scholarly journals Heterologous Hydrogenase Overproduction Systems for Biotechnology—An Overview

2020 ◽  
Vol 21 (16) ◽  
pp. 5890
Author(s):  
Qin Fan ◽  
Peter Neubauer ◽  
Oliver Lenz ◽  
Matthias Gimpel
Keyword(s):  
Production Systems ◽  
Research Area ◽  
H2 Production ◽  
Separation And Purification ◽  
Hydrogenase Maturation ◽  
Oxidation Of Hydrogen ◽  
Catalytic Mechanisms ◽  
High Yields

Hydrogenases are complex metalloenzymes, showing tremendous potential as H2-converting redox catalysts for application in light-driven H2 production, enzymatic fuel cells and H2-driven cofactor regeneration. They catalyze the reversible oxidation of hydrogen into protons and electrons. The apo-enzymes are not active unless they are modified by a complicated post-translational maturation process that is responsible for the assembly and incorporation of the complex metal center. The catalytic center is usually easily inactivated by oxidation, and the separation and purification of the active protein is challenging. The understanding of the catalytic mechanisms progresses slowly, since the purification of the enzymes from their native hosts is often difficult, and in some case impossible. Over the past decades, only a limited number of studies report the homologous or heterologous production of high yields of hydrogenase. In this review, we emphasize recent discoveries that have greatly improved our understanding of microbial hydrogenases. We compare various heterologous hydrogenase production systems as well as in vitro hydrogenase maturation systems and discuss their perspectives for enhanced biohydrogen production. Additionally, activities of hydrogenases isolated from either recombinant organisms or in vivo/in vitro maturation approaches were systematically compared, and future perspectives for this research area are discussed.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2301-2311 ◽  
Author(s):  
Markus Pötter ◽  
Helena Müller ◽  
Frank Reinecke ◽  
Roman Wieczorek ◽  
Florian Fricke ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Azotobacter Vinelandii ◽  
Production Systems ◽  
Complex Structure ◽  
Pcr Analysis ◽  
Unknown Protein ◽  
Considerable Impact ◽  
Unexpected Findings

Analysis of the genome sequence of the polyhydroxyalkanoate- (PHA) accumulating bacterium Ralstonia eutropha strain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20·0, 20·2, 19·6 and 20·2 kDa, respectively. RT-PCR analysis showed that phaP2, phaP3 and phaP4 were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts of R. eutropha had previously been shown by others. Although PhaP2 could not be localized in vivo on poly(3HB) granules, in vitro experiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues in Ralstonia solanacearum, Burkholderia fungorum and Azotobacter vinelandii. These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.

257 DIFFERENCES BETWEEN BOS TAURUS AND BOS INDICUS EMBRYOS: TRANSCRIPTS AMOUNTS

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
S. Wohlres-Viana ◽  
M. M. Pereira ◽  
A. P. Oliveira ◽  
J. H. M. Viana ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Production System ◽  
Production Systems ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Peroxiredoxin 1 ◽  
Vitro Production

The Zebu breeds (Bos indicus) are different from European breeds (Bos taurus) in some aspects of their reproductive physiology, including follicle recruitment, number of follicular waves, and oocyte ultrastructure. On the other hand, embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. The aim of this study was to evaluate the effect of breed (Gyr v. Holstein) within embryo production system (in vivo and in vitro), as well as effect of production systems within breeds on relative abundance of transcripts related to formation, survival, and subsequent development of blastocysts, such as those involved in water and small solutes transport (Aquaporins 3 and 11), blastocoel formation (Na+/K+-ATPase a1 and |52), and cellular stress response (Peroxiredoxin 1). For in vivo embryo production, donors were superstimulated with FSH and inseminated, and embryos were recovered 7 days after AI. For in vitro embryo production, oocytes recovered by ovum pickup were in vitro matured and fertilized and then cultured for 7 days in culture medium under 5% CO2 at 38.5°C. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA, USA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA, USA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA, USA). The cDNA were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed by REST© software. To evaluate breed effect within the production systems, 2 comparisons were performed: (1) in vivo: Gyr v. Holstein and (2) in vitro: Gyr v. Holstein, considering Holstein data as 1.00. To evaluate production system effect within breeds, 2 comparisons were performed: (1) Gyr: in vivo v. in vitro and (2) Holstein: in vivo v. in vitro, considering in vivo produced embryo data as 1.00. The results are shown as mean ± SEM. For in vivo comparison between breeds, Aquaporin 3 (1.66 ± 0.77), Na+/K+-ATPase a1 (1.61 ± 0.56), and Peroxiredoxin 1 (1.61 ± 0.66) were up-regulated (P < 0.05) in Gyr embryos when compared with Holstein embryos, whereas for in vitro comparison, no differences (P > 0.05) were found. For comparisons between production systems within breeds, only Peroxiredoxin 1 (0.31 ± 0.39) was down-regulated (P < 0.01) in in vitro produced Gyr embryos when compared with in vivo counterparts. No differences (P > 0.05) were found between production systems for the Holstein breed. In conclusion, these data suggest that there is a difference on gene expression between Bos taurus and Bos indicus blastocysts, but such difference between breeds can be attenuated by the in vitro production system, indicating an embryo adaptation to the in vitro culture conditions. The data also suggest that the in vitro production system can influence the amount of transcripts in Gyr embryos. Other genes should be evaluated for a better understanding of these differences. Financial support was provided by CNPq and FAPEMIG.

