shiny black (sb), matt black (mb) and matt grey (mg) horizontal test surfaces in experiment 2.

<p>The inset is a photograph of a tabanid fly landed on the matt grey test surface. The number of repetition is 20 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103339#s4" target="_blank">Materials and methods</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103339#s3" target="_blank">Discussion</a>).</p

Degree of linear polarization <i>d</i> of the shiny black (sb), matt black (mb) and matt grey (mg) horizontal test surfaces used in experiments 1 and 2 measured with imaging polarimetry in the red (650 nm), green (550 nm) and blue (450 nm) parts of the spectrum when sun- and skylight (A, Supplementary Fig. S4I) or canopylight originating from trees and bushes (B, Supplementary Fig. S4II) was reflected by the test surfaces.

<p>Columns: averages. Vertical bars: standard deviations. The average is calculated for the whole area of each test surface (corresponding to 2500×4000 = 10 000 000 pixels in the pictures and polarization patterns).</p