scholarly journals Prediction of Aerosol Deposition in the Human Respiratory Tract via Computational Models: A Review with Recent Updates

Atmosphere ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 137 ◽  
Author(s):  
Vu Khac Hoang Bui ◽  
Ju-Young Moon ◽  
Minhe Chae ◽  
Duckshin Park ◽  
Young-Chul Lee
Keyword(s):  
Respiratory Tract ◽  
Particle Deposition ◽  
Computational Models ◽  
Aerosol Particles ◽  
Three Dimensional ◽  
Aerosol Deposition ◽  
Research Area ◽  
Human Respiratory Tract

The measurement of deposited aerosol particles in the respiratory tract via in vivo and in vitro approaches is difficult due to those approaches’ many limitations. In order to overcome these obstacles, different computational models have been developed to predict the deposition of aerosol particles inside the lung. Recently, some remarkable models have been developed based on conventional semi-empirical models, one-dimensional whole-lung models, three-dimensional computational fluid dynamics models, and artificial neural networks for the prediction of aerosol-particle deposition with a high accuracy relative to experimental data. However, these models still have some disadvantages that should be overcome shortly. In this paper, we take a closer look at the current research trends as well as the future directions of this research area.

scholarly journals Spinasterol, 22,23-Dihydrospinasterol and Fernenol from Citrullus Colocynthis L. with Aphicidal Activity against Cabbage Aphid Brevicoryne Brassicae L.

Molecules ◽  
2020 ◽  
Vol 25 (9) ◽  
pp. 2184
Author(s):  
Maqsood Ahmed ◽  
Peiwen Qin ◽  
Mingshan Ji ◽  
Ran An ◽  
Hongxia Guo ◽  
...  
Keyword(s):  
Mass Spectrum ◽  
Brevicoryne Brassicae ◽  
Separation And Purification ◽  
Citrullus Colocynthis ◽  
Cabbage Aphid ◽  
Chemical Structures ◽  
Greenhouse Assay ◽  
Aphicidal Activity

Brevicoryne brassicae is a problematic pest in cabbage and other field crops. Synthetic pesticides are used to control this pest, but they are injurious for human health and the environment. The present study aimed to purify and identify the active compounds from Citrullus colocynthis leaves with an appraisal of their efficacy against B. brassicae. Separation and purification were performed via different chromatographic techniques. Molecular analysis and chemical structures were recognized by mass spectrum (MS) and nuclear magnetic resonance (NMR), respectively. Moreover, in vitro and in vivo aphicidal activity was assessed using various concentrations, i.e., 6.25, 12.5, 25 and 50 µg/mL at 12, 24, 48 and 72 h exposure. The outcome shows that mass spectrum analyses of the purified compounds suggested the molecular formulae are C30H50O and C29H50O, C29H48O. The compounds were characterized as fernenol and a mixture of spinasterol, 22,23-dihydrospinasterol by 1H-NMR and 13C-NMR spectrum analysis. The toxicity results showed that the mixture of spinasterol and 22,23-dihydrospinasterol showed LC50 values of 32.36, 44.49 and 37.50 µg/mL by contact, residual and greenhouse assay at 72 h exposure, respectively. In contrast, fernenol recorded LC50 values as 47.99, 57.46 and 58.67 µg/mL, respectively. On the other hand, spinasterol, 22,23-dihydrospinasterol showed the highest mortality, i.e., 66.67%, 53.33% and 60% while, 30%, 23.33% and 25% mortality was recorded by fernenol after 72 h at 50 µg/mL by contact, residual and greenhouse assay, respectively. This study suggests that spinasterol, 22,23-dihydrospinasterol are more effective against B. brassicae which may be introduced as an effective and suitable substitute of synthetic chemical pesticides.

scholarly journals Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase

2019 ◽  
Vol 60 (5) ◽  
pp. 953-962 ◽  
Author(s):  
Peter J. Harrison ◽  
Kenneth Gable ◽  
Niranjanakumari Somashekarappa ◽  
Van Kelly ◽  
David J. Clarke ◽  
...  
Keyword(s):  
In Vivo Studies ◽  
Serine Palmitoyltransferase ◽  
Biochemical Pathways ◽  
Catalytic Mechanisms ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
The Impact ◽  
Sphingolipid Biosynthesis

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis. Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.

scholarly journals Feline Coronavirus Antivirals: A Review

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1150
Author(s):  
Manon Delaplace ◽  
Hélène Huet ◽  
Adèle Gambino ◽  
Sophie Le Poder
Keyword(s):  
Research Area ◽  
Viral Escape ◽  
Viral Pathogens ◽  
Feline Infectious Peritonitis ◽  
Escape Mutants ◽  
Asymptomatic Infections ◽  
Therapeutic Compounds ◽  
Feline Coronavirus

Feline coronaviruses (FCoV) are common viral pathogens of cats. They usually induce asymptomatic infections but some FCoV strains, named Feline Infectious Peritonitis Viruses (FIPV) lead to a systematic fatal disease, the feline infectious peritonitis (FIP). While no treatments are approved as of yet, numerous studies have been explored with the hope to develop therapeutic compounds. In recent years, two novel molecules (GS-441524 and GC376) have raised hopes given the encouraging results, but some concerns about the use of these molecules persist, such as the fear of the emergence of viral escape mutants or the difficult tissue distribution of these antivirals in certain affected organs. This review will summarize current findings and leads in the development of antiviral therapy against FCoV both in vitro and in vivo, with the description of their mechanisms of action when known. It highlights the molecules, which could have a broader effect on different coronaviruses. In the context of the SARS-CoV-2 pandemic, the development of antivirals is an urgent need and FIP could be a valuable model to help this research area.

scholarly journals 127APOPTOSIS IN BOVINE BLASTOCYSTS FROM FIVE DIFFERENT PRODUCTION SYSTEMS

2004 ◽  
Vol 16 (2) ◽  
pp. 186
Author(s):  
J.O. Gjørret ◽  
P. Maddox-Hyttel
Keyword(s):  
Production Systems ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Embryo Cell ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Tunel Reaction

Regulation of apoptosis may be affected by factors during preimplantation development, and this is possibly related to embryo developmental potential. Here we investigate differences in the incidence of apoptotic nuclei in Day 7 bovine blastocysts produced by two different in vivo and three different in vitro methods. In vivo embryos were produced either by a regular superovulation procedure (reg group; n=29; Laurincik et al., 2003, Mol. Reprod. Dev. 65, 73–85), or by postponement of the LH surge (pp group; n=35; van de Leemput et al., 2001, Therio. 55, 573–592). In vitro embryos were derived from systems using either co-culture (cc group; n=30, Avery and Greve 2000, Mol. Reprod. Dev. 55, 438–445), or culture in synthetic oviduct fluid (SOF) with (S+group; n=35) or without serum (S− group; n=38; Holm et al., 1999, Theriogenology, 52, 683–700). Embryos were collected at approx. 168h post ovulation/insemination and subjected to chromatin staining and detection of DNA degradation by TUNEL reaction. The total number of nuclei, number of nuclei displaying apoptotic morphology (+M), number of nuclei displaying TUNEL reaction (+T), and number of nuclei displaying both markers simultaneously (M&amp;T) were scored according to J.O. Gjørret et al. (2003 Biol. Reprod. 69. in press). Only M&amp;T nuclei were regarded as apoptotic, and +M, +T, and apoptotic (M&amp;T) indices (%) were calculated for the trophoblast (tb), inner cell mass (i) and the total blastocysts (t) in each group. Significant differences were observed for all parameters when all groups were compared (ANOVA, P ranging from 0.024 to&lt;0.0001). Highest number of total nuclei were observed in the S+ group, whereas the lowest indices were observed in the pp group, which had significant lower indices in the i and t than in the reg., S+ and S− groups P&lt;0.05; Tukey’s post test for ANOVA). Highest indices were generally observed in the S− group. The results demonstrate that not only embryo cell numbers but also incidences of apoptotic markers are affected by the mode of production. However, in Day 7 bovine blastocysts high cell number is not consistent with a low incidence of apoptosis. Even though cell numbers appeared comparable in the two in vivo groups, their incidences of apoptosis were different, and the reg group displayed indices comparable to the in vitro groups, highlighting the importance of ovulation protocols when in vivo embryos are used as reference material in general. Table 1

Interaction among endophytic bacteria, sweet sorghum (Sorghum bicolor) cultivars and chemical nitrogen fertilization

2020 ◽  
Author(s):  
Gabriela Heijo ◽  
Cecilia Taulé ◽  
Cintia Mareque ◽  
Adriano Stefanello ◽  
Emanuel M Souza ◽  
...  
Keyword(s):  
Plant Growth ◽  
Nitrogen Fertilization ◽  
Environmental Sustainability ◽  
Sweet Sorghum ◽  
Endophytic Bacteria ◽  
Production Systems ◽  
N Fertilization ◽  
Plant Growth Promoting Bacteria

Abstract The application of new agricultural technologies to attain sustainable production systems is necessary. The use of plant growth-promoting bacteria to improve plant growth and health has been studied for decades. This work aimed to isolate diazotrophic endophytic bacteria associated with sweet sorghum plants and study the interaction of their inoculation in combination with chemical N-fertilization on different sorghum cultivars. A bacterial collection of 181 isolates was constructed and characterized in vitro and in vivo. From that, the strains Enterobacter sp. UYSB89 and Kosakonia sp. UYSB139 were nifH+, produce IAA, defined as true endophytes and able to promote growth of two sweet sorghum under greenhouse conditions. The evaluated cultivars responded differentially to bacterial inoculation, the nitrogen fertilization doses and their interaction. Thus, plant growth is a multifactorial consequence of the interrelation between crop practices and the plant genotypes. This knowledge is a valuable factor in terms of understanding plant-bacteria endophyte interactions to preserve environmental sustainability during the implementation of agronomic practices.

Medical applications of X-ray fluorescence for trace element research

2007 ◽  
Vol 22 (2) ◽  
pp. 130-137 ◽  
Author(s):  
Jimmy Börjesson ◽  
Sören Mattsson
Keyword(s):  
Element Concentration ◽  
Prostate Gland ◽  
Blood Lead ◽  
Research Area ◽  
X Ray ◽  
Bone Lead ◽  
Occupationally Exposed ◽  
Lead Workers

Techniques for estimation of element levels directly in humans (noninvasivein vivo) or in samples (in vitro) from humans are reviewed. Toxic, nonessential, trace elements may cause temporary or permanent damage to various organs and tissues in humans. There is thus a need to control the concentrations. Knowledge of the relations between toxic effects and element concentration may be extracted from measurements in humans as well as in samples from humans and her environment. Applications traditionally include occupationally exposed subjects, but an increasing research area is studies of members of the general population and of patients undergoing therapy for malignant and other diseases. Mostin vivoXRF studies deal with lead in bone and cadmium in kidneys. For retired lead workers, a clear association has been demonstrated between bone lead and blood lead, due to endogenous lead excretion from the skeleton. A study of mercuryin vivoshowed that the technique is capable of detecting mercury in heavily exposed worker’s kidneys.In vivoXRF in cancer and rheumatology patients has helped to understand how platinum and gold are retained in the human body. The newestin vivoapplications include zinc in prostate gland and arsenic in skin.

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